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Sample records for oocytes

  1. [Mitochondrial and oocyte development].

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    Deng, Wei-Ping; Ren, Zhao-Rui

    2007-12-01

    Oocyte development and maturation is a complicated process. The nuclear maturation and cytoplasmic maturation must synchronize which can ensure normal oocyte fertilization and following development. Mitochondrial is the most important cellular organell in cytoplasm, and the variation of its distribution during oocyte maturation, the capacity of OXPHOS generating ATP as well as the content or copy number or transcription level of mitochondrial DNA play an important role in oocyte development and maturation. Therefore, the studies on the variation of mitochondrial distribution, function and mitochondrial DNA could enhance our understanding of the physiology of reproduction and provide new insight to solve the difficulties of assisted reproduction as well as cloning embryo technology.

  2. Oocyte cryopreservation in oncological patients.

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    Porcu, Eleonora; Fabbri, Raffaella; Damiano, Giuseppe; Fratto, Rosita; Giunchi, Susanna; Venturoli, Stefano

    2004-04-05

    The use of chemotherapy and radiotherapy in oncological patients may reduce their reproductive potential. Sperm cryopreservation has been already used in men affected by neoplastic disease. Oocyte cryopreservation might be an important solution for these patients at risk of losing ovarian function. A program of oocyte cryopreservation for oncological patients is also present in our center. From June 1996 to January 2000, 18 patients awaiting chemotherapy and radiotherapy for neoplastic disease were included in our oocyte cryopreservation program. Our experience documents that oocyte storage may be a concrete and pragmatic alternative for oncological patients. The duration of oocyte storage does not seem to interfere with oocyte survival as pregnancies occurred even after several years of gamete cryopreservation in liquid nitrogen.

  3. Diving into the oocyte pool

    DEFF Research Database (Denmark)

    Kristensen, Stine G; Pors, Susanne E; Yding Andersen, Claus

    2017-01-01

    PURPOSE OF REVIEW: The ovarian reserve comprises an enormous surplus of follicles. Despite this, some women produce insufficient numbers of oocytes by conventional fertility treatments. However, recent technical accomplishments may transform assisted reproductive technology (ART) in such a way...... for revitalizing deficient oocytes may transform ART, and potentially enhance both quantity and quality of fertilizable oocytes; hereby augmenting the pregnancy potential of women with poor reproductive performance....

  4. Cryopreservation of unfertilized human oocytes.

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    Stachecki, James J; Cohen, Jacques; Garrisi, John; Munné, Santiago; Burgess, Colleen; Willadsen, Steen M

    2006-08-01

    Previous investigations revealed that choline-based freezing media developed in our laboratory were superior to conventional sodium-based media for storing mouse oocytes. This paper examines the ability of the choline-based medium CJ2 and a modified form of this medium, CJ3, to cryopreserve unfertilized human oocytes. Oocytes that were consented for research and matured overnight, as well as freshly collected, donor, mature metaphase II (MII) oocytes, were cryopreserved using choline-based media and an optimized slow-cooling protocol. The results showed higher survival and fertilization rates when CJ3 supplemented with 0.2 mmol/l sucrose was used as compared with CJ2 supplemented with either 0.1 mmol/l or 0.2 mmol/l sucrose. Freshly collected oocytes were more difficult to cryopreserve than those matured in vitro. Modification of the base medium proved to be one of the key factors in obtaining survival rates over 90%. Fertilization rates, embryo development, and genetic analysis of embryos resulting from control and frozen-thawed oocytes are provided. There appears to be a high correlation between chromosomal anomalies and abnormal morphology in embryos from thawed oocytes.

  5. Clinical benefit of metaphase I oocytes

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    Van der Elst Josiane

    2005-12-01

    Full Text Available Abstract Background We studied the benefit of using in vitro matured metaphase I (MI oocytes for ICSI in patients with a maximum of 6 mature metaphase II (MII oocytes at retrieval. Methods In 2004, 187 ICSI cycles were selected in which maximum 6 MII oocytes and at least one MI oocyte were retrieved. MI oocytes were put in culture to mature until the moment of ICSI, which was performed between 2 to 11 hours after oocyte retrieval (day 0. In exceptional cases, when the patient did not have any mature oocyte at the scheduled time of ICSI, MI oocytes were left to mature overnight and were injected between 19 to 26 hours after retrieval (day 1. Embryos from MI oocytes were chosen for transfer only when no other good quality embryos from MII oocytes were available. Outcome parameters were time period of in vitro maturation (IVM, IVM and fertilization rates, embryo development, clinical pregnancy rates, implantation rates and total MI oocyte utilization rate. Results The overall IVM rate was 43%. IVM oocytes had lower fertilization rates compared to in vivo matured sibling oocytes (52% versus 68%, P Conclusion Fertilization of in vitro matured MI oocytes can result in normal embryos and pregnancy, making IVM worthwhile, particularly when few MII oocytes are obtained at retrieval.

  6. Oocyte development, meiosis and aneuploidy.

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    MacLennan, Marie; Crichton, James H; Playfoot, Christopher J; Adams, Ian R

    2015-09-01

    Meiosis is one of the defining events in gametogenesis. Male and female germ cells both undergo one round of meiotic cell division during their development in order to reduce the ploidy of the gametes, and thereby maintain the ploidy of the species after fertilisation. However, there are some aspects of meiosis in the female germline, such as the prolonged arrest in dictyate, that appear to predispose oocytes to missegregate their chromosomes and transmit aneuploidies to the next generation. These maternally-derived aneuploidies are particularly problematic in humans where they are major contributors to miscarriage, age-related infertility, and the high incidence of Down's syndrome in human conceptions. This review will discuss how events that occur in foetal oocyte development and during the oocytes' prolonged dictyate arrest can influence meiotic chromosome segregation and the incidence of aneuploidy in adult oocytes.

  7. Evolutionary conservation of the mature oocyte proteome

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    Tamar Lotan

    2014-06-01

    Significance: The current study provides the first proteomic profile of an oocyte of a cnidarian organism the starlet sea anemone N. vectensis and gives new insights on the ancient origin of an oocyte proteome template. The comparative analysis with a chordate oocyte suggests that the oocyte proteome predates the divergence of the cnidarian and bilaterian lineages. In addition, the data generated in the study will serve as a valuable resource for further developmental and evolutional studies.

  8. Effect of Collection Technique on Yield of Bovine Oocytes and the Development Potential of Oocytes from Different Grades of Oocytes

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    R.G Sianturi

    2002-10-01

    Full Text Available Oocyte collection technique is important to obtain a maximum number of oocytes to be employed on in vitro production of embryos. In this study, immature bovine oocytes were collected from slaughterhouse ovaries by two techniques: aspiration of 2- to 6-mm follicles and slicing. Following collection, oocyte qualities were classified into four categories (A, B, C, and D on the basis of cumulus attachment. Oocytes of each category were matured in vitro in CO2 incubator for 22-24 hours and cumulus expansion and maturation rates were observed. The total number of oocytes (group A+B+C+D and yield of good quality oocytes (only group A and B recovered per ovary by aspiration were 12.02 and 8.21, and by slicing were 29.38 and 19.65 (P<0.01, respectively. The total cumulus cells expansion rates of A, B, C and D oocytes were 97.1%, 88.3%, 6.0% and 20.6% respectively. Maturation rates for A, B and C categories of oocytes were 91.4%, 82.3% and 35.0% respectively while no matured oocyte was observed for group D oocytes. Maturation rates were significantly different between group A and C and also between B and C but not between A and B (P<0.05. In conclusion, slicing technique recovered more oocytes per ovary (2.4 times than that of aspiration and the best maturation rate was observed from category A oocytes which surrounded by more than 3 layers of cumulus cells. However oocytes of category A and B can be considered as good quality oocytes.

  9. Human oocyte maturation in vitro.

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    Coticchio, Giovanni; Dal-Canto, Mariabeatrice; Guglielmo, Maria-Cristina; Mignini-Renzini, Mario; Fadini, Rubens

    2012-01-01

    Oocytes from medium-sized antral follicles have already completed their growth phase and, if released from the follicular environment and cultured in vitro, are able to resume the meiotic process and mature. However, in vitro maturation (IVM) does not entirely support all the nuclear and cytoplasmic changes that occur physiologically as an effect of the ovulatory stimulus. Regardless, oocyte IVM is widely applied for the breeding of agriculturally important species. In assisted reproduction technology, IVM has been proposed as an alternative treatment to circumvent the drawbacks of standard ovarian stimulation regimens. Initially introduced to eliminate the risks of ovarian hyperstimulation syndrome afflicting women presenting with polycystic ovaries, subsequently IVM has been suggested to represent an additional approach suitable also for normovulatory patients. So far, in children born from IVM cycles, no doubts of an increased incidence of congenital abnormalities have been raised. Many more births would be achieved if novel IVM systems, currently dominated by empiricism, could be conceived according to more physiological criteria. Recent findings shedding new light on the control of meiotic progression, the support of cumulus cells to the oocyte cellular reorganization occurring during maturation, and the modulation of the stimulus that promotes oocyte maturation downstream the mid-cycle gonadotropin signal are likely to provide crucial hints for the development of more efficient IVM systems.

  10. Recent Progress in Cryopreservation of Bovine Oocytes

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    In-Sul Hwang

    2014-01-01

    Full Text Available Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK inhibitor. Use of an antioxidant α-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation.

  11. Cold-induced changes in amphibian oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Angelier, N.; Moreau, N.A.; N' Da, E.A.; Lautredou, N.F. (Centre de Biologie Cellulaire, Ivry-sur-Seine (France))

    1989-08-01

    Female Pleurodeles waltl newts (Amphibia, urodele), usually raised at 20 degrees C, were submitted to low temperatures; oocytes responded to this cold stress by drastic changes both in lampbrush chromosome structure and in protein pattern. Preexisting lateral loops of lampbrush chromosomes were reduced in size and number, while cold-induced loops which were tremendously developed, occurred on defined bivalents of the oocyte at constant, reproducible sites. A comparison of protein patterns in control and stressed oocytes showed two main differences: in stressed oocytes, overall protein synthesis was reduced, except for a set of polypeptides, the cold-stress proteins; second, there was a striking inversion of the relative amount of beta- and gamma-actin found in the oocyte nucleus before and after cold stress. Whereas beta-actin was the predominant form in control oocytes, gamma-actin became the major form in stressed oocytes.

  12. Caffeine delays oocyte aging and maintains the quality of aged oocytes safely in mouse.

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    Zhang, Xia; Liu, Xiaoyan; Chen, Li; Wu, Dan-Ya; Nie, Zheng-Wen; Gao, Ying-Ying; Miao, Yi-Liang

    2017-03-28

    Caffeine, as an oocyte aging inhibitor, was used in many different species to control or delay oocyte aging. However, the safety of caffeine and developmental competence of aged oocytes inhibited by caffeine has not been studied systematically. So we detected the spindle morphology, distribution of cortical granules, zona pellucida hardening and pronucleus formation to assess oocyte quality of caffeine treated oocytes. We found that aged oocytes treated by caffeine maintained weak susceptibility to activating stimuli and regained normal competent after aged further 6 hr. Caffeine maintained the spindle morphology, changed cortical granules distribution of aged oocytes and could not prevent zona pellucida hardening. Furthermore, caffeine increased pronucleus formation of aged oocytes and decreased fragmentation after fertilization. These results suggested that caffeine could maintain the quality of aged oocytes safely in mouse.

  13. Relationship between the number of cells surrounding oocytes and energy states of oocytes.

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    Munakata, Yasuhisa; Ichinose, Tomoya; Ogawa, Kaori; Itami, Nobuhiko; Tasaki, Hidetaka; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2016-10-15

    Lipid content, ATP content, and histone acetylation are thought to reflect the energy state of cells. In addition, the energy state closely associates with growth and developmental ability of oocytes. Oocyte growth is accompanied by active proliferation of the surrounding granulosa cells (GCs), and GCs play a key role in the provision of energy substrates to the oocytes. In the present study, we first examined the relationship among the average number of GCs per follicle, the average number of cumulus cells (CCs) per oocyte, and the average lipid content in oocytes that developed in vivo within individual donor gilts. Second, we validated the relationship between the number of cells surrounding oocytes and the energy states of oocytes by using an IVC system of oocyte granulosa cell complexes (OGCs) derived from early antral follicles. We collected cumulus cells and oocyte complexes (COCs) from antral follicles (3-5 mm in diameter) and found that average lipid content in oocytes significantly correlated with the average number of both GCs/follicle and CCs/oocyte (P cell number of OGCs, as well as the lipid content, ATP content, and acetylation level of H4K12 in oocytes grown in vitro. In addition, glucose consumption by OGCs was calculated from the sample media collected at Days 13 and 14. The lipid content of oocytes grown in vitro, significantly correlated with the number of cells surrounding the oocytes (P number of cells surrounding the oocytes (P number of cells surrounding the oocytes, and glucose uptake by OGCs is crucial for lipid content and ATP content, and H4K12 acetylation in oocytes.

  14. [Controversy in ART: should we cryopreserve oocytes or embryos? Do prefer oocytes].

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    Boyer, P

    2014-09-01

    Since the beginning of IVF, cryopreservation concern spermatozoa or embryos due to the poor efficiency of oocyte freezing. To date, oocyte vitrification allows changing our practice privileging female gamete vitrification instead of human embryo freezing.

  15. Molecular and structural aspects of oocyte maturation

    NARCIS (Netherlands)

    Hölzenspies, J.J.

    2009-01-01

    In the mammalian ovary, oocytes are contained within follicles, specialized structures that facilitate oocyte growth and development. During the reproductive cycle, several follicles are recruited into growth, and through a process of selection, one (human, cow) or several (mouse, pig) of these foll

  16. Apoptosis in mammalian oocytes: a review.

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    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

  17. [FERTILITY PRESERVATION OF WOMEN: OOCYTE VITRIFICATION].

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    Montserrat, Pallas Seijas

    2015-09-01

    Cryopreservation ofhuman oocytes to delay fertility also be an option for women who are going to be subjected to a cancer/autoimmune treatment. It allows for creating a bank of oocytes for donation in assisted reproduction centers. The legislation allows the use of cryopreserved oocytes throughout the reproductive life of women with what conservation could last up to 48-50 years. Oocyte vitrification is a ultrafast freezing method in which cryoprotectants are used to prevent the formation of ice crystals within the cell. Treatment for oocyte vitrification process is similar to IVF treatment, ending at the time of obtaining the ova. The eggs obtained in the laboratory are classified according to maturity and quality. The apartments will be cryopreserved by vitrification technique tanks and maintained in liquid nitrogen until used for reproductive purposes.

  18. Diffused Intra-Oocyte Hydrogen Peroxide Activates Myeloperoxidase and Deteriorates Oocyte Quality.

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    Sana N Khan

    Full Text Available Hydrogen peroxide (H2O2 is a relatively long-lived signaling molecule that plays an essential role in oocyte maturation, implantation, as well as early embryonic development. Exposure to relatively high levels of H2O2 functions efficiently to accelerate oocyte aging and deteriorate oocyte quality. However, little precise information exists regarding intra-oocyte H2O2 concentrations, and its diffusion to the oocyte milieu. In this work, we utilized an L-shaped amperometric integrated H2O2-selective probe to directly and quantitatively measure the real-time intra-oocyte H2O2 concentration. This investigation provides an exact measurement of H2O2 in situ by reducing the possible loss of H2O2 caused by diffusion or reactivity with other biological systems. This experiment suggests that the intra-oocyte H2O2 levels of oocytes obtained from young animals are reasonably high and remained constant during the procedure measurements. However, the intra-oocyte H2O2 concentration dropped significantly (40-50% reduction in response to catalase pre-incubation, suggesting that the measurements are truly H2O2 based. To further confirm the extracellular diffusion of H2O2, oocytes were incubated with myeloperoxidase (MPO, and the diffused H2O2 triggered MPO chlorinating activity. Our results show that the generated hypochlorous acid (HOCl facilitated the deterioration in oocyte quality, a process that could be prevented by pre-incubating the oocytes with melatonin, which was experimentally proven to be oxidized utilizing HPLC methods. This study is the first to demonstrate direct quantitative measurement of intracellular H2O2, and its extracellular diffusion and activation of MPO as well as its impact on oocyte quality. These results may help in designing more accurate treatment plans in assisted reproduction under inflammatory conditions.

  19. The DNA damage response in mammalian oocytes

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    John eCarroll

    2013-06-01

    Full Text Available DNA damage is one of the most common insults that challenge all cells. To cope, an elaborate molecular and cellular response has evolved to sense, respond to and correct the damage. This allows the maintenance of DNA fidelity essential for normal cell viability and the prevention of genomic instability that can lead to tumour formation. In the context of oocytes, the impact of DNA damage is not one of tumour formation but of the maintenance of fertility. Mammalian oocytes are particularly vulnerable to DNA damage because physiologically they may lie dormant in the ovary for many years (>40 in humans until they receive the stimulus to grow and acquire the competence to become fertilized. The implication of this is that in some organisms, such as humans, oocytes face the danger of cumulative genetic damage for decades. Thus, the ability to detect and repair DNA damage is essential to maintain the supply of oocytes necessary for reproduction. Therefore, failure to confront DNA damage in oocytes could cause serious anomalies in the embryo that may be propagated in the form of mutations to the next generation allowing the appearance of hereditary disease. Despite the potential impact of DNA damage on reproductive capacity and genetic fidelity of embryos, the mechanisms available to the oocyte for monitoring and repairing such insults have remained largely unexplored until recently. Here, we review the different aspects of the response to DNA damage in mammalian oocytes. Specifically, we address the oocyte DNA damage response from embryonic life to adulthood and throughout oocyte development.

  20. Fractal organization of feline oocyte cytoplasm.

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    De Vico, G; Peretti, V; Losa, G A

    2005-01-01

    The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display self-similar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400x with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD). The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.

  1. Fractal organization of feline oocyte cytoplasm

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    G De Vico

    2009-06-01

    Full Text Available The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display selfsimilar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400 X with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD. The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.

  2. Age-dependent radiosensitivity of mouse oocytes

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    Koehler, C.

    1976-06-08

    It has been shown that there are three distinct phases of radiosensitivity in oocytes of prepubertal mice: a period of rapidly increasing sensitivity between 0 and 4 days of age; a period of consistent, high sensitivity between 5 and 18 days of age; and a period of decreasing sensitivity from 19 to at least 21 days of age. Two distinct phases have been demonstrated for the rate of population decline of the oocytes of primary follicles: an initial period of rapid loss from 0 to 4 days of age; and a period of much slower loss from 5 through 23 days of age. Correlations have been drawn between the first two phases of radiosensitivity and morphological changes in the oocyte, and between the third phase of radiosensitivity and endocrinological changes in the maturing animal. The reaction of oocytes to radiation has been separated into two categories: immediate death (within 24 hours); and delayed death (over the entire lifespan of the animal). (auth)

  3. Oocyte Maturation Process and Affecting Factors

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    Yurdun Kuyucu

    2009-08-01

    Full Text Available Normal female fertility depends on normally occuring oogenesis and maturation progress. Oogenesis and folliculogenesis are different progresses but occure in a harmony and at the same time. Oogenesis includes the events that take place matur ovum produced from primordial germ cells. Although folliculogenesis includes the stages primordial, primary, secondary, matur (Graaf follicules in the influece of gonadotropines and local growth factors. During oocyte maturation meiosis is distrupted till the puberty. Under LH influence it starts again and first meiosis completes before ovulation. Oocyte maturation can be regarded as the process of coming metaphase II from prophase I of oocyte at the puberty and can be studied as nuclear and cytoplasmic maturation. Meiosis is completed when fertilization occures and zygot is formed. In this article oogenesis, folliculogenesis and oocyte maturation process are summerized with related studies and reiews are revised. [Archives Medical Review Journal 2009; 18(4.000: 227-240

  4. Oocyte Cryopreservation in Human Assited Reproduction

    Institute of Scientific and Technical Information of China (English)

    J Konc; S Cseh; E Varga; R Kriston; K Kanyó

    2006-01-01

    Embryo cryopreservation(CP) has became a very important part of the clinical use of in vitro fertilization. Oocyte CP offers more advantages compared with embryo freezing with regard to less ethical, legal and moral problems. However, the efficiency of this procedure is still low, which prevents its clinical application in wide range. The aim of our paper is to review the basic principles, technical and safety aspects and current status of oocyte cryopreservation in human assisted reproduction.

  5. Bovine cumulus-oocyte disconnection in vitro

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul

    1987-01-01

    Cumulus-oocyte complexes were obtained from cows by aspiration of small (1-6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and processed for transmission electron microscopy after different periods of culture including a 0...... frequency of gap junctions was maintained until 12-18 h of culture where the junctional contact was completely disrupted. This decrease in intercellular communication was parallelled by resumption of oocyte meiosis....

  6. Metabolic Determinants of Mitochondrial Function in Oocytes.

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    Seidler, Emily A; Moley, Kelle H

    2015-11-01

    Mitochondrial production of cellular energy is essential to oocyte function, zygote development and successful continuation of pregnancy. This review focuses on several key functions of healthy oocyte mitochondria and the effect of pathologic states such as aging, oxidative stress and apoptosis on these functions. The effect of these abnormal conditions is presented in terms of clinical presentations, specifically maternal obesity, diminished ovarian reserve and assisted reproductive technologies.

  7. IL-6 and mouse oocyte spindle.

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    Jashoman Banerjee

    Full Text Available Interleukin 6 (IL-6 is considered a major indicator of the acute-phase inflammatory response. Endometriosis and pelvic inflammation, diseases that manifest elevated levels of IL-6, are commonly associated with higher infertility. However, the mechanistic link between elevated levels of IL-6 and poor oocyte quality is still unclear. In this work, we explored the direct role of this cytokine as a possible mediator for impaired oocyte spindle and chromosomal structure, which is a critical hurdle in the management of infertility. Metaphase-II mouse oocytes were exposed to recombinant mouse IL-6 (50, 100 and 200 ng/mL for 30 minutes and subjected to indirect immunofluorescent staining to identify alterations in the microtubule and chromosomal alignment compared to untreated controls. The deterioration in microtubule and chromosomal alignment were evaluated utilizing both fluorescence and confocal microscopy, and were quantitated with a previously reported scoring system. Our results showed that IL-6 caused a dose-dependent deterioration in microtubule and chromosomal alignment in the treated oocytes as compared to the untreated group. Indeed, IL-6 at a concentration as low as 50 ng/mL caused deterioration in the spindle structure in 60% of the oocytes, which increased significantly (P<0.0001 as IL-6 concentration was increased. In conclusion, elevated levels of IL-6 associated with endometriosis and pelvic inflammation may reduce the fertilizing capacity of human oocyte through a mechanism that involves impairment of the microtubule and chromosomal structure.

  8. Computer-assisted oocyte morphometry before ICSI: correlation of oocyte measurements with fertilization and embryo development.

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    Camargos, Maria das Graças R S; Lobach, Veronica N M; Pereira, Francisco A N; Lemos, Cláudia N C D; Reis, Fernando M; Camargos, Aroldo F

    2012-03-01

    The present study aimed to correlate morphometric parameters of the oocytes with the occurrence of fertilization following intracytoplasmic sperm injection (ICSI). In a prospective, controlled cohort design, women (n = 32) who were candidates for ICSI had oocytes (n = 258) collected and submitted to morphometric evaluation using the Cronus3 software program. The morphometric parameters obtained were oocyte diameter, perivitelline space width, zona pellucida thickness, and first polar body diameter. The median oocyte diameter was similar in cases in which fertilization occurred compared with those in which fertilization failed (75.2 and 75.9 μm, respectively; P = .218). The 2 groups also had similar measurements of perivitelline space, zona pellucida, and first polar body. However, the best quality zygotes identified by a morphological score resulted from oocytes with larger diameter (75.6 vs 74.0 μm; P < .01) and narrow perivitelline space (5.3 vs 7.1 μm; P < .01). Embryo development, as assessed by cleavage at second day of culture, was not significantly associated with oocyte morphometric parameters. These findings suggest that morphometric parameters of the oocytes do not correlate with the occurrence of fertilization following ICSI but may assist in selecting oocytes more likely to originate high-quality zygotes.

  9. Nuclear structures in Tribolium castaneum oocytes.

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    Bogolyubov, Dmitry S; Batalova, Florina M; Kiselyov, Artyom M; Stepanova, Irina S

    2013-10-01

    The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs. We also identified the counterparts of 'universal' extrachromosomal nuclear domains, Cajal bodies (CBs) and interchromatin granule clusters (IGCs). Nuclear bodies containing IGC marker protein SC35 display some features unusual for typical IGCs. SC35 domains in T. castaneum oocytes are predominantly fibrillar complex bodies that do not contain trimethyl guanosine (TMG)-capped small nuclear (sn) RNAs. Microinjections of 2'-O-methyl (U)22 probes into the oocytes allowed revealing poly(A)+ RNAs in these nuclear domains. Several proteins related to mRNA export (heterogeneous ribonucleoprotein core protein A1, export adapters Y14 and Aly and export receptor NXF1) were also detected there. We believe that unusual SC35 nuclear domains of T. castaneum oocytes are possibly involved in mRNP but not snRNP biogenesis.

  10. Regulation of oocyte growth in the mouse ovary

    DEFF Research Database (Denmark)

    Krarup, T.; Pedersen, T.; Faber, M.

    1969-01-01

    MICE are born with a finite number of oocytes which develop in foetal life from primordial oogonia and their direct mitotic progeny. After birth no new oocytes are formed, and the total number of oocytes decreases with advancing age. During the first 2 weeks of life this decrease is due to degene......MICE are born with a finite number of oocytes which develop in foetal life from primordial oogonia and their direct mitotic progeny. After birth no new oocytes are formed, and the total number of oocytes decreases with advancing age. During the first 2 weeks of life this decrease is due...

  11. Oocyte cryopreservation for donor egg banking.

    Science.gov (United States)

    Cobo, Ana; Remohí, José; Chang, Ching-Chien; Nagy, Zsolt Peter

    2011-09-01

    Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages

  12. Apoptosis maintains oocyte quality in aging Caenorhabditis elegans females.

    Directory of Open Access Journals (Sweden)

    Sara Andux

    2008-12-01

    Full Text Available In women, oocytes arrest development at the end of prophase of meiosis I and remain quiescent for years. Over time, the quality and quantity of these oocytes decreases, resulting in fewer pregnancies and an increased occurrence of birth defects. We used the nematode Caenorhabditis elegans to study how oocyte quality is regulated during aging. To assay quality, we determine the fraction of oocytes that produce viable eggs after fertilization. Our results show that oocyte quality declines in aging nematodes, as in humans. This decline affects oocytes arrested in late prophase, waiting for a signal to mature, and also oocytes that develop later in life. Furthermore, mutations that block all cell deaths result in a severe, early decline in oocyte quality, and this effect increases with age. However, mutations that block only somatic cell deaths or DNA-damage-induced deaths do not lower oocyte quality. Two lines of evidence imply that most developmentally programmed germ cell deaths promote the proper allocation of resources among oocytes, rather than eliminate oocytes with damaged chromosomes. First, oocyte quality is lowered by mutations that do not prevent germ cell deaths but do block the engulfment and recycling of cell corpses. Second, the decrease in quality caused by apoptosis mutants is mirrored by a decrease in the size of many mature oocytes. We conclude that competition for resources is a serious problem in aging germ lines, and that apoptosis helps alleviate this problem.

  13. Ultrastructure of quiescent oocytes of Cebus albifrons.

    Science.gov (United States)

    Barton, B R; Hertig, A T

    1975-11-01

    Quiescent oocytes of the monkey Cebus albifrons were examined with the electron microscope. In many respects the ultrastructure of these cells was similar to that of other mammalian species. Elongate and oval mitochondria, lamellar Golgi complexes, small profiles of smooth endoplasmic reticulum, and vacuolar organelles were randomly distributed around a round nucleus which usually contained a nucleolus and clumps of heterochromatin. Among the unusual morphological characteristics of these oocytes are 'membranous aggregates', membrane-bound organelles containing a complex of convoluted membranes, some very dense rod-like structures and a droplet of moderate density which resembles lipid. A similar droplet is frequently found in mitochondria. Rough endoplasmic reticulum is abundant in many of these oocytes, forming parallel arrays and concentric rings around the nucleus. Folded membrane complexes, apparent elaborations of smooth endoplasmic reticulum, are frequently found in the cytoplasm in continuity with cisternae of smooth and rough endoplasmic reticulum and associated with vesicles which often contain flocculent material. The morphology of Cebus oocytes suggests a greater rate of steroid and protein synthesis, transport, and storage than is usually indicated by the ultrastructure of other mammalian oocytes.

  14. Polarized light microscopy in mammalian oocytes.

    Science.gov (United States)

    Caamaño, J N; Muñoz, M; Diez, C; Gómez, E

    2010-06-01

    The meiotic spindle structure plays a key role in normal chromosome alignment and segregation during meiosis. Polarized light microscopy (PLM) allows non-invasive evaluation of the meiotic spindle of metaphase oocytes from different animal species. The purpose of this article is to review the use of PLM in animal reproduction, mainly in the assessment of the meiotic spindle in oocytes. A brief overview of the methods to assess the meiotic spindle is presented as well as the principles behind the PLM. The use of PLM to evaluate oocyte quality and spindle morphology is discussed and the results on the viability of the oocytes after being exposed to PLM are presented. Several researchers showed that PLM could be successfully implemented on cryopreservation, nuclear transfer and intracytoplasmic sperm injection procedures as a tool to improve the outcome of these procedures. In addition, PLM can be used to develop studies on oocyte maturation and spindle dynamics. However, the information on the practical use of this technology in farm animals is very limited and further studies are needed to assess the importance of PLM in animal reproduction.

  15. Control of nucleus positioning in mouse oocytes.

    Science.gov (United States)

    Almonacid, Maria; Terret, Marie-Emilie; Verlhac, Marie-Hélène

    2017-08-12

    The position of the nucleus in a cell can instruct morphogenesis in some cases, conveying spatial and temporal information and abnormal nuclear positioning can lead to disease. In oocytes from worm, sea urchin, frog and some fish, nucleus position regulates embryo development, it marks the animal pole and in Drosophila it defines the future dorso-ventral axis of the embryo and of the adult body plan. However, in mammals, the oocyte nucleus is centrally located and does not instruct any future embryo axis. Yet an off-center nucleus correlates with poor outcome for mouse and human oocyte development. This is surprising since oocytes further undergo two extremely asymmetric divisions in terms of the size of the daughter cells (enabling polar body extrusion), requiring an off-centering of their chromosomes. In this review we address not only the bio-physical mechanism controlling nucleus positioning via an actin-mediated pressure gradient, but we also speculate on potential biological relevance of nuclear positioning in mammalian oocytes and early embryos. Copyright © 2017. Published by Elsevier Ltd.

  16. Motility contrast imaging of live porcine cumulus-oocyte complexes

    Science.gov (United States)

    An, Ran; Turek, John; Machaty, Zoltan; Nolte, David

    2013-02-01

    Freshly-harvested porcine oocytes are invested with cumulus granulosa cells in cumulus-oocyte complexes (COCs). The cumulus cell layer is usually too thick to image the living oocyte under a conventional microscope. Therefore, it is difficult to assess the oocyte viability. The low success rate of implantation is the main problem for in vitro fertilization. In this paper, we demonstrate our dynamic imaging technique called motility contrast imaging (MCI) that provides a non-invasive way to monitor the COCs before and after maturation. MCI shows a change of intracellular activity during oocyte maturation, and a measures dynamic contrast between the cumulus granulosa shell and the oocytes. MCI also shows difference in the spectral response between oocytes that were graded into quality classes. MCI is based on shortcoherence digital holography. It uses intracellular motility as the endogenous imaging contrast of living tissue. MCI presents a new approach for cumulus-oocyte complex assessment.

  17. Calcium ion currents mediating oocyte maturation events

    Directory of Open Access Journals (Sweden)

    Tosti Elisabetta

    2006-05-01

    Full Text Available Abstract During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed.

  18. Growth of primordial oocytes in neonatal and adult mammals.

    Science.gov (United States)

    Moniruzzaman, Mohammad; Miyano, Takashi

    2010-12-01

    Mammalian ovaries are endowed with a huge number of small oocytes (primordial oocytes) in primordial follicles. A small number of primordial oocytes start to grow, while others remain quiescent. Little is known about the mechanism regulating the activation of primordial oocytes. Recently, we found that primordial follicles in mature cows and prepubertal pigs took longer to initiate growth in xenografts compared with those in neonatal animals. We think that primordial oocytes in adult mammals are different from those in neonatal mammals. In this review, we summarize the results regarding the activation of primordial oocytes in neonatal and adult ovaries of different species and propose a model in which ovaries of neonatal mammals contain a mixed population of both quiescent and activated primordial oocytes, while almost all primordial oocytes are quiescent in adult females. The dormancy of primordial oocytes may be required to reserve the non-growing oocyte pool for the long reproductive life in mammals. FOXO3 is considered one of the molecules responsible for the dormancy of primordial oocytes in adult ovaries. These quiescent primordial oocytes are activated, perhaps by certain mechanisms involving the interaction between stimulatory and inhibitory factors, to enter the growth phase.

  19. Oocyte Pickup from Live Cows Through Laparoscopic Guided Aspiration

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In this experiment, the bovine follicular oocytes were aspirated from the ovaries of Chinese Holsteins with laparoscope made in China. The results were as following: for identifying the suitable negative aspiration pressure, six different pressures (50, 100, 150, 200, 250 and 300mmHg)were tested. The aspiration pressure of 100mmHg was the best. Its oocyte recovery rate was 37. 2%, and G I , G Ⅱ oocyte rate was 89. 5%. The heifers were picked up by laparoscope once or twice a week. Each heifer was collected with 2. 4 oocytes once a week or 4. 4 oocytes twice a week.Its oocyte recovery rate was 48. 0% and the G Ⅰ ,G Ⅱ oocyte rate was 93. 5%. In addition, 1.9 oocytes were collected from each cow once a week or 5.4 oocytes from each cow twice a week. Its oocyte recovery rate was 51.7% and the G Ⅰ , G Ⅱ oocyte rate was 85. 1%. It showed that it was possible to pick up bovine oocyte twice a week. Two cows were picked up twice a week for several weeks(53 times). 268 follicles were aspirated(5.1 follicles per cow per time), and 141 oocytes were recovered(2.7 oocytes per cow per time). The oocyte recovery rate was 52.5%, and the G Ⅰ , G Ⅱ oocyte rate was 85. 1%. It was advisable to pick up oocytes twice a week continuously. Some cows in estrous cycles were superovulated with PMSG(500IU). Each of them could be recovered 2.3 follicles and 1.1 oocytes, the others were superovulated with FSH(0. 7mg) , each of them could be aspirated with 4.4 follicles and 2.3 oocytes. It was obvious that the effect of OPU(oocyte pick up) by superovulation with FSH was much better than that with PMSG. The best time for OPU with laparoscope was at the beginning of cow's estrous cycles. At the first day of their estrus, each of them could be averagely aspirated with 8 follicles and 5.7 oocytes.

  20. Cryopreservation of hamster oocytes: effects of vitrification or freezing on human sperm penetration of zona-free hamster oocytes.

    Science.gov (United States)

    Critser, J K; Arneson, B W; Aaker, D V; Ball, G D

    1986-08-01

    Three experiments were conducted for evaluation of the efficacy of conventional freezing or vitrification of hamster oocytes for use in a human sperm penetration assay (hSPA). In experiment 1, oocytes were cryopreserved and evaluated for survival on the basis of morphologic criteria. Survival of vitrified oocytes and that of frozen oocytes were not different, whereas all cryopreserved groups had lower survival than noncryopreserved controls. In experiment 2, oocytes were conventionally frozen or vitrified and evaluated in an hSPA. Vitrified oocytes had a lower frequency of sperm penetration than frozen oocytes, and all cryopreserved groups had lower penetration rates than untreated controls. In experiment 3, oocytes were exposed to the cryoprotectant used to vitrify (VS1) or freeze (DMSO) but not cooled prior to evaluation in an hSPA. Exposure to DMSO but not VS1 reduced hSPA values. It is concluded from these experiments that while all cryopreserved oocytes do not survive, at current stages of development conventionally frozen oocytes perform better than vitrified oocytes in the hSPA and losses associated with conventional freezing procedures may be related to cryoprotectant exposure, whereas vitrification losses are more probably due to events associated with rapid cooling and/or warming of the oocytes.

  1. Cryopreservation of in vitro matured oocytes after ex vivo oocyte retrieval from gynecologic cancer patients undergoing radical surgery.

    Science.gov (United States)

    Park, Chan Woo; Lee, Sun Hee; Yang, Kwang Moon; Lee, In Ho; Lim, Kyung Teak; Lee, Ki Heon; Kim, Tae Jin

    2016-06-01

    The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays.

  2. Improving oocyte quality by transfer of autologous mitochondria from fully grown oocytes

    DEFF Research Database (Denmark)

    Kristensen, Stine Gry; Pors, Susanne Elisabeth; Andersen, Claus Yding

    2017-01-01

    options using autologous mitochondria to potentially augment pregnancy potential in ART. Autologous transfer of mitochondria from the patient's own germline cells has attracted much attention as a possible new treatment to revitalize deficient oocytes. IVF births have been reported after transfer...... of oogonial precursor cell-derived mitochondria; however, the source and quality of the mitochondria are still unclear. In contrast, fully grown oocytes are loaded with mitochondria which have passed the genetic bottleneck and are likely to be of high quality. An increased supply of such oocytes could...... with high quality mitochondria can be obtained from natural or stimulated ovaries and potentially be used to improve both quality and quantity of oocytes available for fertility treatment....

  3. Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes.

    Directory of Open Access Journals (Sweden)

    Hyuck Jun Mok

    Full Text Available The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H2O2, a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H2O2-treated oocytes, we identified 35 decreased and 26 increased lipids in H2O2-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA, phosphatidylinositol (PI, phosphatidylserine (PS, and lysophosphatidylserine (LPS significantly decreased both in H2O2-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG was only noted in H2O2-treated oocytes, indicating that the acute effect of H2O2-caused oxidative stress is distinct from aging-associated lipidome alteration. In H2O2-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes.

  4. First Babies from Cryopreserved Oocytes in Hungary

    Institute of Scientific and Technical Information of China (English)

    Konc J; Kanyo K; Varga E; Kriston R; Cseh S

    2006-01-01

    Objective To evaluate the value of oocyte cryopreservation (CP) in our clinical ICSIprogram.Methods Freezing procedure with medium containing 1.5 mol/L propanediol (PrOH)+ 0.3 mol/L succrose and traditional slow-freezingprotocol were used. Thawed oocytes were fertilized with ICSI (4-6 h after thawing), and fertilization was assessed 12-16 h later. Laser assisted hatching was performed on all transferred embryos and embryo transfer was carried out 48-72 h after ICSI.Results Eighty-five eggs were thawed and survival rate of 75.3% (64/85) was obtained.Sixty-four oocytes were inseminated with ICSI, 47fertilized (47/64; 73.4%) and a cleavage rate of 85% (40/47) was obtained. Embryo transfers were performed in 18patients, and 4 (19%) resulted in clinical pregnancies. One of the pregnancies encountered first trimester abortion. Implantation rate were 17.2% (5/29) per embryo and 5. 8% (5/85) per egg thawed. In all cases, chorion biopsy was performed resulting46 XY kariotype.Conclusion Our results provide further evidence of that although egg freezing cannot currently claim to be a routine procedure in human IVF, there will certainly be a place for oocyte CP in reproductive medicine in the future.

  5. Bovine cumulus-oocyte disconnection in vitro

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul

    1987-01-01

    Cumulus-oocyte complexes were obtained from cows by aspiration of small (1-6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and processed for transmission electron microscopy after different periods of culture including a 0...

  6. [Karyosphere capsule in Tribolium castaneum oocytes].

    Science.gov (United States)

    Batalova, F M; Bogoliubov, D S

    2013-01-01

    Structure and composition of the karyosphere (karyosome) capsule were studied in the oocytes of a laboratory insect, Tribolium castaneum, with the use of electron microscopy and immunoelectron cytochemistry. Basing on the study of nuclear structure dynamics, we distinguished 8 stages that characterize the period of oocyte growth. At the diplotene stage, T. castaneum oocyte chromosomes conjoin early into a compact karyosphere, but a significant chromatin condensation does not occur. The process of karyosphere formation is accompanied by the development of an extensive extrachromosome capsule surrounding chromatin. The capsule consists of a material of different morphological types. Significant molecular components of the T. castaneum karyosphere capsule are represented by the proteins of nuclear matrix including F-actin and lamin B. Besides the structural proteins, the Sm proteins of small nuclear (sn) RNPs and mature 2,2,7-trimethyl guanosine (TMG) 5'-capped snRNAs are revealed immunocytochemically in the karyosphere capsule. The obtained data can form a basis for further expansion of ideas on the functions of the karyosphere capsule as a specialized extrachromosomal nuclear domain of the oocytes. We believe that the T. castaneum karyosphere capsule plays not only a structural role, but may be involved directly in the processes related to gene expression.

  7. Ultrastructure and mitochondrial numbers in pre- and postpubertal pig oocytes

    DEFF Research Database (Denmark)

    Pedersen, Hanne Skovsgaard; Callesen, Henrik; Løvendahl, Peter

    2016-01-01

    with different diameters from pre- and postpubertal pigs. The ultrastructure of smaller prepubertal immature oocytes indicated active cells in close contact with cumulus cells. The postpubertal oocytes were more quiescent cell types. The small prepubertal oocytes had a lower total mitochondrial number......, but no differences were observed in mitochondrial densities between groups. Mature postpubertal oocytes adhered to the following characteristics: presence of metaphase II, lack of contact between cumulus cells and oocyte, absence of rough endoplasmic reticulum and Golgi complexes, peripheral location of cortical...

  8. In vitro maturation alters gene expression in bovine oocytes.

    Science.gov (United States)

    Adona, Paulo R; Leal, Cláudia L V; Biase, Fernando H; De Bem, Tiago H; Mesquita, Lígia G; Meirelles, Flávio V; Ferraz, André L; Furlan, Luiz R; Monzani, Paulo S; Guemra, Samuel

    2016-08-01

    Gene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo- and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (≥2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein-protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein-protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence.

  9. Human oocyte chromosome analysis: complicated cases and major pitfalls

    Indian Academy of Sciences (India)

    Bernd Rosenbusch; Michael Schneider; Hans Wilhelm Michelmann

    2008-08-01

    Human oocytes that remained unfertilized in programmes of assisted reproduction have been analysed cytogenetically for more than 20 years to assess the incidence of aneuploidy in female gametes. However, the results obtained so far are not indisputable as a consequence of difficulties in evaluating oocyte chromosome preparations. Because of the lack of guidelines, we decided to summarize for the first time, the possible pitfalls in human oocyte chromosome analysis. Therefore, we screened the material from our previous studies and compiled representative, complicated cases with recommendations for their cytogenetic classification. We point out that maturity and size of the oocyte are important parameters and that fixation artefacts, as well as the particular structure of oocyte chromosomes, may predispose one to misinterpretations. Moreover, phenomena related to oocyte activation and fertilization are illustrated and explained. This compilation may help to avoid major problems in future studies and contribute to a more precise, and uniform assessment of human oocyte chromosomes.

  10. Selection of Ovine Oocytes by Brilliant Cresyl Blue Staining

    Directory of Open Access Journals (Sweden)

    Liqin Wang

    2012-01-01

    Full Text Available Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research on in vitro embryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving the in vitro culture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003, Madison et al. (1992 and De Loos et al. (1992. BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB− are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+ due to insuffcient G6PD activity to decolorize the BCB dye.

  11. Oocyte Degeneration Associated with Follicle Cells in Female Mactra chinensis (Bivalvia: Mactridae)

    OpenAIRE

    Kim, Sung Han; Chung, Ee-Yung; Lee, Ki-Young

    2014-01-01

    Ultrastructural studies of oocyte degeneration in the oocyte, and the functions of follicle cells during oocyte degeneration are described to clarify the reproductive mechanism on oocyte degeneration of Mactra chinensis using cytological methods. Commonly, the follicle cells are attached to the oocyte. Follicle cells play an important role in oocyte degeneration. In particular, the functions of follicle cells during oocyte degeneration are associated with phagocytosis and the intracellular di...

  12. Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation.

    Science.gov (United States)

    Gohin, Maella; Bodinier, Pascal; Fostier, Alexis; Chesnel, Franck; Bobe, Julien

    2011-07-01

    While it is generally well accepted that the ovarian follicular sites of estradiol-17β (E2) synthesis are restricted to somatic cells, the possible contribution of the germinal compartment has received little or no attention in teleosts. In order to demonstrate the expression of ovarian aromatase in the oocyte, cyp19a1a mRNA was studied in ovarian follicles by in situ hybridization. In addition, the expression of cyp19a1a was studied in both somatic and germinal compartments of the ovarian follicle in rainbow trout (Oncorhynchus mykiss) during final oocyte maturation (i.e., maturational competence acquisition and subsequent meiosis resumption) by real-time PCR. The enzymatic activity of ovarian aromatase was also studied in both somatic and germinal compartments of the ovarian follicle. Finally, E2 levels were monitored in follicle-enclosed oocytes throughout the pre-ovulatory period. We were able to demonstrate a significant ovarian aromatase expression and activity in the late vitellogenic oocyte. Furthermore, a dramatic decrease in aromatase expression and activity occurs in the oocyte during late oogenesis, concomitantly with the trend observed in surrounding follicular layers. We also report an unexpected increase of E2 levels in the oocyte during the pre-ovulatory period. To our knowledge, these observations are reported for the first time in any teleost species. Together, our data support the hypothesis of the participation of the germinal compartment in follicular estrogen synthesis and a biological role of E2 during oocyte and/or early embryo development.

  13. Oocyte induction of EGF responsiveness in somatic cells is associated with the acquisition of porcine oocyte developmental competence.

    Science.gov (United States)

    Ritter, Lesley J; Sugimura, Satoshi; Gilchrist, Robert B

    2015-06-01

    Oocytes progressively acquire the competence to support embryo development as oogenesis proceeds with ovarian folliculogenesis. The objectives of this study were to investigate oocyte-secreted factor (OSF) participation in the development of somatic cell epidermal growth factor (EGF) responsiveness associated with oocyte developmental competence. A well-established porcine model was employed using oocytes from small (4 mm) antral follicles, representing low vs moderate developmental competence, respectively. Cumulus-oocyte complexes (COCs) were treated in vitro with inducers of oocyte maturation, and cumulus cell functions and oocyte developmental competence were assessed. COCs from small follicles responded to FSH but, unlike COCs from larger follicles, were incapable of responding to EGF family growth factors known to mediate oocyte maturation in vivo, exhibiting perturbed cumulus expansion and expression of associated transcripts (HAS2 and TNFAIP6). Low and moderate competence COCs expressed equivalent levels of EGF receptor (EGFR) mRNA; however, the former had less total EGFR protein leading to failed activation of phospho-EGFR and phospho-ERK1/2, despite equivalent total ERK1/2 protein levels. Native OSFs from moderate, but not from low, competence oocytes established EGF responsiveness in low competence COCs. Four candidate recombinant OSFs failed to mimic the actions of native OSFs in regulating cumulus expansion. Treatment with OSFs and EGF enhanced oocyte competence but only of the low competence COCs. These data suggest that developmental acquisition by the oocyte of capacity to regulate EGF responsiveness in the oocyte's somatic cells is a major milestone in the oocyte's developmental program and contributes to coordinated oocyte and somatic cell development.

  14. How does vitrification affect oocyte viability in oocyte donation cycles? A prospective study to compare outcomes achieved with fresh versus vitrified sibling oocytes.

    Science.gov (United States)

    Solé, M; Santaló, J; Boada, M; Clua, E; Rodríguez, I; Martínez, F; Coroleu, B; Barri, P N; Veiga, A

    2013-08-01

    How does vitrification affect oocyte viability? Vitrification does not affect oocyte viability in oocyte donation cycles. Oocyte vitrification is performed routinely and successfully in IVF and oocyte donation programs. This is a prospective study performed between June 2009 and February 2012 to compare ongoing pregnancy rates and other indices of viability between fresh and vitrified oocytes. A total of 99 donations with more than 16 oocytes (MII) in which oocytes were allocated both to a synchronous recipient (fresh oocytes) and to an asynchronous recipient (vitrified oocytes) were included. The participants were consenting couples (donors and recipients) from the oocyte donation program. On the day of retrieval, the oocytes allocated to the synchronous recipient were inseminated and those allocated for banking were denuded of cumulus and vitrified. Vitrified oocytes were microinjected with spermatozoa 2 h after warming. Embryo transfer was performed on Day 2 of development in both groups, and the remaining embryos were cryopreserved on Day 3. Clinical pregnancy was defined by a positive fetal heartbeat at 6 weeks. A total of 989 oocytes were warmed and 85.6% survived. No significant differences were observed between fresh and vitrified oocytes: fertilization rate (80.7 versus 78.2%), ongoing embryo rate (71.0 versus 68.2%) or good-quality embryo rate (54.1 versus 49.8%). The mean number of embryos transferred was similar in both groups (1.82 ± 0.44 versus 1.90 ± 0.34). The implantation rate (33.3 versus 34.0%) and the multiple pregnancy rate (27.7 versus 20.8) were also similar between both groups (P > 0.05). The live birth rate per cycle was 38.4% in the recipients of fresh oocytes and 43.4% in the recipients of vitrified oocytes (P > 0.05). Eighty five frozen embryo transfers were also evaluated. Comparing embryos from fresh and vitrified oocytes there were no significant differences in the embryo survival rate (70.1 versus 65.8%), clinical pregnancy rate

  15. Metabolism throughout follicle and oocyte development in mammals.

    Science.gov (United States)

    Collado-Fernandez, Esther; Picton, Helen M; Dumollard, Rémi

    2012-01-01

    Metabolic studies of mammalian embryos started with the development of in vitro culture systems more than 40 years ago. More recently, metabolic studies have begun to shed light on the requirements of growing oocytes/follicles from the earliest stages of folliculogenesis. While growing oocytes preferentially metabolise pyruvate over glucose, the somatic compartment of ovarian follicles is more glycolytic. The metabolic preferences of the oocyte are reflected in the early zygote, which becomes increasingly dependent on glycolytic energy production as development progresses to the blastocyst stage. Furthermore, the intricate metabolic relationship between each oocyte and its somatic surroundings is critical for oocyte growth and developmental competence. Measurements of amino acid turnover in bovine oocytes indicate that glutamine, arginine and leucine are consistently depleted, while alanine is produced, showing similarities with amino acid turnover in preimplantation embryos. Amino acid profiling is a good predictor of embryo quality and might also turn out to be a predictor of oocyte developmental competence. Finally, recent studies have uncovered lipid metabolism in oocytes and early embryos, suggesting that endogenous fatty acids might be used for energy production. Together, metabolic studies have revealed the multiplicity of energetic substrates used by oocytes and early embryos, and suggest that the versatility of the metabolic pathways available for energy production is key for high developmental potential. Metabolic studies of early embryos are now being applied to follicle culture, and the goal of describing the metabolome of the growing oocyte in its follicle is now very attainable.

  16. Knockdown of FOXO3 induces primordial oocyte activation in pigs.

    Science.gov (United States)

    Moniruzzaman, Mohammad; Lee, Jibak; Zengyo, Mai; Miyano, Takashi

    2010-02-01

    Mammalian ovaries are endowed with a large number of primordial follicles, each containing a nongrowing oocyte. Only a small population of primordial oocytes (oocytes in primordial follicles) is activated to enter the growth phase throughout a female's reproductive life. Little is known about the mechanism regulating the activation of primordial oocytes. Here, we found that the primordial oocytes from infant pigs (10- to 20-day-old) grew to full size at 2 months after xenografting to immunodeficient mice, whereas those from prepubertal pigs (6-month-old) survived without initiation of their growth even after 4 months; thereafter, they started to grow and reached full size after 6 months. These results suggest that the mechanism regulating the activation of primordial oocytes in prepubertal pigs is different from that in infant pigs. In this regard, the involvement of FOXO3, a forkhead transcription factor, was studied. In prepubertal pigs, FOXO3 was detected in almost all (94+/-2%) primordial oocyte nuclei, and in infant pigs, 42+/-7% primordial oocytes were FOXO3 positive. At 4 months after xenografting, the percentage of FOXO3-positive primordial oocytes from prepubertal pigs had decreased to the infant level. Further, siRNA was designed to knock down porcine FOXO3. FOXO3-knockdown primordial follicles from prepubertal pigs developed to the antral stage accompanied by oocyte growth at 2 months after xenografting. These results suggest that primordial oocytes are dormant in prepubertal pigs by a FOXO3-related mechanism to establish a nongrowing oocyte pool in the ovary, and that a transient knockdown of the FOXO3 activates the primordial oocytes to enter the growth phase.

  17. Fertilization and Embryo Development of Fresh and Cryopreserved Sibling Oocytes

    Directory of Open Access Journals (Sweden)

    Robert F. Casper

    2010-01-01

    Full Text Available Background: Oocyte cryopreservation is potentially the best way to preserve female fertility forunmarried women or young girls at risk of losing ovarian function. The aim of this study was tocompare fertilization and embryo development in frozen-thawed oocytes to their fresh siblings inwomen undergoing in vitro fertilization (IVF and embryo transfer (ET.Materials and Methods: Eleven infertile women undergoing infertility treatment, between theages of 24 to 37 years (mean ± SD = 31.6 ± 3.5, were included in this study. Mature oocytesfrom each patient were randomized into cryopreserved and fresh groups prior to intracytoplasmicsperm injection (ICSI. One hundred and thirty nine oocytes were retrieved, of which 105 were atmetaphase II (MII. Forty- five fresh MII oocytes were kept in culture whereas their sibling 60 MIIoocytes were cryopreserved using a slow cooling protocol. The frozen oocytes remained in LN2for 2 hours before thawing. ICSI was performed 1-2 hours after thawing for frozen oocytes and 4-5hours after retrieval for fresh oocytes. Fertilization and embryo development were compared.Results: Following thawing, 31 oocytes (51.6 % survived and 22 fertilized (79% while 32 freshoocytes fertilized upon ICSI (71%. The mean ± SE scores for embryos developing from frozenthawedoocytes were significantly lower at 48 and 72 hours post-ICSI than for embryos resultingfrom fresh oocytes (p<0.05.Conclusion: Our data demonstrated that oocyte freezing resulted in acceptable survival ratesfollowing cryopreservation, and similar fertilization rates following ICSI as compared to the freshsibling oocytes. However the number of blastomeres and the embryo quality on day three wassuperior in embryos from fresh oocytes when compared to the frozen oocytes.

  18. Follicular steroid hormones as markers of oocyte quality and oocyte development potential.

    Science.gov (United States)

    Carpintero, Nayara López; Suárez, Onica Armijo; Mangas, Carmen Cuadrado; Varea, Carolina González; Rioja, Rubén Gómez

    2014-07-01

    Various components of follicular fluid are suggested as biochemical predictors of oocyte quality. Previous studies of follicular steroid hormone levels have shown disparate results when related with fertilization outcomes. The objective of the study was to relate the levels of steroid hormones of each individual follicle with oocyte maturation, fertilization results, embryo quality, and pregnancy rates. Prospective cohort study in a university hospital. In 31 patients, who underwent intracytoplasmic sperm injection, it was performed an ultrasound guided aspiration of follicular fluid of the first two mature follicles from each ovary. Follicular levels of estradiol, progesterone, testosterone, and dehydroepiandrosterone sulfate were measured by chemiluminescent immunoassay. Generalized estimating equation model. In follicular fluids with mature oocyte presence, in normal as well as in failed fertilization, there was a positive correlation between follicular testosterone and progesterone (r = 0.794, P = 0.0001 and r = 0.829, P = 0.0001). Progesterone levels were higher in cases of normal fertilization compared to failed fertilization (P = 0.003). B quality embryos came from oocytes immersed in follicular fluids with higher estradiol values and higher estradiol/progesterone and estradiol/testosterone ratios than those of C quality (P = 0.01; P = 0.0009; P = 0.001). Estradiol levels were higher in patients who achieved pregnancy (P = 0.02). The analysis of follicular hormone composition could be considered as an additional tool in oocyte selection.

  19. Cytoplasmic Streaming in the Drosophila Oocyte.

    Science.gov (United States)

    Quinlan, Margot E

    2016-10-06

    Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

  20. Oocyte Maturation Process and Affecting Factors

    OpenAIRE

    Yurdun Kuyucu; Ozgul Tap

    2009-01-01

    Normal female fertility depends on normally occuring oogenesis and maturation progress. Oogenesis and folliculogenesis are different progresses but occure in a harmony and at the same time. Oogenesis includes the events that take place matur ovum produced from primordial germ cells. Although folliculogenesis includes the stages primordial, primary, secondary, matur (Graaf) follicules in the influece of gonadotropines and local growth factors. During oocyte maturation meiosis is distrupted til...

  1. Pregnancy and birth rates after oocyte donation.

    Science.gov (United States)

    Remohí, J; Gartner, B; Gallardo, E; Yalil, S; Simón, C; Pellicer, A

    1997-04-01

    To determine accumulated conception and live birth rates in ovum donation. Retrospective study from a computer database. Pregnancies with one gestational sac observed by ultrasound have been included as conceptional cycles and pregnancies that resulted in one live child were recorded for the analysis of the live birth rates. Life table analysis was applied. Oocyte donation program at the Instituto Valenciano de Infertilidad. Three hundred ninety-seven recipients undergoing a total of 627 ETs were analyzed. Ovarian stimulation and ovum pick-up in donors. Uterine ET in recipients after appropriate exogenous steroid replacement. Accumulated and estimated (95% confidence intervals [CI]) conception and live birth rates in the oocyte donation program as well as considering age and cause of infertility of the recipients. Pregnancy rate after one cycle was 53.4% (CI 50.9% to 55.9%), with a delivery rate of 42.6% (CI 40.1% to 45.1%). Accumulated pregnancy rate increased up to 94.8% (CI 90.6% to 99.0%) after four transfers. Similarly, live birth rates reached 88.7% (CI 88.1% to 89.3%) after four attempts of ET by ovum donation. Cycle fecundity rates were maintained at approximately 50% after each attempt. Implantation rate was 18.3% (430/2,340 replaced embryos). Age and cause of entering the program did not influence the overall results of ovum donation. Oocyte donation is a successful treatment modality for infertile couples that offers even higher success rates than natural conception. No difference in cumulative pregnancy rate was observed regardless of recipient age, indication for oocyte donation, or number of cycles attempted.

  2. In Vitro Maturation of Cumulus-Oocyte Complexes for Efficient Isolation of Oocytes from Outbred Deer Mice

    Science.gov (United States)

    Choi, Jung Kyu; He, Xiaoming

    2013-01-01

    Background The outbred (as with humans) deer mice have been a useful animal model of research on human behavior and biology including that of the reproductive system. One of the major challenges in using this species is that the yield of oocyte isolation via superovulation is dismal according to the literature to date less than ∼5 oocytes per animal can be obtained so far. Objective The goal of this study is to improve the yield of oocyte isolation from outbred deer mice close to that of most laboratory mice by in vitro maturation (IVM) of cumulus-oocyte complexes (COCs). Methods Oocytes were isolated by both superovulation and IVM. For the latter, COCs were obtained by follicular puncture of antral follicles in both the surface and inner cortical layers of ovaries. Immature oocytes in the COCs were then cultured in vitro under optimized conditions to obtain metaphase II (MII) oocytes. Quality of the oocytes from IVM and superovulation was tested by in vitro fertilization (IVF) and embryo development. Results Less than ∼5 oocytes per animal could be isolated by superovulation only. However, we successfully obtained 20.3±2.9 oocytes per animal by IVM (16.0±2.5) and superovulation (4.3±1.3) in this study. Moreover, IVF and embryo development studies suggest that IVM oocytes have even better quality than that from superovulation The latter never developed to beyond 2-cell stage as usual while 9% of the former developed to 4-cells. Significance We have successfully established the protocol for isolating oocytes from deer mice with high yield by IVM. Moreover, this is the first ever success to develop in vitro fertilized deer mice oocytes beyond the 2-cell stage in vitro. Therefore, this study is of significance to the use of deer mice for reproductive biology research. PMID:23457518

  3. In vitro maturation of cumulus-oocyte complexes for efficient isolation of oocytes from outbred deer mice.

    Directory of Open Access Journals (Sweden)

    Jung Kyu Choi

    Full Text Available BACKGROUND: The outbred (as with humans deer mice have been a useful animal model of research on human behavior and biology including that of the reproductive system. One of the major challenges in using this species is that the yield of oocyte isolation via superovulation is dismal according to the literature to date less than ∼5 oocytes per animal can be obtained so far. OBJECTIVE: The goal of this study is to improve the yield of oocyte isolation from outbred deer mice close to that of most laboratory mice by in vitro maturation (IVM of cumulus-oocyte complexes (COCs. METHODS: Oocytes were isolated by both superovulation and IVM. For the latter, COCs were obtained by follicular puncture of antral follicles in both the surface and inner cortical layers of ovaries. Immature oocytes in the COCs were then cultured in vitro under optimized conditions to obtain metaphase II (MII oocytes. Quality of the oocytes from IVM and superovulation was tested by in vitro fertilization (IVF and embryo development. RESULTS: Less than ∼5 oocytes per animal could be isolated by superovulation only. However, we successfully obtained 20.3±2.9 oocytes per animal by IVM (16.0±2.5 and superovulation (4.3±1.3 in this study. Moreover, IVF and embryo development studies suggest that IVM oocytes have even better quality than that from superovulation The latter never developed to beyond 2-cell stage as usual while 9% of the former developed to 4-cells. SIGNIFICANCE: We have successfully established the protocol for isolating oocytes from deer mice with high yield by IVM. Moreover, this is the first ever success to develop in vitro fertilized deer mice oocytes beyond the 2-cell stage in vitro. Therefore, this study is of significance to the use of deer mice for reproductive biology research.

  4. Interactions between oocytes and cumulus cells during in vitro maturation of porcine cumulus-oocyte complexes in a chemically defined medium: effect of denuded oocytes on cumulus expansion and oocyte maturation.

    Science.gov (United States)

    Appeltant, R; Somfai, T; Nakai, M; Bodó, S; Maes, D; Kikuchi, K; Van Soom, A

    2015-03-01

    The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P  0.05) but did enhance cumulus expansion of oocytectomized complexes (P medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture through gap junctions. On the basis of these findings, future research could investigate if coculture with DOs during IVM is beneficial for fertilization and embryo development. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Effect of Ultrasound on Parthenogenic Activation of Mouse Oocyte

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    Hamid Gurabi

    2010-01-01

    Full Text Available Objective: Artificial stimulation of mouse oocyte, in the absence of sperm contribution,can induce its parthenogenic activation of oocyte. Ultrasound is one of the newest methodsfor artificial activation of mammal oocytes, and its successful utilization in pig oocyteactivation has been recently reported. Our objective was to assess the effect of ultrasoundon mouse oocyte activation.Materials and Methods: Our groups included1 control group, 3 experimental groups consistingof 1, 2 and 3 repetitions of ultrasound exposure, and 3 sham groups handled similarto experimental groups but ultrasound system was off during treatments.In experimental groups, adult female NMRI mice at the interval between pregnant mareserum gonadotropin (PMSG and human corionic gonadotropin (hCG injections, wereexposed to continuous ultrasound with 3.28 MHz frequency and peak intensity (Ipk = 355mW/cm2.Sixteen hours after injection of hCG, the mice were euthanized and their oocytes werecollected; thereafter, parthenogenic oocytes were counted.Results: Data analysis using the ANOVA test shows a significant increase in the number ofparthenogenic oocytes in mice with 3 overall exposures to ovarian ultrasound (p<0.05.A significant decrease in the number of metaphase II (MII oocytes numbers was alsoseen in mice treated with ultrasound (p<0.05.Conclusion: Ultrasound is thought to induce pores generation in oocyte membranes andprovides an easier inward transport of Ca++ into oocytes. This phenomenon can inducemeiosis resumption in immature oocytes. With increased exposure repetitions from 1 to 3times and greater Ca++ arrival, oocytes can be parthenogenetically activated.

  6. Selection of Ovine Oocytes by Brilliant Cresyl Blue Staining

    OpenAIRE

    Liqin Wang; Jiapeng Lin; Juncheng Huang; Jing Wang; Yuncheng Zhao; Tong Chen

    2012-01-01

    Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research on in vitro embryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving the in vitro culture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable exper...

  7. PTK2b function during fertilization of the mouse oocyte

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Jinping [Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 (United States); McGinnis, Lynda K. [Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Carlton, Carol [Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Beggs, Hilary E. [Department of Ophthalmology, University of California, San Francisco, CA (United States); Kinsey, William H., E-mail: wkinsey@kumc.edu [Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

    2014-08-01

    Highlights: • PTK2b is expressed in oocytes and is activated following fertilization. • PTK2b suppression in oocytes prevents fertilization, but not parthenogenetic activation. • PTK2b suppression prevents the oocyte from fusing with or incorporating bound sperm. • PTK2b suppressed oocytes that fail to fertilize do not exhibit calcium oscillations. - Abstract: Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

  8. Cryopreservation of in vitro matured oocytes after ex vivo oocyte retrieval from gynecologic cancer patients undergoing radical surgery

    Science.gov (United States)

    Park, Chan Woo; Lee, Sun Hee; Yang, Kwang Moon; Lee, In Ho; Lim, Kyung Teak; Lee, Ki Heon

    2016-01-01

    Objective The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). Methods Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. Results A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. Conclusion Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays. PMID:27358831

  9. The dormant and the fully competent oocyte: comparing the transcriptome of human oocytes from primordial follicles and in metaphase II

    DEFF Research Database (Denmark)

    Grøndahl, Marie Louise; Borup, Rehannah; Vikeså, Jonas

    2013-01-01

    Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human...... oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were...

  10. Hydrogen sulfide donor protects porcine oocytes against aging and improves the developmental potential of aged porcine oocytes.

    Directory of Open Access Journals (Sweden)

    Tereza Krejcova

    Full Text Available Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS, cystathionine-γ-lyase (CSE and 3-mercaptopyruvate sulfurtransferase (MPST. We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging.

  11. Anti-Mullerian hormone : a marker for oocyte quantity, oocyte quality and embryo quality?

    NARCIS (Netherlands)

    Fong, S. Lie; Baart, E. B.; Martini, E.; Schipper, I.; Visser, J. A.; Themmen, A. P. N.; de Jong, F. H.; Fauser, B. J. C. M.; Laven, J. S. E.

    2008-01-01

    Serum anti-Mullerian hormone (AMH) concentrations decline with increasing age and constitute a Sensitive marker for ovarian ageing. In addition, basal serum AMH concentrations predict ovarian response during IVF cycles. Concomitantly, oocyte quantity and embryo quality decrease with advancing age. H

  12. Maternal factors required for oocyte developmental competence in mice: transcriptome analysis of non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) oocytes.

    Science.gov (United States)

    Ma, Jun-Yu; Li, Mo; Luo, Yi-Bo; Song, Shuhui; Tian, Dongmei; Yang, Jin; Zhang, Bing; Hou, Yi; Schatten, Heide; Liu, Zhonghua; Sun, Qing-Yuan

    2013-06-15

    During mouse antral follicle development, the oocyte chromatin gradually transforms from a less condensed state with no Hoechst-positive rim surrounding the nucleolus (NSN) to a fully condensed chromatin state with a Hoechst-positive rim surrounding the nucleolus (SN). Compared with SN oocytes, NSN oocytes display a higher gene transcription activity and a lower rate of meiosis resumption (G2/M transition), and they are mostly arrested at the two-cell stage after in vitro fertilization. To explore the differences between NSN and SN oocytes, and the maternal factors required for oocyte developmental competence, we compared the whole-transcriptome profiles between NSN and SN oocytes. First, we found that the NSN and SN oocytes were different in their metabolic pathways. In the phosphatidylinositol signaling pathway, the SN oocytes tend to produce diacylglycerol, whereas the NSN oocytes tend to produce phosphatidylinositol (3,4,5)-trisphosphate. For energy production, the SN oocytes and NSN oocytes differed in the gluconeogenesis and in the synthesis processes. Second, we also found that the key genes associated with oocyte meiosis and/or preimplantation embryo development were differently expressed in the NSN and SN oocytes. Our results illustrate that during the NSN-SN transition, the oocytes change their metabolic activities and accumulate maternal factors for further oocyte maturation and post-fertilization embryo development.

  13. The effect of follicular fluid hormones on oocyte recovery after ovarian stimulation: FSH level predicts oocyte recovery

    Directory of Open Access Journals (Sweden)

    Rinaudo Paolo F

    2009-04-01

    Full Text Available Abstract Background Ovarian stimulation for assisted reproductive technology (ART overcomes the physiologic process to develop a single dominant follicle. However, following stimulation, egg recovery rates are not 100%. The objective of this study is to determine if the follicular fluid hormonal environment is associated with oocyte recovery. Methods This is a prospective study involving patients undergoing ART by standard ovarian stimulation protocols at an urban academic medical center. A total of 143 follicular fluid aspirates were collected from 80 patients. Concentrations of FSH, hCG, estradiol, progesterone, testosterone and prolactin were determined. A multivariable regression analysis was used to investigate the relationship between the follicular fluid hormones and oocyte recovery. Results Intrafollicular FSH was significantly associated with oocyte recovery after adjustment for hCG (Adjusted odds ratio (AOR = 1.21, 95%CI 1.03–1.42. The hCG concentration alone, in the range tested, did not impact the odds of oocyte recovery (AOR = 0.99, 95%CI 0.93–1.07. Estradiol was significantly associated with oocyte recovery (AOR = 0.98, 95% CI 0.96–0.99. After adjustment for progesterone, the strength of association between FSH and oocyte recovery increased (AOR = 1.84, 95%CI 1.45–2.34. Conclusion The relationship between FSH and oocyte recovery is significant and appears to work through mechanisms independent of the sex hormones. FSH may be important for the physiologic event of separation of the cumulus-oocyte complex from the follicle wall, thereby influencing oocyte recovery. Current methods for inducing the final stages of oocyte maturation, with hCG administration alone, may not be optimal. Modifications of treatment protocols utilizing additional FSH may enhance oocyte recovery.

  14. Oxidative stress and ageing of the post-ovulatory oocyte.

    Science.gov (United States)

    Lord, Tessa; Aitken, R John

    2013-12-01

    With extended periods of time following ovulation, the metaphase II stage oocyte experiences deterioration in quality referred to as post-ovulatory oocyte ageing. Post-ovulatory ageing occurs both in vivo and in vitro and has been associated with reduced fertilization rates, poor embryo quality, post-implantation errors and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been established, the molecular mechanisms controlling this process are not well defined. This review analyses the relationships between biochemical changes exhibited by the ageing oocyte and the symptoms associated with the ageing phenotype. We also discuss molecular events that are potentially involved in orchestrating post-ovulatory ageing with a particular focus on the role of oxidative stress. We propose that oxidative stress may act as the initiator for a cascade of events that create the aged oocyte phenotype. Specifically, oxidative stress has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor, impair calcium homoeostasis, induce mitochondrial dysfunction and directly damage multiple intracellular components of the oocyte such as lipids, proteins and DNA. Finally, this review addresses current strategies for delaying post-ovulatory oocyte ageing with a particular focus on the potential use of compounds such as caffeine or selected antioxidants in the development of more refined media for the preservation of oocyte integrity during IVF procedures.

  15. Perinatal outcomes in 375 children born after oocyte donation

    DEFF Research Database (Denmark)

    Malchau, Sara S; Loft, Anne; Larsen, Elisabeth C;

    2013-01-01

    To describe perinatal outcomes in children born after oocyte donation (OD) compared with in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and spontaneous conception (SC).......To describe perinatal outcomes in children born after oocyte donation (OD) compared with in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and spontaneous conception (SC)....

  16. Follicle-stimulating hormone accelerates mouse oocyte development in vivo.

    Science.gov (United States)

    Demeestere, Isabelle; Streiff, Agathe K; Suzuki, João; Al-Khabouri, Shaima; Mahrous, Enas; Tan, Seang Lin; Clarke, Hugh J

    2012-07-01

    During folliculogenesis, oocytes grow and acquire developmental competence in a mutually dependent relationship with their adjacent somatic cells. Follicle-stimulating hormone (FSH) plays an essential and well-established role in the differentiation of somatic follicular cells, but its function in the development of the oocyte has still not been elucidated. We report here that oocytes of Fshb(-/-) mice, which cannot produce FSH, grow at the same rate and reach the same size as those of wild-type mice. Consistent with this observation, the granulosa cells of Fshb(-/-) mice express the normal quantity of mRNA encoding Kit ligand, which has been implicated in oocyte growth. Oocytes of Fshb(-/-) mice also accumulate normal quantities of cyclin B1 and CDK1 proteins and mitochondrial DNA. Moreover, they acquire the ability to complete meiotic maturation in vitro and undergo transition from non-surrounded nucleolus to surrounded nucleolus. However, these events of late oocyte development are significantly delayed. Following in vitro maturation and fertilization, only a small number of embryos derived from oocytes of Fshb(-/-) mice reach the blastocyst stage. Administration of equine chorionic gonadotropin, which provides FSH activity, 48 h before in vitro maturation increases the number of blastocysts obtained subsequently. These results indicate that FSH is not absolutely required for oocyte development in vivo but that this process occurs more rapidly in its presence. We suggest that FSH may coordinate the development of the germline and somatic compartments of the follicle, ensuring that ovulation releases a developmentally competent egg.

  17. Induction and inhibition of oocyte maturation by EDCs in zebrafish

    Directory of Open Access Journals (Sweden)

    Tokumoto Mika

    2005-12-01

    Full Text Available Abstract Background Oocyte maturation in lower vertebrates is triggered by maturation-inducing hormone (MIH, which acts on unidentified receptors on the oocyte surface and induces the activation of maturation-promoting factor (MPF in the oocyte cytoplasm. We previously described the induction of oocyte maturation in fish by an endocrine-disrupting chemical (EDC, diethylstilbestrol (DES, a nonsteroidal estrogen. Methods In this study, stimulatory and inhibitory effects of EDCs and natural steroids on oocyte maturation were examined in zebrafish. For effective agents, some details about the mechanism in induction or inhibition of maturation were examined. Possible groups of DES interacting with the MIH receptor are discussed based on relative potency of steroids to induce maturation. Results Among agents tested, tamoxifen (TAM and its metabolite 4-hydroxytamoxifen (4-OHT showed stimulatory activity similar to DES. The time courses of the change in germinal vesicle breakdown and an intracellular molecular event (the synthesis of cyclin B induced by TAM were indistinguishable from those induced by MIH. In contrast, pentachlorophenol (PCP had a potent inhibitory effect on MIH-induced oocyte maturation. PCP inhibited not only MIH-induced maturation but also DES- and TAM-induced maturation. Methoxychlor also inhibited maturation when oocytes were pre-treated with this agent. Conclusion These results suggest that EDCs act as agonists or antagonists in the induction of oocyte maturation in fish.

  18. Hormonal control of mammalian oocyte meiosis at diplotene stage.

    Science.gov (United States)

    Zhang, Meijia; Xia, Guoliang

    2012-04-01

    Mammalian oocytes grow and undergo meiosis within ovarian follicles. Fully grown oocytes are arrested at the first meiotic prophase by a mural granulosa origin "arrester" until a surge of luteinizing hormone (LH) from the pituitary at the mid-cycle stimulates the immature oocyte to resume meiosis. Recent evidence indicates that natriuretic peptide precursor type C (NPPC) produced by mural granulosa cells stimulates the generation of cyclic guanosine 3',5'-monophosphate (cGMP) by cumulus cell natriuretic peptide receptor 2 (NPR2), which diffuses into oocyte via gap junctions and inhibits oocyte phosphodiesterase 3A (PDE3A) activity and cyclic adenosine 3',5'-monophosphate (cAMP) hydrolysis and maintains meiotic arrest with a high intraoocyte cAMP level. This cAMP is generated through the activity of the Gs G-protein by the G-protein-coupled receptor, GPR3 and GPR12, and adenylyl cyclases (ADCY) endogenous to the oocyte. Further studies suggest that endocrine hormones, such as follicle-stimulating hormone (FSH), LH, 17β-estradiol (E2) and oocyte-derived paracrine factors (ODPFs), participate in oocyte meiosis possibly by the regulation of NPPC and/or NPR2. A detailed investigation of NPPC and NPR2 expression in follicle cells will elucidate the precise molecular mechanisms of gonadotropins, and control the arrest as well as resumption of meiosis.

  19. Oocyte-to-embryo transition: kinase cabal plots regime change.

    Science.gov (United States)

    Greenstein, David; Lee, Laura A

    2006-02-07

    The transition from oocyte to embryo is among the most enthralling events in developmental biology. Recent studies of this transition in the nematode Caenorhabditis elegans have revealed how conserved kinases administer the destruction of key oocyte meiotic regulators to create an embryo.

  20. Functional Topography of the Fully Grown Human Oocyte

    Science.gov (United States)

    Monti, Manuela; Calligaro, Alberto; Behr, Barry; Pera, Renee Rejo; Redi, Carlo Alberto; Wossidlo, Mark

    2017-01-01

    In vivo maturation (IVM) of human oocytes is a technique used to increase the number of usable oocytes for in vitro fertilization (IVF) and represents a necessity for women with different ovarian pathologies. During IVM the oocytes progress from the germinal vesicle stage (GV) through the metaphase II and during this journey both nuclear and cytoplasmic rearrangements must be obtained to increase the probability to get viable and healthy zygotes/embryos after IVF. As the successful clinical outcomes of this technique are a reality, we wanted to investigate the causes behind oocytes maturation arrest. For obvious ethical reasons, we were able to analyze only few human immature oocytes discarded and donated to research by transmission electron microscopy showing that, as in the mouse, they have different chromatin and cytoplasmic organizations both essential for further embryo development.

  1. Oocyte donation in patients without ovarian function.

    Science.gov (United States)

    Devroey, P; Wisanto, A; Camus, M; Van Waesberghe, L; Bourgain, C; Liebaers, I; Van Steirteghem, A C

    1988-08-01

    The clinical, hormonal and cytogenetic findings in 36 women with primary ovarian failure, referred for oocyte or embryo donations are reported. Fifteen women were suffering from ovarian dysgenesis and 11 from premature menopause. Six of these 26 patients showed X-chromosome abnormalities. One patient had a Noonan syndrome. The remaining 10 had surgical menopause. The mean duration of their infertility was 6.5 +/- 3.2 years (+/- SD). All patients had elevated serum gonadotrophins within the menopausal range. Hypothalamic, pituitary and thyroid function were found to be intact. In one of the 15 ovarian biopsies on the patients with chromosomal competent ovarian failure, primordial follicles were found. Hysterosalpingograms revealed a normal uterine cavity in all patients. In view of oocyte donation, careful evaluation of the obstetric risk was mandatory in the six patients with X-chromosome aberrations and in the patient with the Noonan syndrome, because of their short stature and possible concomitant cardiovascular and renal disease. After substitution therapy with oestradiol valerate and natural progesterone, 13 pregnancies were established, seven patients delivered (one set of twins), eight healthy children were born, three pregnancies aborted and three pregnancies are progressing normally.

  2. Organisation of Xenopus oocyte and egg cortices.

    Science.gov (United States)

    Chang, P; Pérez-Mongiovi, D; Houliston, E

    1999-03-15

    The division of the Xenopus oocyte cortex into structurally and functionally distinct "animal" and "vegetal" regions during oogenesis provides the basis of the organisation of the early embryo. The vegetal region of the cortex accumulates specific maternal mRNAs that specify the development of the endoderm and mesoderm, as well as functionally-defined "determinants" of dorso-anterior development, and recognisable "germ plasm" determinants that segregate into primary germ cells. These localised elements on the vegetal cortex underlie both the primary animal-vegetal polarity of the egg and the organisation of the developing embryo. The animal cortex meanwhile becomes specialised for the events associated with fertilisation: sperm entry, calcium release into the cytoplasm, cortical granule exocytosis, and polarised cortical contraction. Cortical and subcortical reorganisations associated with meiotic maturation, fertilisation, cortical rotation, and the first mitotic cleavage divisions redistribute the vegetal cortical determinants, contributing to the specification of dorso-anterior axis and segregation of the germ line. In this article we consider what is known about the changing organisation of the oocyte and egg cortex in relation to the mechanisms of determinant localisation, anchorage, and redistribution, and show novel ultrastructural views of cortices isolated at different stages and processed by the rapid-freeze deep-etch method. Cortical organisation involves interactions between the different cytoskeletal filament systems and internal membranes. Associated proteins and cytoplasmic signals probably modulate these interactions in stage-specific ways, leaving much to be understood.

  3. Dormancy and activation of human oocytes from primordial and primary follicles: molecular clues to oocyte regulation.

    Science.gov (United States)

    Ernst, E H; Grøndahl, M L; Grund, S; Hardy, K; Heuck, A; Sunde, L; Franks, S; Andersen, C Y; Villesen, P; Lykke-Hartmann, K

    2017-08-01

    Do specific transcriptome dynamics in human oocytes from primordial and primary follicles identify novel pathways in oocyte activation? The transcriptomic profiles in oocytes from primordial and primary follicles, respectively, revealed several new canonical pathways as putative mediators of oocyte dormancy and activation. Cellular signaling pathways including PI3K/AKT and AKT/mTOR as well as TGF-β and IGF signaling are known to regulate the primordial-to-primary transition in mammalian follicle development. We performed a class comparison study on human oocytes from primordial (n = 436) and primary (n = 182) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy. RNA was extracted from oocytes from primordial and primary follicles isolated by Laser Capture Microdissection, and submitted to the HiSeq Illumina platform. Data mapping, quality control, filtering and expression analysis were performed using Tophat (2.0.4), Cufflinks (2.0.2), BWA (0.6.2) and software R. Modeling of complex biological systems was performed using the IPA® software. Finally, qPCR and immunohistochemistry were employed to explore expression and localization of selected genes and products in human ovarian tissue. We found 223 and 268 genes down-regulated and up-regulated, respectively, in the oocytes during the human primordial-to-primary follicle transition (P 2). IPA® enrichment analysis revealed known pathways ('mTOR Signaling', 'PI3K/AKT Signaling' and 'PTEN Signaling') as well as enriched canonical pathways not previously associated with human ovarian follicle development such as 'ErB Signaling' and 'NGF Signaling' in the down-regulated category and 'Regulation of eIF4 and P70S6K Signaling' and 'HER-2 Signaling in Breast Cancer' in the up-regulated group. Additionally, immunohistochemistry on human ovarian tissue explored the intraovarian localization of VASA, FOXO1 and eIF4E. http://users-birc.au.dk/biopv/published_data/ernst_2017/. This is a

  4. Developmental competence of oocytes after ICSI in the rhesus monkey.

    Science.gov (United States)

    Nusser, K D; Mitalipov, S; Widmann, A; Gerami-Naini, B; Yeoman, R R; Wolf, D P

    2001-01-01

    Oocyte quantity and quality are critical to assisted reproductive technology (ART), yet few assessments beyond counting metaphase II (MII) oocytes exist. In this study, 30 +/- 2 oocytes per cycle were recovered from rhesus monkeys subjected to follicular stimulation with human gonadotrophins, of which 15 +/- 1 were MII. Oocyte quality was investigated by monitoring the developmental potential of oocytes subjected to intracytoplasmic sperm injection (ICSI). Despite uniform fertilization rates (71 +/- 4%), progression of embryos to blastocysts varied when expressed as a monthly average, from 20 to 85%, with lows from February to April and again in October, which could be attributed to developmental failure of a significant number of oocyte cohorts (14 of 55). Blastocyst rates, after elimination of failed cohorts, were uniform over time (59 +/- 4%). Neither culture conditions, the number of follicular stimulations, nor the individual sperm or oocyte donor were associated specifically with developmental failure, suggesting that intrinsic differences between stimulation cycles account for the observed variation in developmental potential. The in-vivo developmental competence of ICSI-produced embryos grown to blastocysts in vitro was also assessed. Two ongoing pregnancies and the birth of a normal female, 'Blastulina', represent landmarks in efforts to expand the use of ART in the rhesus monkey.

  5. Strategies to support human oocyte development in vitro.

    Science.gov (United States)

    Telfer, Evelyn E; McLaughlin, Marie

    2012-01-01

    Many young cancer patients are now being given the option to store ovarian cortical biopsies before undergoing potentially damaging chemo- or radiotherapy. This tissue mainly contains large numbers of immature primordial follicles. Currently the only option to restore fertility using this tissue is by transplantation which may not be a viable option for all patients. Greater options to realise the potential of this tissue to restore fertility could be achieved by the development of in vitro systems that support oocyte development. The ability to develop human oocytes from the most immature stages of follicles (primordial) through to maturation and fertilisation in vitro would revolutionise fertility preservation practice. This has been achieved in mouse where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. However, developing IVG systems to support complete development of human oocytes has been more difficult because of differences in scale of timing and size. Our lab has been working on a multi-step culture system to support growth and development of bo-vine and human oocytes from primordial through to fully grown, using fresh and cryopreserved ovarian cortical tissue. This review outlines the approaches being taken to obtain complete in vitro development of human oocytes and strategies for assessing the health and viability of IVG oocytes.

  6. Mitochondrial dynamics and their intracellular traffic in porcine oocytes.

    Science.gov (United States)

    Yamochi, T; Hashimoto, S; Amo, A; Goto, H; Yamanaka, M; Inoue, M; Nakaoka, Y; Morimoto, Y

    2016-08-01

    Meiotic maturation of oocytes requires a variety of ATP-dependent reactions, such as germinal vesicle breakdown, spindle formation, and rearrangement of plasma membrane structure, which is required for fertilization. Mitochondria are accordingly expected be localized to subcellular sites of energy utilization. Although microtubule-dependent cellular traffic for mitochondria has been studied extensively in cultured neuronal (and some other somatic) cells, the molecular mechanism of their dynamics in mammalian oocytes at different stages of maturation remains obscure. The present work describes dynamic aspects of mitochondria in porcine oocytes at the germinal vesicle stage. After incubation of oocytes with MitoTracker Orange followed by centrifugation, mitochondria-enriched ooplasm was obtained using a glass needle and transferred into a recipient oocyte. The intracellular distribution of the fluorescent mitochondria was then observed over time using a laser scanning confocal microscopy equipped with an incubator. Kinetic analysis revealed that fluorescent mitochondria moved from central to subcortical areas of oocytes and were dispersed along plasma membranes. Such movement of mitochondria was inhibited by either cytochalasin B or cytochalasin D but not by colcemid, suggesting the involvement of microfilaments. This method of visualizing mitochondrial dynamics in live cells permits study of the pathophysiology of cytoskeleton-dependent intracellular traffic of mitochondria and associated energy metabolism during meiotic maturation of oocytes.

  7. Gene expression profiles of single human mature oocytes in relation to age

    DEFF Research Database (Denmark)

    Grøndahl, M L; Andersen, Claus Yding; Bogstad, J

    2010-01-01

    The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes.......The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes....

  8. In vitro maturation and artificial activation of donkey oocytes.

    Science.gov (United States)

    Zhao, Gaoping; Wu, Kaifeng; Cui, Liang; Zhao, Lixia; Liu, Yiyi; Tan, Xiuwen; Zhou, Huanmin

    2011-09-01

    Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.

  9. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization

    Directory of Open Access Journals (Sweden)

    Graham C. Gilchrist

    2016-03-01

    Full Text Available Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV oocytes, metaphase II (MII oocytes, and presumptive zygotes (PZ. Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR. Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05. To determine whether changes in specific primary miRNA (pri-miRNA transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.

  10. Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Desrosiers, R.R.; Romanik, E.A.; O' Connor, C.M. (Worcester Foundation for Experimental Biology, Shrewsbury, MA (USA))

    1990-12-05

    The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-(methyl-3H)methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-(methyl-3H)methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly.

  11. Oocytes Polar Body Detection for Automatic Enucleation

    Directory of Open Access Journals (Sweden)

    Di Chen

    2016-02-01

    Full Text Available Enucleation is a crucial step in cloning. In order to achieve automatic blind enucleation, we should detect the polar body of the oocyte automatically. The conventional polar body detection approaches have low success rate or low efficiency. We propose a polar body detection method based on machine learning in this paper. On one hand, the improved Histogram of Oriented Gradient (HOG algorithm is employed to extract features of polar body images, which will increase success rate. On the other hand, a position prediction method is put forward to narrow the search range of polar body, which will improve efficiency. Experiment results show that the success rate is 96% for various types of polar bodies. Furthermore, the method is applied to an enucleation experiment and improves the degree of automatic enucleation.

  12. Relationship between respiration rate and weight of loach oocytes.

    Science.gov (United States)

    Ozernyuk, N D; Zotin, A I

    1975-01-01

    It is shown that the constant k in the equation QO2 equals apk and the constant b in the equation qo2 equals aP-b change during the oogenesis of the loach. Hence, the growth of oocytes differs considerably from the growth of animals, where the constants k and b do not change with increase in weight. It is suggested that the relationship between the respiration rate and weight of the oocytes is due to the change in the amount of mitochondria in the oocytes.

  13. Survival of oocytes recovered from vitrified sheep ovarian tissues.

    Science.gov (United States)

    Al-aghbari, A M; Menino, A R

    2002-05-15

    The objective of this work was to develop an effective vitrification technique for cryopreserving oocytes in sheep ovarian tissues. Ovaries were surgically recovered from 15 pubertal ewes and the ovarian cortex was cut into sections. Ovarian tissues were placed in equilibration medium consisting of 4% (v/v) ethylene glycol (EG) and 20% (v/v) FBS in TCM-199 on ice for 30 min and transferred to vitrification solution (35% EG, 5% polyvinylpyrrolidone, 0.4M trehalose and 20% FBS in TCM-199) for 5 min. Ovarian tissues were vitrified by dropping the tissue on the surface of a steel cube cooled by liquid nitrogen. Cumulus-enclosed oocyte complexes (COC) were also collected and vitrified following the procedure used for ovarian tissues. After 2-3 weeks of storage in liquid nitrogen, ovarian tissues and COC were thawed at 37 degrees C in 0.3M trehalose and COC in ovarian tissues were mechanically and enzymatically isolated. Vitrified COC and freshly collected COC were washed twice in maturation medium (TCM-199 supplemented with 0.255 mM pyruvate and 10% heat-treated estrus cow serum) and cultured in 50 microl drops of maturation medium under paraffin oil for 23-25h at 39 degrees C in a humidified atmosphere of 5% CO(2) in air. After culture, cumulus cells were removed by hyaluronidase treatment and vortexing and oocytes were fixed and stained. No significant differences were observed between vitrified oocytes, oocytes recovered from vitrified ovarian tissues and non-vitrified control oocytes in the percentage of oocytes with acceptable staining per total number of oocytes fixed or with visible chromatin per total number of oocytes with acceptable staining. However, fewer (P0.05) were observed due to treatment in the percentages of oocytes developing to metaphase II. These results demonstrate that sheep oocytes can be successfully cryopreserved by vitrification of ovarian tissues and exhibit in vitro maturation rates similar to that of vitrified and non-vitrified oocytes.

  14. Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

    Directory of Open Access Journals (Sweden)

    Seung Eun Lee

    2014-05-01

    Full Text Available Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68 or control oocytes (44 h IVM; 42.14±4.40 significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68 (p<0.05. Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK, and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1 compared with untreated, 24 h-aged IVM oocytes (p<0.05. Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS activity and DNA fragmentation (p<0.05, and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05 and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1, anti-apoptosis (BCL2L1 and BIRC5; p<0.05, and development (NANOG and SOX2; p<0.05 genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3 compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

  15. Effect of oocyte selection, estradiol and antioxidant treatment on in vitro maturation of oocytes collected from prepubertal Boer goats

    Directory of Open Access Journals (Sweden)

    George W. Smith

    2010-02-01

    Full Text Available Development of improved procedures for in vitro maturation of oocytes collected from prepubertal goats has applications for in vitro embryo production and accompanying strategies for genetic improvement. The objective of described studies was to determine the effects of oocyte grade, in vitro maturation time, antioxidant supplementation and concentrations of estradiol in the maturation medium on in vitro maturation of oocytes harvested from 1-6 mm follicles present on the ovaries (obtained from an abattoir of 1-6 month-old prepubertal Boer goats. Rates of progression to metaphase II were greater for grade 1 oocytes (>3 compact layers of cumulus cells and evenly granulated cytoplasm than grade 2 oocytes (in vitro maturation in the presence of high concentrations of estradiol (10 and 100 mg/mL on progression to metaphase II was observed, and no effect was observed in response to 1 mg/mL estradiol treatment as compared with control. Results suggest that oocyte selection and beta-mercaptoethanol supplementation can positively influence progression to metaphase II of oocytes harvested from ovaries of prepubertal goats, whereas high concentrations of estradiol are inhibitory to in vitro maturation.

  16. Mouse Oocytes Enable LH-Induced Maturation of the Cumulus-Oocyte Complex via Promoting EGF Receptor-Dependent Signaling

    Science.gov (United States)

    Su, You-Qiang; Sugiura, Koji; Li, Qinglei; Wigglesworth, Karen; Matzuk, Martin M.; Eppig, John J.

    2010-01-01

    LH triggers the maturation of the cumulus-oocyte complex (COC), which is followed by ovulation. These ovarian follicular responses to LH are mediated by epidermal growth factor (EGF)-like growth factors produced by granulosa cells and require the participation of oocyte-derived paracrine factors. However, it is not clear how oocytes coordinate with the EGF receptor (EGFR) signaling to achieve COC maturation. The aim of the present study was to test the hypothesis that oocytes promote the expression of EGFR by cumulus cells, thus enabling them to respond to the LH-induced EGF-like peptides. Egfr mRNA and protein expression were dramatically reduced in cumulus cells of mutant mice deficient in the production of the oocyte-derived paracrine factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). Moreover, microsurgical removal of oocytes from wild-type COCs dramatically reduced expression of Egfr mRNA and protein, and these levels were restored by either coculture with oocytes or treatment with recombinant GDF9 or GDF9 plus recombinant BMP15. Blocking Sma- and Mad-related protein (SMAD)2/3 phosphorylation in vitro inhibited Egfr expression in wild-type COCs and in GDF9-treated wild-type cumulus cells, and conditional deletion of Smad2 and Smad3 genes in granulosa cells in vivo resulted in the reduction of Egfr mRNA in cumulus cells. These results indicate that oocytes promote expression of Egfr in cumulus cells, and a SMAD2/3-dependent pathway is involved in this process. At least two oocyte-derived growth factors, GDF9 and BMP15, are required for EGFR expression by cumulus cells. PMID:20382892

  17. Vitrification, in vitro fertilization, and development of Atg7 deficient mouse oocytes.

    Science.gov (United States)

    Bang, Soyoung; Lee, Geun-Kyung; Shin, Hyejin; Suh, Chang Suk; Lim, Hyunjung Jade

    2016-03-01

    Autophagy contributes to the clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified mouse oocytes show acute increases in autophagy during warming. Herein, we investigate the potential role of Atg7 in oocyte vitrification by using an oocyte-specific deletion model of the Atg7 gene, a crucial upstream gene in the autophagic pathway. Oocyte-specific Atg7 deficient mice were generated by crossing Atg7 floxed mice and Zp3-Cre transgenic mice. The oocytes were vitrified-warmed and then subjected to in vitro fertilization and development. The rates of survival, fertilization, and development were assessed in the Atg7 deficient oocytes in comparison with the wildtype oocytes. Light chain 3 (LC3) immunofluorescence staining was performed to determine whether this method effectively evaluates the autophagy status of oocytes. The survival rate of vitrified-warmed Atg7(f/f) ;Zp3-Cre (Atg7(d/d) ) metaphase II (MII) oocytes was not significantly different from that of the wildtype (Atg7(f/f) ) oocytes. Fertilization and development in the Atg7(d/d) oocytes were significantly lower than the Atg7(f/f) oocytes, comparable to the Atg5(d/d) oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed Atg7(d/d) MII oocytes when compared to fresh Atg7(d/d) oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes. We confirmed that the LC3-positive signal is nearly absent in Atg7(d/d) oocytes. While autophagy is induced during the warming process after vitrification of MII oocytes, the Atg7 gene is not essential for survival of vitrified-warmed oocytes. Thus, induction of autophagy during warming of vitrified MII oocytes seems to be a natural response to manage cold or other cellular stresses.

  18. Oocyte Degeneration Associated with Follicle Cells in Female Mactra chinensis (Bivalvia: Mactridae).

    Science.gov (United States)

    Kim, Sung Han; Chung, Ee-Yung; Lee, Ki-Young

    2014-12-01

    Ultrastructural studies of oocyte degeneration in the oocyte, and the functions of follicle cells during oocyte degeneration are described to clarify the reproductive mechanism on oocyte degeneration of Mactra chinensis using cytological methods. Commonly, the follicle cells are attached to the oocyte. Follicle cells play an important role in oocyte degeneration. In particular, the functions of follicle cells during oocyte degeneration are associated with phagocytosis and the intracellular digestion of products. In this study, morphologically similar degenerated phagosomes (various lysosomes), which were observed in the degenerated oocytes, appeared in the follicle cells. After the spawning of the oocytes, the follicle cells were involved in oocyte degeneration through phagocytosis by phagolysosomes. Therefore, it can be assumed that follicle cells reabsorb phagosomes from degenerated oocytes. In this study, the presence of lipid granules, which occurred from degenerating yolk granules, gradually increased in degenerating oocytes. The function of follicle cells can accumulate reserves of lipid granules and glycogen in the cytoplasm, which can be employed by the vitellogenic oocyte. Based on observations of follicle cells attached to degenerating oocytes after spawning, the follicle cells of this species are involved in the lysosomal induction of oocyte degeneration for the reabsorption of phagosomes (phagolysosomes) in the cytoplasm for nutrient storage, as seen in other bivalves.

  19. Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

    Directory of Open Access Journals (Sweden)

    Yokoi Hayato

    2011-04-01

    Full Text Available Abstract Background Large volumes of lymph can be collected from the eye-sacs of bubble-eye goldfish. We attempted to induce vitellogenin (Vtg in the eye-sac lymph of bubble-eye goldfish and develop a method for visualizing Vtg incorporation by zebrafish oocytes using FITC-labeling. Methods Estrogen efficiently induced Vtg in the eye-sac lymph of goldfish. After FITC-labeled Vtg was prepared, it was injected into mature female zebrafish. Results Incorporation of FITC-labeled Vtg by zebrafish oocytes was detected in in vivo and in vitro experiments. The embryos obtained from zebrafish females injected with FITC-labeled Vtg emitted FITC fluorescence from the yolk sac and developed normally. Conclusion This method for achieving Vtg incorporation by zebrafish oocytes could be useful in experiments related to the development and endocrinology of zebrafish oocytes.

  20. Vitrification of Bovine Oocytes by Open Pulled Straw

    Institute of Scientific and Technical Information of China (English)

    ZHU Shi-en; ZENG Shen-ming; WU Tong-yi; MENG Qing-gang; ZHANG Zhong-cheng; CHEN Yong-fu

    2002-01-01

    Bovine oocytes cultured in vitro for 6 hours or 22 hours were cryopreserved in different vitrification solutions (EFS40, EFS50, EDFS30 or EDFS40) by the two-step method with OPS (open pulled straw).The best results were achieved by using EDFS30 to cryopreserve the oocytes either for in vitro fertilization or for chemical activation. The blastocyst rates were 12% and 17% in 6 hour and 22 hour cultures respectively following in vitro fertilization. If frozen-thawed oocytes were continued in culture up to 24 hours, and were activated by chemicals, the blastocyst rates were 22% and 24% in 6-hour and 22-hour groups respectively.There were no statistical differences between frozen and fresh oocytes (P > 0.05).

  1. Cryopreservation of Oocytes and Embryos in Human Assisted Reproduction

    Directory of Open Access Journals (Sweden)

    Konc J

    2005-01-01

    Full Text Available Cryopreservation has become an integral component of assisted reproductive technology. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals prior to cancer treatments. While the efficiency of oocyte and embryo freezing technology has increased over time, there is still room for improvement, since even under ideal circumstances the clinical pregnancy rate from frozen embryo transfer is approximately two-thirds of that from the fresh transfer of embryos. Thus, studies connected with cryopreservation of human oocytes and embryos are very important to the expansion of effective clinical services. This review gives a summary of the theoretical and technical aspects of oocyte and embryo cryopreservation.

  2. RASD1 Knockdown Results in Failure of Oocyte Maturation

    OpenAIRE

    Youngeun Lee; Kyeoung-Hwa Kim; Hyemin Yoon; Ok-Hee Lee; Eunyoung Kim; Miseon Park; Hoon Jang; Kwonho Hong; Hyuk Song; Jung Jae Ko; Woo Sik Lee; Kyung-Ah Lee; Eun Mi Chang; Youngsok Choi

    2016-01-01

    Background: Ras dexamethasone-induced protein (RASD1) is a member of Ras superfamily of small GTPases. RASD1 regulates various signaling pathways involved in iron homeostasis, growth hormone secretion, and circadian rhythm. However, RASD1 function in oocyte remains unknown. Methods: Using immunohistochemistry, immunofluorescence, and quantitative real-time RT-PCR, RASD1 expression in mouse ovary and RASD1 role in oocyte maturation-related gene expression, spindle formation, and chromosome ali...

  3. Kinetochore microtubule establishment is defective in oocytes from aged mice

    OpenAIRE

    Shomper, Maria; Lappa, Christina; FitzHarris, Greg

    2014-01-01

    Errors in chromosome segregation in mammalian oocytes increase in number with advancing maternal age, and are a major cause of pregnancy loss. Why chromosome segregation errors are more common in oocytes from older females remains poorly understood. In mitosis, accurate chromosome segregation is enabled by attachment of kinetochores to microtubules from appropriate spindle poles, and erroneous attachments increase the likelihood of mis-segregation. Whether attachment errors are responsible fo...

  4. In vivo induction of oocyte maturation and ovulation in zebrafish.

    Directory of Open Access Journals (Sweden)

    Toshinobu Tokumoto

    Full Text Available The maturation of fish oocytes is a well-characterized system induced by progestins via non-genomic actions. In a previous study, we demonstrated that diethylstilbestrol (DES, a non-steroidal estrogen, induces fish oocyte maturation via the membrane progestin receptor (mPR. Here, we attempted to evaluate the effect of DES as an environmental endocrine disrupting chemical (EDC upon fish oocyte maturation using live zebrafish. DES triggered oocyte maturation within several hours in vivo when administrated directly into the surrounding water. The natural teleost maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP also induced oocyte maturation in vivo. Steroids such as testosterone, progesterone or 17alpha-hydroxyprogesterone were also effective in vivo. Further studies indicated that externally applied 17,20beta-DHP even induced ovulation. In contrast to 17,20beta -DHP, DES induced maturation but not ovulation. Theoretically this assay system provides a means to distinguish pathways involved in the induction of ovulation, which are known to be induced by genomic actions from the pathway normally involved in the induction of oocyte maturation, a typical non-genomic action-dependent pathway. In summary, we have demonstrated the effect of EDCs on fish oocyte maturation in vivo. To address the effects, we have explored a conceptually new approach to distinguish between the genomic and non-genomic actions induced by steroids. The assay can be applied to screens of progestin-like effects upon oocyte maturation and ovulation for small molecules of pharmacological agents or EDCs.

  5. Investigation of DNA repair in human oocytes and preimplantation embryos

    OpenAIRE

    Jaroudi, S.

    2010-01-01

    DNA repair genes are expressed in mammalian embryos and in human germinal vesicles, however, little is known about DNA repair in human preimplantation embryos. This project had three aims: 1) to produce a DNA repair profile of human MII oocytes and blastocysts using expression arrays and identify repair pathways that may be active before and after embryonic genome activation; 2) to design an in vitro functional assay that targeted mismatch repair and which could be applied to human oocytes...

  6. Frequency of aneuploidy related to age in porcine oocytes.

    Science.gov (United States)

    Hornak, Miroslav; Jeseta, Michal; Musilova, Petra; Pavlok, Antonin; Kubelka, Michal; Motlik, Jan; Rubes, Jiri; Anger, Martin

    2011-04-27

    It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.

  7. Resveratrol protects mouse oocytes from methylglyoxal-induced oxidative damage.

    Science.gov (United States)

    Liu, Yu; He, Xiao-Qin; Huang, Xin; Ding, Lu; Xu, Lin; Shen, Yu-Ting; Zhang, Fei; Zhu, Mao-Bi; Xu, Bai-Hui; Qi, Zhong-Quan; Wang, Hai-Long

    2013-01-01

    Methylglyoxal, a reactive dicarbonyl compound, is mainly formed from glycolysis. Methylglyoxal can lead to the dysfunction of mitochondria, the depletion of cellular anti-oxidation enzymes and the formation of advanced glycation ends. Previous studies showed that the accumulation of methylglyoxal and advanced glycation ends can impair the oocyte maturation and reduce the oocyte quality in aged and diabetic females. In this study, we showed that resveratrol, a kind of phytoalexin found in the skin of grapes, red wine and other botanical extracts, can alleviate the adverse effects caused by methylglyoxal, such as inhibition of oocyte maturation and disruption of spindle assembly. Besides, methylglyoxal-treated oocytes displayed more DNA double strands breaks and this can also be decreased by treatment of resveratrol. Further investigation of these processes revealed that methylglyoxal may affect the oocyte quality by resulting in excessive reactive oxygen species production, aberrant mitochondrial distribution and high level lipid peroxidation, and resveratrol can block these cytotoxic changes. Collectively, our results showed that resveratrol can protect the oocytes from methylglyoxal-induced cytotoxicity and this was mainly through the correction of the abnormity of cellular reactive oxygen species metabolism.

  8. Presence and integration of HBV DNA in mouse oocytes

    Institute of Scientific and Technical Information of China (English)

    Tian-Hua Huang; Qing-Jian Zhang; Qing-Dong Xie; Li-Ping Zeng; Xi-Fan Zeng

    2005-01-01

    AIM: Hepatitis B is a worldwide public health problem. To explore the feasibility of hepatitis B virus (HBV) vertical transmission via oocytes, the presence and integration of HBV DNA in mouse oocytes were studied. METHODS: Genomic DNA was isolated and metaphases were prepared, respectively from mouse oocytes cocultured with pBR322-HBV DNA plasmids. PCR, Southern blot, dot hybridization and fluorescence in situ hybridization (FISH) were performed to explore the existence and integration of HBV DNA in oocytes.RESULTS: PCR detected positive bands in the tested samples, and then Southern blot revealed clear hybridization signals in PCR products. Final washing solutions were collected for dot hybridization and no signal for HBV DNA was observed, which excluded the possibility that contamination of washing solutions gave rise to positive results of PCR and Southern blot. FISH demonstrated that 36 of 1 000 metaphases presented positive signals. CONCLUSION: HBV DNA sequences are able to pass through the zona and oolemma to enter into oocytes and tointegrate into their chromosomes. HBV DNA sequences might be brought into embryo via oocytes as vectors when they are fertilized with normal spermatozoa.

  9. Frequency of aneuploidy related to age in porcine oocytes.

    Directory of Open Access Journals (Sweden)

    Miroslav Hornak

    Full Text Available It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH, combined with whole genome amplification (WGA, to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.

  10. Improvement of an electrical activation protocol for porcine oocytes.

    Science.gov (United States)

    Zhu, Jie; Telfer, Evelyn E; Fletcher, Judy; Springbett, Anthea; Dobrinsky, John R; De Sousa, Paul A; Wilmut, Ian

    2002-03-01

    Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage to establish an effective activation protocol for pig nuclear transfer. This evaluation included 1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, 2) an investigation of interactions among oocyte age, applied voltage field strength, electrical pulse number, and pulse duration, and 3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimized protocol based on an in vitro system of oocyte maturation and embryo culture. In the first study, addition of amino acids in maturation medium was beneficial for the developmental competence of activated oocytes. In the second study, the developmental response of activated oocytes was dependent on interactions between oocyte age at activation and applied voltage field strength, voltage field strength and pulse number, and pulse number and duration. The formation of parthenogenetic blastocysts was optimal when activation was at 44 h of maturation using three 80-microsec consecutive pulses of 1.0 kV/cm DC. Approximately 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying a potential for further in vivo development.

  11. The Influence of Seasons on Oocyte Parameters in ICSI Cycles

    Directory of Open Access Journals (Sweden)

    Seddigheh Esmaeilzadeh

    2009-03-01

    Full Text Available Objective: This study aimed to evaluate the effect of seasonal variability on the assisted reproductive technique (ART success rate.Materials and methods: This study was a descriptive – analytical survey performed on 91 infertile women undergoing  intracytoplasmic sperm injection – embryo transfer (ICSI-ET in different seasons. The patients aged less than 35 years old and had normal LH/FSH ratio. All patients entered long protocol down regulation treatment cycle and the picked up oocytes were transferred to GIII medium in the infertility laboratory. The cumulus characteristics, oocyte parameters including number of the retrieved oocytes, morphological characteristics, fertilization and degeneration rate and number of cleaved embryos were recorded. Data were analyzed by SPSS software.  Results: The number of embryos was significantly higher in autumn. Abnormal morphological parameters (color, size, zona thickness and the degeneration rate were significantly higher in spring. The number of retrieved oocytes, MI, MII oocytes and fertilization rate had no significant seasonal variations.Conclusion: The results of this study showed a significant seasonal variation in morphological parameters of the oocytes, degeneration rate and the number of formed embryos.

  12. Reorganization of cytoskeletal proteins of mouse oocytes mediated by integrins

    Institute of Scientific and Technical Information of China (English)

    YUE Limin; ZHANG Lei; HE Yaping; ZHANG Jinhu; ZHENG Jie; HE Yanfang; ZHENG Yu; ZHANG Jie; ZHANG Li

    2004-01-01

    To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for superovulation. The zona-free oocytes were incubated separately with specific ligand of integrins,an active RGD peptide, in vitro for certain period of time. RGE peptide and mouse capacitated sperm were used as controls. Freshly ovulated oocytes and those treated with different factors were immunostained with FITC-labeled anti-actin antibody, then detected with confocal microscope. The results demonstrated that freshly ovulated mouse oocytes, oocytes incubated for 2 h in vitro and those treated with control RGE peptide for 15 min showed hardly visible fluorescene or only thin fluorescence in plasma membrane region. Oocytes coincubated with sperms for 15 min and those treated with active RGD peptide for 10 min, 30 min and 2 hours respectively had strong and thick fluorescence in the plasma membrane and cortical region of oocytes, and some of them showed asymmetrically fluorescent distribution. It is proved that integrins on membrane are ligated directly with cytoskeletal protein. Integrins binding with their ligands regulate reorganization of cytoskelal protein, which may be involved in transmembrane signaling in egg activation.

  13. Reorganization of cytoskeletal proteins of mouse oocytes mediated by integrins

    Institute of Scientific and Technical Information of China (English)

    YUE; Limin; ZHANG; Lei; HE; Yaping; ZHANG; Jinhu; ZHENG; Ji

    2004-01-01

    To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for superovulation. The zona-free oocytes were incubated separately with specific ligand of integrins,an active RGD peptide, in vitro for certain period of time. RGE peptide and mouse capacitated sperm were used as controls. Freshly ovulated oocytes and those treated with different factors were immunostained with FITC-labeled anti-actin antibody, then detected with confocal microscope. The results demonstrated that freshly ovulated mouse oocytes, oocytes incubated for 2 h in vitro and those treated with control RGE peptide for 15 min showed hardly visible fluorescene or only thin fluorescence in plasma membrane region. Oocytes coincubated with sperms for 15 min and those treated with active RGD peptide for 10 min, 30 min and 2 hours respectively had strong and thick fluorescence in the plasma membrane and cortical region of oocytes, and some of them showed asymmetrically fluorescent distribution. It is proved that integrins on membrane are ligated directly with cytoskeletal protein. Integrins binding with their ligands regulate reorganization of cytoskelal protein, which may be involved in transmembrane signaling in egg activation.

  14. Txndc9 Is Required for Meiotic Maturation of Mouse Oocytes

    Directory of Open Access Journals (Sweden)

    Fanhua Ma

    2017-01-01

    Full Text Available Txndc9 (thioredoxin domain containing protein 9 has been shown to be involved in mammalian mitosis; however, its function in mammalian oocyte meiosis remains unclear. In this study, we initially found that Txndc9 is expressed during meiotic maturation of mouse oocytes and higher expression of Txndc9 mRNA and protein occurred in germinal vesicle (GV stage. By using confocal scanning, we observed that Txndc9 localized at both nucleus and cytoplasm, especially at spindle microtubules. Specific depletion of Txndc9 by siRNA in mouse oocyte resulted in decreasing the rate of first polar body extrusion and increasing abnormal spindle assemble. Moreover, knockdown of Txndc9 in germinal vesicle (GV stage oocytes led to higher level of reactive oxygen species (ROS and lower level of antioxidant glutathione (GSH as compared with control oocytes, which indicated that Txndc9 may be involved in mediating the redox balance. In summary, our results demonstrated that Txndc9 is crucial for mouse oocyte maturation by regulating spindle assembly, polar body extrusion, and redox status.

  15. Non-meiotic chromosome instability in human immature oocytes.

    Science.gov (United States)

    Daina, Gemma; Ramos, Laia; Rius, Mariona; Obradors, Albert; Del Rey, Javier; Giralt, Magda; Campillo, Mercedes; Velilla, Esther; Pujol, Aïda; Martinez-Pasarell, Olga; Benet, Jordi; Navarro, Joaquima

    2014-02-01

    Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25-45 years of age) and 24 IVF oocyte donors (18-33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.

  16. Total number of oocytes and zygotes are predictive of live birth pregnancy in fresh donor oocyte in vitro fertilization cycles.

    Science.gov (United States)

    Hariton, Eduardo; Kim, Keewan; Mumford, Sunni L; Palmor, Marissa; Bortoletto, Pietro; Cardozo, Eden R; Karmon, Anatte E; Sabatini, Mary E; Styer, Aaron K

    2017-08-01

    To evaluate the association of oocyte donor-recipient characteristics, oocyte donor response, and live birth pregnancy rate following fresh donor oocyte IVF-ET. Retrospective cohort study. Academic reproductive medicine practice. Two hundred thirty-seven consecutive fresh donor oocyte IVF-ET cycles from January 1, 2007 to December 31, 2013 at the Massachusetts General Hospital Fertility Center. None. Live birth rate per cycle initiated. The mean (±SD) age of oocyte donors and recipients was 27.0 ± 3.7 and 41.4 ± 4.6 years, respectively. Oocyte donor demographic/reproductive characteristics, ovarian reserve testing, and peak serum E2 during ovarian stimulation were similar among cycles which did and did not result in live birth, respectively. Overall implantation, clinical pregnancy, and live birth pregnancy rates per cycle initiated were 40.5%, 60.8%, and 54.9%, respectively. The greatest probability of live birth was observed in cycles with >10 oocytes retrieved, mature oocytes, oocytes with normal fertilization (zygote-two pronuclear stage), and cleaved embryos. The number of oocytes (total and mature), zygotes, and cleaved embryos are associated with live birth following donor oocyte IVF cycles. These findings suggest that specific peri-fertilization factors may be predictive of pregnancy outcomes following donor oocyte IVF cycles. Copyright © 2017 American Society for Reproductive Medicine. All rights reserved.

  17. Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes.

    Science.gov (United States)

    Holubcová, Zuzana; Blayney, Martyn; Elder, Kay; Schuh, Melina

    2015-06-05

    Aneuploidy in human eggs is the leading cause of pregnancy loss and several genetic disorders such as Down syndrome. Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg's progenitor cell. The basis for particularly error-prone chromosome segregation in human oocytes is not known. We analyzed meiosis in more than 100 live human oocytes and identified an error-prone chromosome-mediated spindle assembly mechanism as a major contributor to chromosome segregation defects. Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. Instead, spindle assembly was mediated by chromosomes and the small guanosine triphosphatase Ran in a process requiring ~16 hours. This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs.

  18. Oocyte development and fecundity type of the Brazilian Snapper Lutjanus alexandrei Moura & Lindeman, 2007 (Perciformes: Lutjanidae).

    Science.gov (United States)

    Fernandes, C A F; Oliveira, P G V; Oliveira, C H B; Hazin, F H V; Travassos, P

    2016-02-01

    Lutjanid species exhibit multiple spawning behaviour during an extended spawning season in warm months, asynchronous oocyte development and indeterminate fecundity. Although early studies have contributed to knowledge of the reproductive cycle of many species within the group, they have not considered aspects about the number of cortical alveoli oocyte stage throughout maturity phases along spawning season. The latter aspect is also considered very important to confirm indeterminate fecundity hypothesis. In the present study, were analyzed 154 Brazilian snapper Lutjanus alexandrei female gonads obtained from artisanal fisheries in Pernambuco State (Brazil) between October 2010 and March 2011. Were measured oocyte size frequency distribution for maturity phases (developing, spawning capable and actively spawning), and oocyte development stage (unyolked oocytes, cortical alveoli, primary, secondary and tertiary vitellogenic oocytes and hydrated oocytes), and also the oocyte stage frequency during spawning season. The frequency of cortical alveoli oocyte stage was constantly found in the spawning period (>37%), showing slight variation throughout maturity phases. The absence of gap in the oocyte size frequency distribution between primary and secondary oocyte growth stages during spawning season is a strong indicator of continuous oocyte recruitment from reserve stocks. In addition, co-occurrence of tertiary vitellogenic oocytes, hydrated oocytes, post-ovulatory follicles and yellow-brown bodies in the histological sections of ovaries reinforce indeterminate fecundity hypothesis.

  19. Kinetochore microtubule establishment is defective in oocytes from aged mice.

    Science.gov (United States)

    Shomper, Maria; Lappa, Christina; FitzHarris, Greg

    2014-01-01

    Errors in chromosome segregation in mammalian oocytes increase in number with advancing maternal age, and are a major cause of pregnancy loss. Why chromosome segregation errors are more common in oocytes from older females remains poorly understood. In mitosis, accurate chromosome segregation is enabled by attachment of kinetochores to microtubules from appropriate spindle poles, and erroneous attachments increase the likelihood of mis-segregation. Whether attachment errors are responsible for age-related oocyte aneuploidy is unknown. Here we report that oocytes from naturally aged mice exhibit substantially increased chromosome misalignment, and fewer kinetochore pairs that make stable end-on attachments to the appropriate spindle poles compared with younger oocytes. The profile of mis-attachments exhibited is consistent with the types of chromosome segregation error observed in aged oocytes. Loss of chromosome cohesion, which is a feature of oocytes from older females, causes altered kinetochore geometry in meiosis-I. However, this has only a minor impact upon MT attachment, indicating that cohesion loss is not the primary cause of aneuploidy in meiosis-I. In meiosis-II, on the other hand, age-related cohesion loss plays a direct role in errors, since prematurely individualized sister chromatids misalign and misattach to spindle MTs. Thus, whereas cohesion loss leading to precocious sister chromatid separation is a direct cause of errors in meiosis-II, cohesion loss plays a more minor role in the etiology of aneuploidy in meiosis-I. Our data introduce altered MT-kinetochore interactions as a lesion that explains aneuploidy in meiosis-I in older females.

  20. Mural granulosa cell gene expression associated with oocyte developmental competence

    Directory of Open Access Journals (Sweden)

    Jiang Jin-Yi

    2010-03-01

    Full Text Available Abstract Background Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte. Methods Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC. Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array. Results The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox and nerve growth factor receptor associated protein 1 (Ngfrap1, which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2, which is involved in the regulation of extracellular matrix organization and biogenesis. Conclusions The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and

  1. Ovarian ageing: the role of mitochondria in oocytes and follicles.

    Science.gov (United States)

    May-Panloup, Pascale; Boucret, Lisa; Chao de la Barca, Juan-Manuel; Desquiret-Dumas, Valérie; Ferré-L'Hotellier, Véronique; Morinière, Catherine; Descamps, Philippe; Procaccio, Vincent; Reynier, Pascal

    2016-11-01

    There is a great inter-individual variability of ovarian ageing, and almost 20% of patients consulting for infertility show signs of premature ovarian ageing. This feature, taken together with delayed childbearing in modern society, leads to the emergence of age-related ovarian dysfunction concomitantly with the desire for pregnancy. Assisted reproductive technology is frequently inefficacious in cases of ovarian ageing, thus raising the economic, medical and societal costs of the procedures. Ovarian ageing is characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. Mitochondria play a central role in follicular atresia and could be the main target of the ooplasmic factors determining oocyte quality adversely affected by ageing. Indeed, the oocyte is the richest cell of the body in mitochondria and depends largely on these organelles to acquire competence for fertilization and early embryonic development. Moreover, the oocyte ensures the uniparental transmission and stability of the mitochondrial genome across the generations. This review focuses on the role played by mitochondria in ovarian ageing and on the possible consequences over the generations. PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning mitochondria and ovarian ageing, in animal and human species. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA'; 'ovarian reserve', 'oocyte', 'ovary' or 'cumulus cells'; and 'ageing' or 'ovarian ageing'. These keywords were combined with other search phrases relevant to the topic. References from these articles were used to obtain additional articles. There is a close relationship, in mammalian models and humans, between mitochondria and the decline of oocyte quality with ageing. Qualitatively, ageing-related mitochondrial (mt) DNA instability, which leads to the accumulation of mtDNA mutations in the oocyte, plays a key role in

  2. In vitro developmental competence of bovine oocytes: Effect of corpus luteum and follicle size

    Science.gov (United States)

    Karami Shabankareh, Hamed; Shahsavari, Mohammad Hamed; Hajarian, Hadi; Moghaddam, Gholamali

    2015-01-01

    Background: Previous studies reported many discrepancies about the effects of corpus luteum (CL) and ovarian follicle size on the developmental competence of oocytes. Objective: The aim of this study was to investigate the effects of CL and different size of follicle on the developmental potential of bovine oocytes. Materials and Methods: After ovarian classification based on presence or absence of CL, sample follicles were placed in three groups according to their diameter; small (S; 3–6 mm), medium (M; 6–9 mm), and large (L; 10–20 mm). Collected oocytes in each group were subjected to the in vitro embryo production processes. Results: Results showed that, the percentages of blastocyst obtained from oocytes originating from small and medium follicles of ovaries bearing a CL (CL+S-oocytes and CL+M-oocytes, respectively) were lower (p<0.001) than those of small and medium follicles of ovaries not bearing a CL (CL-S-oocytes and CL-M-oocytes, respectively) (30.8% and 33.6% vs. 36.9% and 38.7% respectively). Although, the percentages of blastocyst obtained from CL-M-oocytes and CL-L-oocytes were greater (p< 0.001) than those of CL+S-oocytes and CL+M-oocytes. There were no significant differences in the percentages of blastocyst formation between controls (C-oocytes), CL-S-oocytes and CL+L-oocytes. Conclusion: According to the results of this study, the negative effect of CL on the developmental competence of bovine oocyte depends on the follicle size. Therefore, oocytes originating from large grown follicles were not influenced by negative effects of CL as much as those originating from small and medium follicles did. PMID:26644789

  3. Time-Lapse Dynamics of the Mouse Oocyte Chromatin Organisation during Meiotic Resumption

    Directory of Open Access Journals (Sweden)

    Martina Belli

    2014-01-01

    Full Text Available In the mammalian oocyte, distinct patterns of centromeres and pericentromeric heterochromatin localisation correlate with the gamete’s developmental competence. Mouse antral oocytes display two main types of chromatin organisation: SN oocytes, with a ring of Hoechst-positive chromatin surrounding the nucleolus, and NSN oocytes lacking this ring. When matured to MII and fertilised, only SN oocytes develop beyond the 2-cell, and reach full term. To give detailed information on the dynamics of the SN or NSN chromatin during meiosis resumption, we performed a 9 hr time-lapse observation. The main significant differences recorded are: (1 reduction of the nuclear area only in SN oocytes; (2 ~17 min delay of GVBD in NSN oocytes; (3 chromatin condensation, after GVBD, in SN oocytes; (4 formation of 4-5 CHCs in SN oocytes; (5 increase of the perivitelline space, ~57 min later in NSN oocytes; (6 formation of a rosette-like disposition of CHCs, ~84 min later in SN oocytes; (7 appearance of the MI plate ~40 min later in NSN oocytes. Overall, we described a pathway of transition from the GV to the MII stage that is punctuated of discrete recordable events showing their specificity and occurring with different time kinetics in the two types of oocytes.

  4. Time-Lapse Dynamics of the Mouse Oocyte Chromatin Organisation during Meiotic Resumption

    Science.gov (United States)

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

    In the mammalian oocyte, distinct patterns of centromeres and pericentromeric heterochromatin localisation correlate with the gamete's developmental competence. Mouse antral oocytes display two main types of chromatin organisation: SN oocytes, with a ring of Hoechst-positive chromatin surrounding the nucleolus, and NSN oocytes lacking this ring. When matured to MII and fertilised, only SN oocytes develop beyond the 2-cell, and reach full term. To give detailed information on the dynamics of the SN or NSN chromatin during meiosis resumption, we performed a 9 hr time-lapse observation. The main significant differences recorded are: (1) reduction of the nuclear area only in SN oocytes; (2) ~17 min delay of GVBD in NSN oocytes; (3) chromatin condensation, after GVBD, in SN oocytes; (4) formation of 4-5 CHCs in SN oocytes; (5) increase of the perivitelline space, ~57 min later in NSN oocytes; (6) formation of a rosette-like disposition of CHCs, ~84 min later in SN oocytes; (7) appearance of the MI plate ~40 min later in NSN oocytes. Overall, we described a pathway of transition from the GV to the MII stage that is punctuated of discrete recordable events showing their specificity and occurring with different time kinetics in the two types of oocytes. PMID:24864231

  5. Changes of spontaneous parthenogenetic activation and development potential of golden hamster oocytes during the aging process.

    Science.gov (United States)

    Jiang, Han; Wang, Ce; Guan, Jiyu; Wang, Lingyan; Li, Ziyi

    2015-01-01

    The golden hamster is an excellent animal experimental model for oocyte research. The hamster oocytes are very useful in clinical examination of human spermatozoan activity. Non-fertile oocytes can lead to time-dependent processes of aging, which will affect the results of human spermatozoa examination. As a consequence there is a need to investigate the aging and anti-aging processes of golden hamster oocytes. In order to study the aging processes and parthenogenetic activation of golden hamster oocytes, in vivo oocytes, oocytes cultured with or without cumulus cells, and oocytes treated with Trichostatin A (TSA) or caffeine were collected and investigated. We found that: (1) spontaneous parthenogenetic activation, developmental potential (cleavage rate), and zona pellucida (ZP) hardening undergo age-dependent changes in in vivo, in vitro, and after TSA or caffeine treatment; (2) in vivo, oocytes became spontaneously parthenogenetic 25 h post-hCG treatment; (3) in vitro, cumulus cells did not significantly increase the parthenogenetic activation rate of cultured hamster oocytes; and (4) TSA or caffeine could delay spontaneous oocyte parthenogenetic activation and the aging processes by at least 5h, but also accelerated the hardening of the ZP. These results define the conditions for the aging and anti-aging processes in golden hamster oocytes. TSA and caffeine play roles in controlling spontaneous activation, which could facilitate the storage and use of golden hamster oocytes for studying processes relevant to human reproduction.

  6. Transcriptomic features of Pecten maximus oocyte quality and maturation

    Science.gov (United States)

    Milan, Massimo; Huvet, Arnaud; Corporeau, Charlotte; Suquet, Marc; Planas, Josep V.; Moreira, Rebeca; Figueras, Antonio; Novoa, Beatriz; Patarnello, Tomaso; Bargelloni, Luca

    2017-01-01

    The king scallop Pecten maximus is a high valuable species of great interest in Europe for both fishery and aquaculture. Notably, there has been an increased investment to produce seed for enhancement programmes of wild scallop populations. However, hatchery production is a relatively new industry and it is still underdeveloped. Major hurdles are spawning control and gamete quality. In the present study, a total of 14 scallops were sampled in the bay of Brest (Brittany, France) to compare transcriptomic profiles of mature oocytes collected by spawning induction or by stripping. To reach such a goal, a microarray analysis was performed by using a custom 8x60K oligonucleotide microarray representing 45,488 unique scallop contigs. First we identified genes that were differentially expressed depending on oocyte quality, estimated as the potential to produce D-larvae. Secondly, we investigated the transcriptional features of both stripped and spawned oocytes. Genes coding for proteins involved in cytoskeletal dynamics, serine/threonine kinases signalling pathway, mRNA processing, response to DNA damage, apoptosis and cell-cycle appeared to be of crucial importance for both oocyte maturation and developmental competence. This study allowed us to dramatically increase the knowledge about transcriptional features of oocyte quality and maturation, as well as to propose for the first time putative molecular markers to solve a major bottleneck in scallop aquaculture. PMID:28253290

  7. Attitudes and intention to donate oocytes for research.

    Science.gov (United States)

    Purewal, Satvinder; van den Akker, Olga

    2010-03-01

    In 2007, the Human Fertilization and Embryology Authority permitted oocyte donation for research through voluntary donation or within an oocyte share model. The aims of this study were to investigate volunteer (nonpatient) women's attitudes and intentions to donate using components of the Theory of Planned Behavior and their attitudes toward parenthood through structural equation modeling. Questionnaires. Online. A total of 253 nonpatient women. Attitudes towards oocyte donation for research and reasons for parenthood scale. Of the 253 respondents, 94 were potential donors, 98 were possible donors, and 61 were non-donors. Most potential donors (68%) reported no preference towards donating their oocytes for research or an infertile couple. Structural equation modeling revealed that age (beta = -.03) and components of the TPB (beta = .16) had a statistically significant direct effect on intentions to donate for research. Attitudes toward parenthood was not linked to intentions to donate for research. There appears to be a strong altruistic motive along with the theoretical underpinnings of positive attitudes, feeling supported, and accepting the consequences of oocyte donation for research, suggesting these have the potential to inform recruitment practices and tailor clinical services. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  8. Kif4 Is Essential for Mouse Oocyte Meiosis.

    Science.gov (United States)

    Camlin, Nicole J; McLaughlin, Eileen A; Holt, Janet E

    2017-01-01

    Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.

  9. Translational regulation of MOS messenger RNA in pig oocytes.

    Science.gov (United States)

    Dai, Yanfeng; Newman, Barbara; Moor, Robert

    2005-11-01

    The temporal and spatial translation control of stored mRNA in oocytes is regulated by elements in their 3'-untranslated region (3'-UTR). The MOS 3'-UTR in pig oocytes is both heterogeneous (180, 480, or 530 nucleotides), and it contains multiple U-rich elements and extensive A-rich sequences (CA13CA5CA5CA6). We have examined the role of these potential regulatory elements by fusing wild-type or mutant MOS 3'-UTRs to luciferase mRNA and then injecting these chimeric transcripts into oocytes. We draw six main conclusions. First, the length of the MOS 3'-UTR tightly controls the level of translation of luciferase during oocyte maturation. Second, two U-rich (U5A) elements and the hexanucleotide signal (AAUAAA) are required for translation. Third, mutations, duplications, or relocations of the A-rich sequence reduce or block translation. Fourth, the relative importance of the A-rich and U-rich elements in controlling the level of translation differs. Fifth, none of our MOS 3'-UTR manipulations relieved translational repression before germinal vesicle breakdown. Sixth, all the MOS mRNA variants underwent polyadenylation during maturation. Whereas mutations to the hexanucleotide signal block both polyadenylation and translation, mutations to either the A-rich sequence or the U-rich elements block translation without fully blocking polyadenylation. We conclude that MOS mRNA translation in pig oocytes is subject to a more extensive series of controls than that in lower vertebrates.

  10. Factors influencing the biochemical markers for predicting mammalian oocyte quality.

    Science.gov (United States)

    Ola, Safiriyu Idowu; Sun, Qing-Yuan

    2012-01-01

    The need for accurate selection of the best oocytes for in vitro fertilization protocols and thus, production of embryos has driven the search for oocyte quality markers from morphological criteria to biochemical parameters. Current studies are focused on the biochemical constituents of the follicular fluid and gene expression profiling of the cumulus cells. These parameters are, however, affected by factors that must be considered before making a judgment of the oocyte's quality. These includes factors such as the type of hormonal stimulation protocol, age of oocyte donor and heat stress on the donor, all of which have been reported to influence the concentrations of many hormones, apolipoproteins, metabolites, fatty acids and growth factors in the follicular fluid and the expression of several genes in the cumulus cells. Another important point to note is species variation in the response to these extraneous influences, which thus calls for species targeted investigations. As reports are still scanty and investigations assumed to be very keen, we employed this review paper to bring attention of researchers and clinicians to those factors that may come to bear on the outcome of their investigations on oocyte and embryo quality.

  11. The Effects of Progesterone on Oocyte Maturation and Embryo Development

    Directory of Open Access Journals (Sweden)

    Saeed Zavareh

    2013-01-01

    Full Text Available Oocyte maturation and embryo development are controlled by intra-ovarian factors suchas steroid hormones. Progesterone (P4 exists in the follicular fluid that contributes tonormal mammalian ovarian function and has several critical functions during embryodevelopment and implantation, including endometrial receptivity, embryonic survivalduring gestation and transformation of the endometrial stromal cells to decidual cells.It is well known that the physiological effects of P4 during the pre-implantation stages ofsome mammal’s embryos are mediated by P4 receptors and their gene expression is determined.The effects of P4 on oocytes and embryo development have been assessed bysome investigations, with contradictory results. P4, a dominant steroid in follicular fluidat approximately 18 hours after the luteinizing hormone (LH surge may have a criticalrole in maturation of oocytes at the germinal stage. However, it has been shown that differentconcentrations of P4 could not improve in vitro maturation rates of germinal vesicles(GV in cumulus oocyte complexes (COCs and cumulus denuded oocytes (CDOs.Culture media supplemented with P4 significantly improved mouse embryo development.In addition, an in vivo experimental design has shown high blastocyst survival andimplantation rates in P4-treated mice.In this review we explain some of the findings that pertain to the effects of P4 onoocyte maturation and embryo development both in vitro and in vivo.

  12. PTK2b function during fertilization of the mouse oocyte.

    Science.gov (United States)

    Luo, Jinping; McGinnis, Lynda K; Carlton, Carol; Beggs, Hilary E; Kinsey, William H

    2014-08-01

    Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development. Published by Elsevier Inc.

  13. Kif4 Is Essential for Mouse Oocyte Meiosis

    Science.gov (United States)

    Camlin, Nicole J.; McLaughlin, Eileen A.; Holt, Janet E.

    2017-01-01

    Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age. PMID:28125646

  14. High-throughput optofluidic system for the laser microsurgery of oocytes

    Science.gov (United States)

    Chandsawangbhuwana, Charlie; Shi, Linda Z.; Zhu, Qingyuan; Alliegro, Mark C.; Berns, Michael W.

    2012-01-01

    This study combines microfluidics with optical microablation in a microscopy system that allows for high-throughput manipulation of oocytes, automated media exchange, and long-term oocyte observation. The microfluidic component of the system transports oocytes from an inlet port into multiple flow channels. Within each channel, oocytes are confined against a microfluidic barrier using a steady fluid flow provided by an external computer-controlled syringe pump. This allows for easy media replacement without disturbing the oocyte location. The microfluidic and optical-laser microbeam ablation capabilities of the system were validated using surf clam (Spisula solidissima) oocytes that were immobilized in order to permit ablation of the 5 μm diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.

  15. Obstetric and neonatal outcome after oocyte donation in 106 women with Turner syndrome

    DEFF Research Database (Denmark)

    Hagman, Anna; Loft, Anne; Wennerholm, Ulla-Britt

    2013-01-01

    What are the obstetric and neonatal outcomes of deliveries after oocyte donation (OD) in women with Turner syndrome (TS)?......What are the obstetric and neonatal outcomes of deliveries after oocyte donation (OD) in women with Turner syndrome (TS)?...

  16. A new approach for the oocyte genotoxicity assay: adaptation of comet assay on mouse cumulus-oocyte complexes.

    Science.gov (United States)

    Greco, F; Perrin, J; Auffan, M; Tassistro, V; Orsière, T; Courbiere, B

    2015-07-01

    Conventional genotoxicity tests are technically difficult to apply to oocytes, and results obtained on somatic cells cannot be extrapolated to gametes. We have previously described a comet assay (original-CA) on denuded mouse oocytes, but, in vivo, oocytes are not isolated from their surrounding follicular cells. Our objective was to develop a comet assay on cumulus-oocyte complexes (COC-CA) for a more physiological approach to study the genotoxicity of environmental factors on oocytes. For COC-CA, whole COC were exposed directly to exogenous agents after ovulation and removal from oviducts. Three conditions were studied: a negative control group, and two positive control groups, one of which was exposed to hydrogen peroxide (H2O2) and the other group was incubated with cerium dioxide nanoparticles (CeO2 NPs). With both tests, DNA damage was significant in the presence of both H2O2 and CeO2 NPs compared with the negative control. COC-CA offers an interesting tool for assaying the genotoxicity of environmental agents towards germinal cells. Furthermore, COC-CA is less time-consuming and simplifies the protocol of the original-CA, because COC-CA is easier to perform without the washing-out procedure.

  17. Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes

    Science.gov (United States)

    Wigglesworth, Karen; Lee, Kyung-Bon; O’Brien, Marilyn J.; Peng, Jia; Matzuk, Martin M.; Eppig, John J.

    2013-01-01

    Coordinated regulation of oocyte and ovarian follicular development is essential for fertility. In particular, the progression of meiosis, a germ cell-specific cell division that reduces the number of chromosomes from diploid to haploid, must be arrested until just before ovulation. Follicular somatic cells are well-known to impose this arrest, which is essential for oocyte–follicle developmental synchrony. Follicular somatic cells sustain meiotic arrest via the natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system, and possibly also via high levels of the purine hypoxanthine in the follicular fluid. Upon activation by the ligand NPPC, NPR2, the predominant guanylyl cyclase in follicular somatic cells, produces cyclic guanosine monophosphate (cGMP), which maintains meiotic arrest after transfer to the oocyte via gap junctions. Here we report that both the NPPC/NPR2 system and hypoxanthine require the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme required for the production of guanylyl metabolites and cGMP. Furthermore, oocyte-derived paracrine factors, particularly the growth differentiation factor 9–bone morphogenetic protein 15 heterodimer, promote expression of Impdh and Npr2 and elevate cGMP levels in cumulus cells. Thus, although the somatic compartment of ovarian follicles plays an essential role in the maintenance of oocyte meiotic arrest, as has been known for many years, this function of the somatic cells is surprisingly regulated by signals from the oocyte itself. PMID:23980176

  18. Translation in the mammalian oocyte in space and time.

    Science.gov (United States)

    Susor, Andrej; Jansova, Denisa; Anger, Martin; Kubelka, Michal

    2016-01-01

    A hallmark of oocyte development in mammals is the dependence on the translation and utilization of stored RNA and proteins rather than the de novo transcription of genes in order to sustain meiotic progression and early embryo development. In the absence of transcription, the completion of meiosis and early embryo development in mammals relies significantly on maternally synthesized RNAs. Post-transcriptional control of gene expression at the translational level has emerged as an important cellular function in normal development. Therefore, the regulation of gene expression in oocytes is controlled almost exclusively at the level of mRNA and protein stabilization and protein synthesis. This current review is focused on the recently emerged findings on RNA distribution related to the temporal and spatial translational control of the meiotic progression of the mammalian oocyte.

  19. Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics

    Directory of Open Access Journals (Sweden)

    Jennifer R. Prentice

    2011-01-01

    Full Text Available The preservation of the female portion of livestock genetics has become an international priority; however, in situ conservation strategies are extremely expensive. Therefore, efforts are increasingly focusing on the development of a reliable cryopreservation method for oocytes, in order to establish ova banks. Slow freezing, a common method for cryopreservation of oocytes, causes osmotic shock (solution effect and intracellular ice crystallization leading to cell damage. Vitrification is an alternative method for cryopreservation in which cells are exposed to a higher concentration of cryoprotectants and frozen with an ultra rapid freezing velocity, resulting in an ice crystal free, solid glass-like structure. Presently, vitrification is a popular method for cryopreservation of embryos. However, vitrification of oocytes is still challenging due to their complex structure and sensitivity to chilling.

  20. Cryopreservation of embryos and oocytes in human assisted reproduction.

    Science.gov (United States)

    Konc, János; Kanyó, Katalin; Kriston, Rita; Somoskői, Bence; Cseh, Sándor

    2014-01-01

    Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized.

  1. Oocyte Maturation and Development [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Marie-Hélène Verlhac

    2016-03-01

    Full Text Available Sexual reproduction is essential for many organisms to propagate themselves. It requires the formation of haploid female and male gametes: oocytes and sperms. These specialized cells are generated through meiosis, a particular type of cell division that produces cells with recombined genomes that differ from their parental origin. In this review, we highlight the end process of female meiosis, the divisions per se, and how they can give rise to a functional female gamete preparing itself for the ensuing zygotic development. In particular, we discuss why such an essential process in the propagation of species is so poorly controlled, producing a strong percentage of abnormal female gametes in the end. Eventually, we examine aspects related to the lack of centrosomes in female oocytes, the asymmetry in size of the mammalian oocyte upon division, and in mammals the direct consequences of these long-lived cells in the ovary.

  2. Cryopreservation of Mammalian oocyte for conservation of animal genetics.

    Science.gov (United States)

    Prentice, Jennifer R; Anzar, Muhammad

    2010-09-21

    The preservation of the female portion of livestock genetics has become an international priority; however, in situ conservation strategies are extremely expensive. Therefore, efforts are increasingly focusing on the development of a reliable cryopreservation method for oocytes, in order to establish ova banks. Slow freezing, a common method for cryopreservation of oocytes, causes osmotic shock (solution effect) and intracellular ice crystallization leading to cell damage. Vitrification is an alternative method for cryopreservation in which cells are exposed to a higher concentration of cryoprotectants and frozen with an ultra rapid freezing velocity, resulting in an ice crystal free, solid glass-like structure. Presently, vitrification is a popular method for cryopreservation of embryos. However, vitrification of oocytes is still challenging due to their complex structure and sensitivity to chilling.

  3. Hormonal Regulation of Oocyte Development and Maturation of Amphioxus

    Institute of Scientific and Technical Information of China (English)

    方永强; 赵维信; 林秋明

    1994-01-01

    Using the electron microscopic technique and radioimmunoassay,the hormonal regulationof the oocyte development and maturation of the amphioxus has been investigated.The results indicate thatthe exogenous GnRH-A can stimulate the oocyte development and maturation as well as the spawning of theamphioxus.It has further been revealed by radioimmunoassay that GnRH-A can increase the content of sexsteroid hormone,especially,estradiol-17β,by 1—3 times.The increase of estradiol-17β coordinates withthe synthesis of oocyte into yolk substance.The results further justify that the hormonal regulation in theoocyte development and maturation of amphioxus is similar to that of vertebrates.This is of great theoreticalsignificance for reproduetive endocrine physiology of amphioxus.

  4. Cryopreservation of Embryos and Oocytes in Human Assisted Reproduction

    Directory of Open Access Journals (Sweden)

    János Konc

    2014-01-01

    Full Text Available Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification of human embryos and oocytes are summarized.

  5. Roles of MAP kinase signaling pathway in oocyte meiosis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases expressed widely in eukaryotic cells. MAPK is activated by a cascade of protein kinase phosphorylation and plays pivotal roles in regulating meiosis process in oocytes. As an important physical substrate of MAPK, p90rsk mediates numerous MAPK functions. MAPK was activated at G2/M transition during meiosis. Its activity reached the peak at MⅠ stage and maintained at this level until the time before the pronuclear formation after fertilization. There is complex interplay between MAPK and MPF in the meiosis regulation. Furthermore, other intracellular signal transducers, such as cAMP, protein kinase C and protein phosphotase, ect., also regulated the activity of MAPK at different stages during meiosis in oocytes. In the present article, the roles of MAPK signaling pathway in oocyte meiosis are reviewed and discussed.

  6. Live imaging of GFP-labeled proteins in Drosophila oocytes.

    Science.gov (United States)

    Pokrywka, Nancy Jo

    2013-03-29

    The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes. The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent and provides a useful system for investigating cytoskeleton function at these stages. Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

  7. Regulation of ALF promoter activity in Xenopus oocytes.

    Directory of Open Access Journals (Sweden)

    Dan Li

    Full Text Available BACKGROUND: In this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter. PRINCIPAL FINDINGS: The promoter from the ALF transcription factor gene was cloned from X. laevis genomic DNA using a PCR-based genomic walking approach. The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues. Homology between the X. laevis ALF promoter sequence and those from human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis was relatively low, making it difficult to use such comparisons to identify putative regulatory elements. However, microinjected promoter constructs were very active in oocytes and the minimal promoter could be narrowed by PCR-mediated deletion to a region as short as 63 base pairs. Additional experiments using a series of site-specific promoter mutants identified two cis-elements within the 63 base pair minimal promoter that were critical for activity. Both elements (A and B were specifically recognized by proteins present in crude oocyte extracts based on oligonucleotide competition assays. The activity of promoter constructs in oocytes and in transfected somatic Xenopus XLK-WG kidney epithelial cells was quite different, indicating that the two cell types are not functionally equivalent and are not interchangeable as assay systems. CONCLUSIONS: Overall the results provide the first detailed characterization of the organization of a germ cell-specific Xenopus promoter and demonstrate the feasibility of using immature frog oocytes as an assay system for dissecting the biochemistry of germ cell gene regulation.

  8. Aflatoxin B1 is toxic to porcine oocyte maturation.

    Science.gov (United States)

    Liu, Jun; Wang, Qiao-Chu; Han, Jun; Xiong, Bo; Sun, Shao-Chen

    2015-07-01

    As a toxic secondary metabolite of Aspergillus species, Aflatoxin B1 (AFB1) is a major food and feed contaminant in tropical and sub-tropical regions with high temperature and humidity. It has been reported to be toxic to the female reproductive system in laboratory and domestic animals. In the present study, the influence of acute exposure to AFB1 (10 and 50 μM, 44h) on porcine oocyte maturation and its possible mechanism were investigated. The maturation rates of oocytes decreased significantly in the presence of 50 μM of AFB1. Cell cycle analysis showed that most oocytes were arrested at germinal vesicle breakdown or meosis I stage. However, actin assembly, spindle structure and chromosome alignment were not disrupted after exposure to 50 μM AFB1. Further study showed that DNA methylation levels increased in treated oocytes (50 μM). Histone methylation levels were also analysed after treatment (50 μM): H3K27me3 and H3K4me2 levels decreased, whereas H3K9me3 level increased, indicating that epigenetic modification was affected. AFB1 treatment (50 μM) also induced oxidative stress and further led to autophagy, as shown by accumulation of reactive oxygen species, up-regulated LC3 protein expression and increased mRNA levels of ATG3, ATG5 and ATG7. Annexin V-FITC staining assay revealed that AFB1 treatment (50 μM) resulted in oocyte early apoptosis, which was confirmed by increased Bak, Bax, Bcl-xl mRNA levels. Collectively, our results suggest that AFB1 disrupts porcine oocyte maturation through changing epigenetic modifications as well as inducing oxidative stress, excessive autophagy and apoptosis.

  9. Cholesterol depletion disorganizes oocyte membrane rafts altering mouse fertilization.

    Directory of Open Access Journals (Sweden)

    Jorgelina Buschiazzo

    Full Text Available Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1 a decrease of the fertilization rate and index; and (2 a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.

  10. Influence of co-culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus-enclosed porcine oocytes in a defined system.

    Science.gov (United States)

    Appeltant, Ruth; Somfai, Tamás; Kikuchi, Kazuhiro; Maes, Dominiek; Van Soom, Ann

    2016-04-01

    Co-culture of cumulus-oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte-secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β-mercaptoethanol. Cumulus-oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co-culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus-enclosed porcine oocytes in a defined system.

  11. Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: a model for utilization of primordial oocytes.

    Science.gov (United States)

    Kaneko, Hiroyuki; Nakai, Michiko; Tanihara, Fuminori; Noguchi, Junko; Kikuchi, Kazuhiro

    2013-11-01

    Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1(nu)). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Acentrosomal spindle assembly and chromosome segregation during oocyte meiosis.

    Science.gov (United States)

    Dumont, Julien; Desai, Arshad

    2012-05-01

    The ability to reproduce relies in most eukaryotes on specialized cells called gametes. Gametes are formed by the process of meiosis in which, after a single round of replication, two successive cell divisions reduce the ploidy of the genome. Fusion of gametes at fertilization reconstitutes diploidy. In most animal species, chromosome segregation during female meiosis occurs on spindles assembled in the absence of the major microtubule-organizing center, the centrosome. In mammals, oocyte meiosis is error prone and underlies most birth aneuploidies. Here, we review recent work on acentrosomal spindle formation and chromosome alignment/separation during oocyte meiosis in different animal models.

  13. Artificial intelligence techniques for embryo and oocyte classification.

    Science.gov (United States)

    Manna, Claudio; Nanni, Loris; Lumini, Alessandra; Pappalardo, Sebastiana

    2013-01-01

    One of the most relevant aspects in assisted reproduction technology is the possibility of characterizing and identifying the most viable oocytes or embryos. In most cases, embryologists select them by visual examination and their evaluation is totally subjective. Recently, due to the rapid growth in the capacity to extract texture descriptors from a given image, a growing interest has been shown in the use of artificial intelligence methods for embryo or oocyte scoring/selection in IVF programmes. This work concentrates the efforts on the possible prediction of the quality of embryos and oocytes in order to improve the performance of assisted reproduction technology, starting from their images. The artificial intelligence system proposed in this work is based on a set of Levenberg-Marquardt neural networks trained using textural descriptors (the local binary patterns). The proposed system was tested on two data sets of 269 oocytes and 269 corresponding embryos from 104 women and compared with other machine learning methods already proposed in the past for similar classification problems. Although the results are only preliminary, they show an interesting classification performance. This technique may be of particular interest in those countries where legislation restricts embryo selection. One of the most relevant aspects in assisted reproduction technology is the possibility of characterizing and identifying the most viable oocytes or embryos. In most cases, embryologists select them by visual examination and their evaluation is totally subjective. Recently, due to the rapid growth in our capacity to extract texture descriptors from a given image, a growing interest has been shown in the use of artificial intelligence methods for embryo or oocyte scoring/selection in IVF programmes. In this work, we concentrate our efforts on the possible prediction of the quality of embryos and oocytes in order to improve the performance of assisted reproduction technology

  14. Effect of stage of follicular growth during superovulation on developmental competence of bovine oocytes

    DEFF Research Database (Denmark)

    Humblot, P; Holm, Peter; Lonergan, P

    2005-01-01

    measured as blastocyst rates (57.6 after in vitro production while no differences were found between oocytes recovered earlier at the first three time points (39.3-41.5. We conclude that oocytes recovered late in the preovulatory period are more developmentally competent than oocytes recovered at the pre...

  15. Developmental competence of oocytes recovered from postmortem ovaries of the endangered Indian blackbuck (Antilope cervicapra).

    Science.gov (United States)

    Sambasiva Rao, Brahmasani; Uma Mahesh, Yelisetti; Lakshmikantan, Uthanda Raman; Suman, Komjeti; Venu Charan, Katari; Shivaji, Sisinthy

    2010-12-01

    The ability to rescue gametes from endangered or wildlife species and to subsequently produce viable embryos holds tremendous potential as a means to increase the population size of endangered or wildlife species. The objective of this study was to assess the meiotic and developmental competence of oocytes recovered from postmortem ovaries of the Indian blackbuck. Oocytes collected from the ovaries of dead blackbucks were allowed to mature in vitro and then tested for developmental potential by activation with ionomycin followed by treatment with 6-dimethylaminopurine. The average number of oocytes recovered per ovary was 10.9, and recovery of the oocytes did not depend on the presence or absence of the corpus luteum, on the side, size and weight of the ovaries or on the type of oocytes recovered. The proportion of good quality oocytes showing cumulus expansion and extrusion of the first polar body were 79.3% and 46.1% when cultured with gonadotropins. In vitro maturation studies indicated that the proportion of oocytes that reached MII stage was significantly higher when good quality oocytes (68%) were used compared with fair quality oocytes (48%) when cultured in the presence of gonadotropins. Furthermore, fifty eight percent of the in vitro matured oocytes cleaved, and thirteen percent of the cleaved oocytes developed into blastocysts. These findings suggest that the oocytes recovered from postmortem ovaries of the blackbuck can be utilized for production of embryos.

  16. The signaling pathways by which the Fas/FasL system accelerates oocyte aging.

    Science.gov (United States)

    Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He

    2016-02-01

    In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis.

  17. Effect of stage of follicular growth during superovulation on developmental competence of bovine oocytes

    DEFF Research Database (Denmark)

    Humblot, P; Holm, P; Lonergan, P;

    2005-01-01

    measured as blastocyst rates (57.6 after in vitro production while no differences were found between oocytes recovered earlier at the first three time points (39.3-41.5. We conclude that oocytes recovered late in the preovulatory period are more developmentally competent than oocytes recovered at the pre...

  18. Fertilization and embryo quality of mature oocytes with specific morphological abnormalities.

    Science.gov (United States)

    Yu, Eun Jeong; Ahn, Hyojeong; Lee, Jang Mi; Jee, Byung Chul; Kim, Seok Hyun

    2015-12-01

    To investigate fertilization and embryo quality of dysmorphic mature oocytes with specific morphological abnormalities obtained from intracytoplasmic sperm injection (ICSI). The fertilization rate (FR) and embryo quality were compared among 58 dysmorphic and 42 normal form oocytes (control 1) obtained from 35 consecutive ICSI cycles, each of which yielded at least one dysmorphic mature oocyte, performed over a period of 5 years. The FR and embryo quality of 441 normal form oocytes from another 119 ICSI cycles that did not involve dysmorphic oocytes served as control 2. Dysmorphic oocytes were classified as having a dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body (PB). The overall FR was significantly lower in dysmorphic oocytes than in normal form oocytes in both the control 1 and control 2 groups. However, embryo quality in the dysmorphic oocyte group and the normal form oocyte groups at day 3 was similar. The FR and embryo quality were similar in the oocyte groups with a single abnormality and multiple abnormalities. Specific abnormalities related with a higher percentage of top-quality embryos were dark cytoplasm (66.7%), abnormal PB (50%), and cytoplasmic vacuoles (25%). The fertilization potential of dysmorphic oocytes in our study was lower, but their subsequent embryonic development and embryo quality was relatively good. We were able to define several specific abnormalities related with good or poor embryo quality.

  19. Corona cell RNA sequencing from individual oocytes revealed transcripts and pathways linked to euploid oocyte competence and live birth.

    Science.gov (United States)

    Parks, Jason C; Patton, Alyssa L; McCallie, Blair R; Griffin, Darren K; Schoolcraft, William B; Katz-Jaffe, Mandy G

    2016-05-01

    Corona cells surround the oocyte and maintain a close relationship through transzonal processes and gap junctions, and may be used to assess oocyte competence. In this study, the corona cell transcriptome of individual cumulus oocyte complexes (COCs) was investigated. Isolated corona cells were collected from COCs that developed into euploid blastocysts and were transferred in a subsequent frozen embryo transfer. Ten corona cell samples underwent RNA-sequencing to generate unique gene expression profiles. Live birth was compared with negative implantation after the transfer of a euploid blastocyst using bioinformatics and statistical analysis. Individual corona cell samples produced a mean of 21.2 million sequence reads, and 307 differentially expressed transcrpits (P corona cell transcriptome was successfully generated using RNA-sequencing. Key genes and signalling pathways were identified in association with implantation outcome after the transfer of a euploid blastocyst in a frozen embryo transfer. These data could provide novel biomarkers for the non-invasive assessment of embryo viability.

  20. The fertilization ability and developmental competence of bovine oocytes grown in vitro.

    Science.gov (United States)

    Makita, Miho; Ueda, Mayuko; Miyano, Takashi

    2016-08-25

    In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4-0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts.

  1. Effects of thioglycolic acid on parthenogenetic activation of Xenopus oocytes.

    Directory of Open Access Journals (Sweden)

    Zhuoran Wang

    Full Text Available BACKGROUND: Existing in Permanent-wave solutions (PWS, thioglycolic acid (TGA is widely used in hairdressing industry for its contribution to hair styling. However, the toxicity of TGA, especially its reproductive toxicity, gradually calls the attention of more and more researchers. METHOD: In this work, xenopus oocytes were pretreated with different concentration of TGA, and then activated by calcium ionophore A23187. During culture, the oocytes activation rates were taken note at different time after adding calcium ionophore A23187. At the end of the culture period, the nuclear status was detected under confocal microscope. In addition, some other samples were collected for Western-Blotting analysis. RESULT: TGA significantly inhibited the oocytes activation rate and pronuclear formation. It may be resulted from the inhibition of the degradation of p-ERK1, Mos and CyclinB2. CONCLUSION: TGA inhibits in vitro parthenogenetic activation of xenopus oocytes with inhibited the degradation of proteins involved in mitogenic-activated protein kinase (MAPK and maturation-promoting factor (MPF pathways.

  2. Ecdysteroids and oocyte development in the black fly Simulium vittatum

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    Hagedorn Henry H

    2002-04-01

    Full Text Available Abstract Background Oocyte development was studied in the autogenous black fly, Simulium vittatum (Diptera, Nematocera, a vector of Onchocerca volvulus, the causative agent of onchocerciasis. Results Oocyte growth was nearly linear between adult eclosion and was complete by 72 hours at 21°C. The oocyte became opaque at 14 hours after eclosion indicating the initiation of protein yolk deposition. The accumulation of vitellogenin was measured using SDS-PAGE. The density of the yolk protein bands at about 200 and 65 kDa increased during the first and second days after eclosion. The amount of protein in the 200 kDa band of vitellogenin, determined using densitometry, rapidly increased between 12 and 25 hours after eclosion. Ecdysteroid levels were measured using a competitive ELISA. Ecdysteroid levels increased rapidly and subsequently declined during the first day after eclosion. Conclusion These data show a correlation between the appearance of vitellogenin in the oocyte, and the rise in ecdysteroids. A possible relationship to molting of the nematode, Onchocerca volvulus, is discussed.

  3. High hydrostatic pressure treatment of porcine oocytes induces parthenogenetic activation

    DEFF Research Database (Denmark)

    Lin, Lin; Pribenszky, Csaba; Molnár, Miklós

    2010-01-01

    An innovative technique called high hydrostatic pressure (HHP) treatment has recently been reported to improve the cryosurvival of gametes and embryos in certain mammalian species, including the mouse, pig, and cattle. In the present study the parthenogenetic activation (PA) of pig oocytes caused...

  4. Pharmaceutical Options for Triggering of Final Oocyte Maturation in ART

    DEFF Research Database (Denmark)

    Castillo, Juan Carlos; Humaidan, Peter; Bernabéu, Rafael

    2014-01-01

    Since the pioneering days of in vitro fertilization, hCG has been the gold standard to induce final follicular maturation. We herein reviewed different pharmaceutical options for triggering of final oocyte maturation in ART. The new upcoming agent seems to be GnRHa with its potential advantages o...

  5. Intra-follicular interactions affecting mammalian oocyte maturation

    NARCIS (Netherlands)

    van Tol, H.T.A.

    2009-01-01

    Nuclear oocyte maturation is defined as reinitiation and progression of the first meiotic division and subsequently formation of the methaphase II (MII) plate. Concomitantly with nuclear maturation, cytoplasmic maturation which is essential for proper fertilization and early embryo development is co

  6. [Progress in proteomics of mammalian oocyte and early embryo].

    Science.gov (United States)

    Chen, Lingsheng; Xu, Ping; Shi, Deshun; Li, Xiangping

    2014-07-01

    The development of female germ cell is the cornerstone for animal reproduction. Mammalian oocyte and early embryo have many distinct phenomena and mechanisms during their growth and development, involving series dynamic changes of protein synthesis/degradation and phosphorylation. Research on the regulatory mechanism of oocyte division, maturation, and developmental principle of pre-implantation embryo is an important topic in the field of animal developmental biology. Proteomics using all of proteins expressed by a cell or tissue as research object, systematically identify, quantify and study the function of all these proteins. With the rapid development of protein separation and identification technology, proteomics provide some new methods and the research contents on fields of oogenesis, differentiation, maturation and quality control, such as protein quantification, modification, location and interaction important information which other omics technology can not provide. These information will contribute to uncover the molecular mechanisms of mammalian oocyte maturation and embryonic development. And it is great significant for improving the culture system of oocyte in vitro maturation, the efficiency of embryo production in vitro, somatic cell clone and transgenic animal production.

  7. Successful cryopreservation of buffalo ovaries using in situ oocyte cryopreservation

    Directory of Open Access Journals (Sweden)

    Saber Mohammed Abd-Allah

    2009-12-01

    Full Text Available To improve the efficiency and efficacy of cryopreservation of ovaries, we developed a new method termed in situ oocyte (ISO cryopreservation. ISO cryopreservation is a multistep procedure that involves aspiration of follicular fluid and then perfusion of antral follicles and diffusion of whole buffalo ovaries with cryoprotectant agent (CPA, rapid cooling, storage, thawing and, finally, dilution and removal of the CPA with return to physiological environment. Our study compared ISO cryo ovaries with cryo-diffused ovaries. We systematically examined the effects of ISO cryo and diffuse cryo on ovaries by morphological examination and with viability tests. The percentages of morphologically normal and viable follicular oocytes from ISO cryo were significantly higher than those that resulted from the cryo-diffused method (p<0.01. The quality of follicular oocytes from ISO cryo ovaries appeared better than that achieved from cryo-diffused ovaries. In conclusion, this study shows that ISO cryo is highly efficient for cryopreservation of oocytes and ovarian tissue.

  8. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

    Science.gov (United States)

    Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronucl...

  9. Aurelia aurita (Cnidaria) oocytes' contact plate structure and development.

    Science.gov (United States)

    Adonin, Leonid S; Shaposhnikova, Tatyana G; Podgornaya, Olga

    2012-01-01

    One of the A. aurita medusa main mesoglea polypeptides, mesoglein, has been described previously. Mesoglein belongs to ZP-domain protein family and therefore we focused on A.aurita oogenesis. Antibodies against mesoglein (AB RA47) stain the plate in the place where germinal epithelium contacts oocyte on the paraffin sections. According to its position, we named the structure found the "contact plate". Our main instrument was AB against mesoglein. ZP-domain occupies about half of the whole amino acid sequence of the mesoglein. Immunoblot after SDS-PAGE and AU-PAGE reveals two charged and high M(r) bands among the female gonad germinal epithelium polypeptides. One of the gonads' polypeptides M(r) corresponds to that of mesogleal cells, the other ones' M(r) is higher. The morphological description of contact plate formation is the subject of the current work. Two types of AB RA47 positive granules were observed during progressive oogenesis stages. Granules form the contact plate in mature oocyte. Contact plate of A.aurita oocyte marks its animal pole and resembles Zona Pellucida by the following features: (1) it attracts spermatozoids; (2) the material of the contact plate is synthesized by oocyte and stored in granules; (3) these granules and the contact plate itself contain ZP domain protein(s); (4) contact plate is an extracellular structure made up of fiber bundles similar to those of conventional Zona Pellucida.

  10. Chromatin structure and ATRX function in mouse oocytes.

    Science.gov (United States)

    De La Fuente, Rabindranath; Baumann, Claudia; Viveiros, Maria M

    2012-01-01

    Differentiation of chromatin structure and function during oogenesis is essential to confer the mammalian oocyte with meiotic and developmental potential. Errors in chromosome segregation during female meiosis and subsequent transmission of an abnormal chromosome complement (aneuploidy) to the early conceptus are one of the leading causes of pregnancy loss in women. The chromatin remodeling protein ATRX (α-thalassemia mental retardation X-linked) has recently emerged as a critical factor involved in heterochromatin formation at mammalian centromeres during meiosis. In mammalian oocytes, ATRX binds to centromeric heterochromatin domains where it is required for accurate chromosome segregation. Loss of ATRX function induces abnormal meiotic chromosome morphology, reduces histone H3 phosphorylation, and promotes a high incidence of aneuploidy associated with severely reduced fertility. The presence of centromeric breaks during the transition to the first mitosis in the early embryo indicates that the role of ATRX in chromosome segregation is mediated through an epigenetic mechanism involving the maintenance of chromatin modifications associated with pericentric heterochromatin (PCH) formation and chromosome condensation. This is consistent with the existence of a potential molecular link between centromeric and PCH in the epigenetic control of centromere function and maintenance of chromosome stability in mammalian oocytes. Dissecting the molecular mechanisms of ATRX function during meiosis will have important clinical implications towards uncovering the epigenetic factors contributing to the onset of aneuploidy in the human oocyte.

  11. GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary.

    Directory of Open Access Journals (Sweden)

    Chen Jiang

    2017-01-01

    Full Text Available Folliculogenesis is a progressive and highly regulated process, which is essential to provide ova for later reproductive life, requires the bidirectional communication between the oocyte and granulosa cells. This physical connection-mediated communication conveys not only the signals from the oocyte to granulosa cells that regulate their proliferation but also metabolites from the granulosa cells to the oocyte for biosynthesis. However, the underlying mechanism of establishing this communication is largely unknown. Here, we report that oocyte geranylgeranyl diphosphate (GGPP, a metabolic intermediate involved in protein geranylgeranylation, is required to establish the oocyte-granulosa cell communication. GGPP and geranylgeranyl diphosphate synthase (Ggpps levels in oocytes increased during early follicular development. The selective depletion of GGPP in mouse oocytes impaired the proliferation of granulosa cells, primary-secondary follicle transition and female fertility. Mechanistically, GGPP depletion inhibited Rho GTPase geranylgeranylation and its GTPase activity, which was responsible for the accumulation of cell junction proteins in the oocyte cytoplasm and the failure to maintain physical connection between oocyte and granulosa cells. GGPP ablation also blocked Rab27a geranylgeranylation, which might account for the impaired secretion of oocyte materials such as Gdf9. Moreover, GGPP administration restored the defects in oocyte-granulosa cell contact, granulosa cell proliferation and primary-secondary follicle transition in Ggpps depletion mice. Our study provides the evidence that GGPP-mediated protein geranylgeranylation contributes to the establishment of oocyte-granulosa cell communication and then regulates the primary-secondary follicle transition, a key phase of folliculogenesis essential for female reproductive function.

  12. Chromosome Cohesion Established by Rec8-Cohesin in Fetal Oocytes Is Maintained without Detectable Turnover in Oocytes Arrested for Months in Mice.

    Science.gov (United States)

    Burkhardt, Sabrina; Borsos, Máté; Szydlowska, Anna; Godwin, Jonathan; Williams, Suzannah A; Cohen, Paula E; Hirota, Takayuki; Saitou, Mitinori; Tachibana-Konwalski, Kikuë

    2016-03-07

    Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis [1]. Rec8-containing cohesin, bound to Smc3/Smc1α or Smc3/Smc1β, maintains bivalent cohesion in mammalian meiosis [2-6]. In females, meiotic DNA replication and recombination occur in fetal oocytes. After birth, oocytes arrest at the prolonged dictyate stage until recruited to grow into mature oocytes that divide at ovulation. How cohesion is maintained in arrested oocytes remains a pivotal question relevant to maternal age-related aneuploidy. Hypothetically, cohesin turnover regenerates cohesion in oocytes. Evidence for post-replicative cohesion establishment mechanism exists, in yeast and invertebrates [7, 8]. In mouse fetal oocytes, cohesin loading factor Nipbl/Scc2 localizes to chromosome axes during recombination [9, 10]. Alternatively, cohesion is maintained without turnover. Consistent with this, cohesion maintenance does not require Smc1β transcription, but unlike Rec8, Smc1β is not required for establishing bivalent cohesion [11, 12]. Rec8 maintains cohesion without turnover during weeks of oocyte growth [3]. Whether the same applies to months or decades of arrest is unknown. Here, we test whether Rec8 activated in arrested mouse oocytes builds cohesion revealed by TEV cleavage and live-cell imaging. Rec8 establishes cohesion when activated during DNA replication in fetal oocytes using tamoxifen-inducible Cre. In contrast, no new cohesion is detected when Rec8 is activated in arrested oocytes by tamoxifen despite cohesin synthesis. We conclude that cohesion established in fetal oocytes is maintained for months without detectable turnover in dictyate-arrested oocytes. This implies that women's fertility depends on the longevity of cohesin proteins that established cohesion in utero.

  13. A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

    Science.gov (United States)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

  14. Effect of corpus luteum: quality and recovery rate of buffalo (Bubalus bubalis) oocytes

    OpenAIRE

    SAHOO, Lakshman; Singla, Suresh K.

    2013-01-01

    Recovery rate and quality of oocytes were investigated from abattoir collected ovaries with and without corpora lutea (CL). The oocytes were categorized into four grades: grade-A, grade-B, grade-C, and grade-D based on the presence of the cumulus cells complex around the oocytes. The number of oocytes per ovary were 1.7 ±0.1 and 1.5 ±0.1, obtained from ovaries without CL and with CL, respectively. Recovery rate of different grades of oocytes from ovaries with CL and without CL were almost ide...

  15. Detection of condensin I and II in maturing pig oocytes.

    Science.gov (United States)

    Lisková, Lucie; Susor, Andrej; Pivonková, Katerina; Sasková, Adéla; Karabínová, Pavla; Kubelka, Michal

    2010-01-01

    The multiprotein complexes known as condensins (I and II) are major players in chromosome dynamics in mitotic and meiotic cells. Here, we report for the first time the detection of different condensin subunits from both complexes in mammalian oocytes. Using immunoblotting analysis we examined expression levels of condensin subunits during meiotic maturation of porcine oocytes. The expression of the core subunit structural maintenance of chromosomes 2 (SMC2), identical in both condensin complexes, did not change significantly during maturation. Similarly, there was no significant change in the expression of the chromosome associated protein (CAP)-H and CAP-H2 subunits, components of condensin I and II, respectively. Conversely, the expression profiles of CAP-G, CAP-D2 (condensin I) and CAP-D3 (condensin II) were more interesting. At least two isoforms of the CAP-D2 subunit were detected, along with three isoforms of the CAP-D3 and CAP-G subunits. We suggest that this diverse migration of subunit isoforms is due to post-translational modification. Earlier, it was reported that non-SMC proteins are phosphorylated by cyclin-dependent kinase 1. In the present study, we analysed the phosphorylation status of the three subunits in oocyte extracts using alkaline phosphatase treatment and we found that at least the fastest migrating form of CAP-D3 was likely to be phosphorylated in maturing porcine oocytes. In addition, the localisation of CAP-H and CAP-H2 subunits was examined using immunofluorescence staining with specific antibodies, as well as following microinjection of their enhanced green fluorescent protein-tagged mRNA into germinal vesicle-stage oocytes. CAP-H was found in the cytoplasm, whereas CAP-H2 was localised within the nucleus.

  16. Aurora kinase A controls meiosis I progression in mouse oocytes.

    Science.gov (United States)

    Saskova, Adela; Solc, Petr; Baran, Vladimir; Kubelka, Michal; Schultz, Richard M; Motlik, Jan

    2008-08-01

    Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G(2) and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G(2) to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.

  17. Epigenetic reprogramming of breast cancer cells with oocyte extracts

    Directory of Open Access Journals (Sweden)

    Kumari Rajendra

    2011-01-01

    Full Text Available Abstract Background Breast cancer is a disease characterised by both genetic and epigenetic alterations. Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. In this study we apply the reprogramming capacity of oocytes to cancer cells in order to study breast oncogenesis. Results We show that breast cancer cells can be directly reprogrammed by amphibian oocyte extracts. The reprogramming effect, after six hours of treatment, in the absence of DNA replication, includes DNA demethylation and removal of repressive histone marks at the promoters of tumour suppressor genes; also, expression of the silenced genes is re-activated in response to treatment. This activity is specific to oocytes as it is not elicited by extracts from ovulated eggs, and is present at very limited levels in extracts from mouse embryonic stem cells. Epigenetic reprogramming in oocyte extracts results in reduction of cancer cell growth under anchorage independent conditions and a reduction in tumour growth in mouse xenografts. Conclusions This study presents a new method to investigate tumour reversion by epigenetic reprogramming. After testing extracts from different sources, we found that axolotl oocyte extracts possess superior reprogramming ability, which reverses epigenetic silencing of tumour suppressor genes and tumorigenicity of breast cancer cells in a mouse xenograft model. Therefore this system can be extremely valuable for dissecting the mechanisms involved in tumour suppressor gene silencing and identifying molecular activities capable of arresting tumour growth. These applications can ultimately shed light on the contribution of epigenetic alterations in breast cancer and advance the development of epigenetic therapies.

  18. Aurora B regulates spindle bipolarity in meiosis in vertebrate oocytes.

    Science.gov (United States)

    Shao, Hua; Ma, Chunqi; Zhang, Xuan; Li, Ruizhen; Miller, Ann L; Bement, William M; Liu, X Johné

    2012-07-15

    Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine(122) is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.

  19. Cryoprotectants up-regulate expression of mouse oocyte AQP7, which facilitates water diffusion during cryopreservation.

    Science.gov (United States)

    Tan, Ya-Jing; Xiong, Yun; Ding, Guo-Lian; Zhang, Dan; Meng, Ye; Huang, He-Feng; Sheng, Jian-Zhong

    2013-04-01

    To investigate the effects of cryoprotectants on the expression of AQP7 in oocytes. Experimental animal study. University-based research laboratory. Adult female C57BL/6J mice. In metaphase II (MII) oocytes obtained from adult female C57BL/6J mice and from donations by fertile women, the mouse oocytes were treated with human tubal fluid medium containing 8% ethylene glycol (EG), 9.5% dimethylsulfoxide (DMSO), and 0.5 M sucrose, respectively; 293T cells transfected with GFP-hAQP7 expression vector were treated with the same solutions. AQP7 expression in oocytes examined by reverse-transcriptase-nested polymerase chain reaction and immunofluorescence, changes in the volume of mouse oocytes treated with different solutions calculated to determine their permeability to water, and survival rates of vitrified oocytes. AQP7 is expressed in human and mouse oocytes. Cryoprotectants, including EG, DMSO, and sucrose, up-regulated AQP7 expression in mouse oocytes and 293T cells transfected with GFP-hAQP7 expression vector. Compared with other cryoprotectants, DMSO stimulated higher expression of AQP7, and this was associated with faster cell volume recovery and lower survival rates of vitrified oocytes. DMSO up-regulates AQP7 expression in mouse oocytes more than EG. This may facilitate water diffusion and reduce the time for oocytes to reach osmotic balance with the cryoprotectant solution during cryopreservation. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  20. Checkpoint for DNA integrity at the first mitosis after oocyte activation.

    Science.gov (United States)

    Liu, Lin; Trimarchi, James R; Smith, Peter J S; Keefe, David L

    2002-06-01

    Activation of oocytes, arrested at the meiosis II (MII) in mammals, initiates meiotic release, mitotic divisions, and development. Unlike most somatic cell types, MII arrested female germ cells lack an efficient DNA integrity checkpoint control. Here we present evidence showing a unique checkpoint for DNA integrity at first mitosis after oocyte activation. Mouse oocytes carrying intact DNA cleaved normally after meiotic release, whereas 50% of oocytes harboring damaged DNA manifested cytofragmentation, a morphological hallmark of apoptosis. If not activated, DNA-damaged MII oocytes did not show apoptotic fragmentation. Further, activated, enucleated oocytes or enucleated fertilized oocytes also underwent cytofragmentation, implicating cytoplasmic coordination of the fragmentation process, independent of the nucleus. Depolymerization of either actin filaments or microtubules induced no cytofragmentation, but inhibited fragmentation upon oocyte activation. During the process of fragmentation, microtubule networks formed, then microtubule asters congregated at discrete locations, around which fragmented cellular bodies formed. Mitotic spindles, however, were not formed inactivated oocytes with damaged or absent DNA; in contrast, normal mitotic spindles were formed in activated oocytes with intact DNA. These results demonstrate that damaged DNA or absence of DNA leads to cytofragmentation after oocyte activation. Further, we found a mechanism of cytoskeletal involvement in the process of cytofragmentation. In addition, possible implication of the present findings in somatic cell cloning and human clinical embryology is discussed.

  1. CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte

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    Naoko Ohnami

    2012-05-01

    When a sperm and oocyte unite into one cell upon fertilization, membranous fusion between the sperm and oocyte occurs. In mice, Izumo1 and a tetraspanin molecule CD9 are required for sperm-oocyte fusion as one of the oocyte factors, and another tetraspanin molecule CD81 is also thought to involve in this process. Since these two tetraspanins often form a complex upon cell-cell interaction, it is probable that such a complex is also formed in sperm-oocyte interaction; however, this possibility is still under debate among researchers. Here we assessed this problem using mouse oocytes. Immunocytochemical analysis demonstrated that both CD9 and CD81 were widely distributed outside the oocyte cell membrane, but these molecules were separate, forming bilayers, confirmed by immunobiochemical analysis. Electron-microscopic analysis revealed the presence of CD9- or CD81-incorporated extracellular structures in those bilayers. Finally, microinjection of in vitro-synthesized RNA showed that CD9 reversed a fusion defect in CD81-deficient oocytes in addition to CD9-deficient oocytes, but CD81 failed in both oocytes. These results suggest that both CD9 and CD81 independently work upon sperm-oocyte fusion as extracellular components.

  2. Role of animal pole protuberance and microtubules during meiosis in sea cucumber Apostichopus japonicus oocytes

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    Pang, Zhenguo; Chang, Yaqing; Sun, Huiling; Yu, Jiaping

    2010-05-01

    Fully grown oocytes of Apostichopus japonicus have a cytoplasmic protuberance where the oocyte attaches to the follicle. The protuberance and the oolamina located on the opposite side of the oocyte indicate the animal-vegetal axis. Two pre-meiotic centrosomes are anchored to the protuberance by microtubules between centrosomes and protuberance. After meiosis reinitiation induced by DTT solution, the germinal vesicle (GV) migrates towards the protuberance. The GV breaks down after it migrates to the oocyte membrane on the protuberance side. The protuberance then contracts back into the oocyte and the first polar body extrudes from the site of the former protuberance. The second polar body forms beneath the first. Thus the oocyte protuberance indicates the presumptive animal pole well before maturation of the oocyte.

  3. Mitochondria Synthesize Melatonin to Ameliorate Its Function and Improve Mice Oocyte's Quality under in Vitro Conditions.

    Science.gov (United States)

    He, Changjiu; Wang, Jing; Zhang, Zhenzhen; Yang, Minghui; Li, Yu; Tian, Xiuzhi; Ma, Teng; Tao, Jingli; Zhu, Kuanfeng; Song, Yukun; Ji, Pengyun; Liu, Guoshi

    2016-06-14

    The physiology of oocyte in vitro maturation remains elusive. Generally, the oocytes have a very low maturation rate under in vitro conditions. In the current study, we found that melatonin promotes the maturation of oocytes in which mitochondria play a pivotal role. It was identified that; (1) mitochondria are the major sites for melatonin synthesis in oocytes and they synthesize large amounts of melatonin during their maturation; (2) melatonin improves mitochondrial function by increased mtDNA copy, mitochondrial membrane potential (ΔΨm) and mitochondrial distribution and ATP production in oocytes; (3) the meiotic spindle assembly is enhanced; (4) melatonin reduces ROS production and inhibits 8-oxodG formation, thereby protecting potential DNA mutation from oxidative damage. As a result, melatonin improves the quality of oocytes, significantly accelerates the developmental ability of IVF embryo. The results provide novel knowledge on the physiology of oocyte's maturation, especially under in vitro conditions.

  4. An ideal oocyte activation protocol and embryo culture conditions for somatic cell nuclear transfer using sheep oocytes.

    Science.gov (United States)

    Patel, Hiren; Chougule, Shruti; Chohan, Parul; Shah, Naval; Bhartiya, Deepa

    2014-10-01

    Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development. Results demonstrate that between electric stimulation and chemical stimulation using calcium ionomycin and ionophore, best oocyte activation was obtained using calcium ionomycin (5 microM for 5 min) which resulted in 83% cleavage followed by 7% of early blastocyst which further increased to 15% when a cumulus bed was also introduced during embryo culture. Sequential modified Charles Rosenkrans 2 (mCR2) medium was used for embryo culture in which glucose levels were increased from 1 mM to 5 mM from Day 3 onwards. SCNT using cumulus cells as donor somatic cell, calcium ionomycin to activate the reconstructed oocyte and embryo culture on a cumulus bed in sequential mCR2 medium, resulted in the development of 6% embryos to early blastocyst stage. Such technological advances will make SCNT a viable option to make patient-specific pluripotent stem cell lines in near future.

  5. Roles of trifluoperazine and verapamil in the oocyte maturation and cumulus expansion of bovine cumulus—oocyte complexes

    Institute of Scientific and Technical Information of China (English)

    SunQingyuan; FengHuailiang; 等

    1994-01-01

    Bovine cumulus-oocyte complexes were cultured in the maturation medium containing 4 different concentrations of verapamil and trifluoperazine to testify the necessity of extracellular Ca2+ and Ca2+-calmodulin complex for the resumption and completion of meiosis as well as cumulus expansion.Ultrastructure of the treated oocytes was also observed to investigate the cytoplasm maturation.The results showed that verapamil didn't influence the cumulus expansion,meiosis resumption and completion and cytoplasm maturation significantly.TFP inhibited cumulus expansion in a dose-dependent manner.25um trifluoperazine significantly inhibited the GVBD and maturation (P<0.01),wherease 1um TFP had no effect,Both oocytes and cumulus cells treated with 25um TFP severely degenerated.Our observtions suggest that the resumption and completion of meiosis and cumulus expansion are Ca2+-CaM dependent and blocking membrane Ca2+ channel does not influence oocyte germinal vesicle breakdown,nuclear and cytoplasm maturation significantly in cattle.

  6. Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts from In Vitro Maturation of Bovine Oocytes

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    Jolanta Opiela

    2014-01-01

    Full Text Available The objective of the present study was to evaluate the effect of hyaluronan (HA during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC, and obtained blastocysts. COCs were matured in vitro in control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001 was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01. Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higher Bax mRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

  7. Winter hibernation and UCHL1-p34cdc2 association in toad oocyte maturation competence.

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    Zhichao Kuang

    Full Text Available Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1 is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34(cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13(suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34(cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte's dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34(cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.

  8. Characterization of the effects of metformin on porcine oocyte meiosis and on AMP-activated protein kinase activation in oocytes and cumulus cells.

    Science.gov (United States)

    Bilodeau-Goeseels, Sylvie; Magyara, Nora; Collignon, Coralie

    2014-05-01

    The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.

  9. Mechanisms by which a lack of germinal vesicle (GV) material causes oocyte meiotic defects: a study using oocytes manipulated to replace GV with primary spermatocyte nuclei.

    Science.gov (United States)

    Zhang, Jie; Cui, Wei; Li, Qing; Wang, Tian-Yang; Sui, Hong-Shu; Wang, Jun-Zuo; Luo, Ming-Jiu; Tan, Jing-He

    2013-10-01

    Oocytes with germinal vesicles (GVs) replaced with somatic nuclei exhibit meiotic abnormalities. Although this suggests an exclusive role for GV material in meiosis, mechanisms by which a lack of GV material causes meiotic defects are unknown. Knowledge of these mechanisms will help us to understand meiotic control, nuclear-cytoplasmic interactions, and cellular reprogramming. This study showed that although oocytes with prometaphase I chromosomes replaced with primary spermatocyte nuclei (PSN) did not, oocytes with GV replaced with PSN (PSG oocytes) did display meiotic defects. Among the defects, insufficient chromosome condensation with chromosome bridges was associated with spindle abnormalities. Abnormal spindle migration, cortical nonpolarization, and the aberrant spindle caused randomly positioning of cleavage furrows, leading to large first polar bodies (PB1) and unequal allocation of chromosomes and mitogen-activated protein kinases (MAPK) between oocyte and PB1. Spindle assembly checkpoint was activated but did not stop the incorrect division. The unequal MAPK allocation resulted in differences in pronuclear formation and PB1 degeneration; oocytes receiving more MAPK were more capable of forming pronuclear rudiments, whereas PB1 receiving more MAPK degenerated sooner than those that received less. Because none of the PSG oocytes or the enucleated GV oocytes injected with sperm heads showed cortical polarization in spite of chromosome localization close to the oolemma and because the PSG oocytes receiving more MAPK could form only pronuclear rudiments and not normal pronuclei, we suggest that the GV material plays essential roles in polarization and pronuclear formation on top of those played by chromosomes or MAPK. In conclusion, using PSG oocytes as models, this study has revealed the primary pathways by which a lack of GV material cause meiotic defects, laying a foundation for future research on the role of GV material in oocyte meiotic control.

  10. Expression and localization of aquaporin 1b during oocyte development in the Japanese eel (Anguilla japonica

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    Matsubara Hajime

    2011-05-01

    Full Text Available Abstract To elucidate the molecular mechanisms underling hydration during oocyte maturation, we characterized the structure of Japanese eel (Anguilla japonica novel-water selective aquaporin 1 (AQP1b that thought to be involved in oocyte hydration. The aqp1b cDNA encodes a 263 amino acid protein that includes the six potential transmembrane domains and two Asn-Pro-Ala motifs. Reverse transcription-polymerase chain reaction showed transcription of Japanese eel aqp1b in ovary and testis but not in the other tissues. In situ hybridization studies with the eel aqp1b cRNA probe revealed intense eel aqp1b signal in the oocytes at the perinucleolus stage and the signals became faint during the process of oocyte development. Light microscopic immunocytochemical analysis of ovary revealed that the Japanese eel AQP1b was expressed in the cytoplasm around the yolk globules which were located in the peripheral region of oocytes during the primary yolk globule stage; thereafter, the immunoreactivity was observed throughout the cytoplasm of oocyte as vitellogenesis progressed. The immunoreactivity became localized around the large membrane-limited yolk masses which were formed by the fusion of yolk globules during the oocyte maturation phase. These results together indicate that AQP1b, which is synthesized in the oocyte during the process of oocyte growth, is essential for mediating water uptake into eel oocytes.

  11. Developmental potential of prepubertal mouse oocytes is compromised due mainly to their impaired synthesis of glutathione.

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    Guang-Zhong Jiao

    Full Text Available Although oocytes from prepubertal animals are found less competent than oocytes from adults, the underlying mechanisms are poorly understood. Using the mouse oocyte model, this paper has tested the hypothesis that the developmental potential of prepubertal oocytes is compromised due mainly to their impaired potential for glutathione synthesis. Oocytes from prepubertal and adult mice, primed with or without eCG, were matured in vitro and assessed for glutathione synthesis potential, oxidative stress, Ca(2+ reserves, fertilization and in vitro development potential. In unprimed mice, abilities for glutathione synthesis, activation, male pronuclear formation, blastocyst formation, cortical granule migration and polyspermic block were all compromised significantly in prepubertal compared to adult oocytes. Cysteamine and cystine supplementation to maturation medium significantly promoted oocyte glutathione synthesis and blastocyst development but difference due to maternal age remained. Whereas reactive oxygen species (ROS levels increased, Ca(2+ storage decreased significantly in prepubertal oocytes. Levels of both catalytic and modifier subunits of the γ-glutamylcysteine ligase were significantly lower in prepubertal than in adult oocytes. Maternal eCG priming improved all the parameters and eliminated the age difference. Together, the results have confirmed our hypothesis by showing that prepubertal oocytes have a decreased ability to synthesize glutathione leading to an impaired potential to reduce ROS and to form male pronuclei and blastocysts. The resulting oxidative stress decreases the intracellular Ca(2+ store resulting in impaired activation at fertilization, and damages the microfilament network, which affects cortical granule redistribution leading to polyspermy.

  12. Vitrification of Human Germinal Vesicle Oocytes: before or after In Vitro Maturation?

    Science.gov (United States)

    Kasapi, Evangelia; Asimakopoulos, Byron; Chatzimeletiou, Katerina; Petousis, Stamatios; Panagiotidis, Yannis; Prapas, Nikos; Nikolettos, Nikos

    2017-01-01

    The use of immature oocytes derived from stimulated cycles could be of great importance, particularly for urgent fertility preservation cases. The current study aimed to determine whether in vitro maturation (IVM) was more successful before or after vitrification of these oocytes. This prospective study was performed in a private in vitro fertilization (IVF) center. We collected 318 germinal vesicle (GV) oocytes from 104 stimulated oocyte donation cycles. Oocytes were divided into two groups according to whether vitrification was applied at the GV stage (group 1) or in vitro matured to the metaphase II (MII) stage and then vitrified (group 2). In the control group (group 3), oocytes were in vitro matured without vitrification. In all three groups, we assessed survival rate after warming, maturation rate, and MII-spindle/chromosome configurations. The chi-square test was used to compare rates between the three groups. Statistical significance was defined at Pvitro before (50.0%) or after (41.2%) vitrification. However, a higher incidence of normal spindle/chromosome configurations existed in the in vitro matured oocytes which were not subjected to vitrification (fresh oocytes, 77.9%). In stimulated cycles, vitrification of in vitro matured MII oocytes rather than GV oocytes seems to be more efficient. This approach needs to be verified in nonstimulated fertility preservation cases.

  13. Use of Rat Estrus Serum for in Vitro Maturation of Bovine Oocytes

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    AR Rafati

    2007-04-01

    Full Text Available Introduction: Superovulation produces complications in some patients, so invitro maturation of oocytes is used to decrease or eliminate these complications and improve IVF. Moreover, IVM is used for different aspects of reproductive researches. Slaughterhouse ovaries are the main source of oocytes for IVM and IVF studies. Different media has been introduced and experimented for in vitro maturation of oocytes. Animal's serum at estrus stage contains different hormones and proteins which are essential for oocyte maturation. The aim of this study was to compare three culture media for in vitro maturation (IVM of bovine oocytes; 1(controlTCM-199, 2HCG and follicular fluid (FF and 3 antibiotic. Methods: Rat estrus serum (RSS or fetal bovine serum (FBS was added to control medium. Total of 1789 compact cumulus oocyte complexes (COCs were aspirated from ovaries of slaughtered animals. Oocytes were randomly cultured in mentioned media and incubated in 38.5◦c, 5% CO2 and 95% humidity for 24 hours. The maturation of oocytes was judged according to cumulus cell expansion or randomly orcein stained oocytes and observation of polar bodies. Results: The results showed that maturation rate was significantly higher in second and third group (90.2%, 78.7% as compared to the control group (p<0.001. There was no significant difference between second and third groups (90.2 % vs. 86.6%. Conclusion: RSS is as effective as FBS for IVM of bovine oocytes and can be used as an alternative.

  14. Nuclear replacement of in vitro-matured porcine oocytes by a serial centrifugation and fusion method.

    Science.gov (United States)

    Maedomari, N; Kikuchi, K; Nagai, T; Fahrudin, M; Kaneko, H; Noguchi, J; Nakai, M; Ozawa, M; Somfai, T; Nguyen, L V; Ito, J; Kashiwazaki, N

    2010-08-01

    The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.

  15. Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing--a review article.

    Science.gov (United States)

    Chen, S U; Lien, Y R; Chao, K H; Ho, H N; Yang, Y S; Lee, T Y

    2003-04-28

    Embryo freezing has been a successful practice, but oocyte cryopreservation formerly achieved poorer results. This was mainly due to low rates of survival, fertilization, and development. The major dissimilarities for oocytes to embryos are the character of the plasma membrane, the presence of cortical granules, at the metaphase of meiosis II with the spindle system. In addition, the oocytes must be fertilized by sperm at the appropriate time. To improve the survival rate, a refined slow freezing method with increased sucrose concentration would dehydrate oocytes more sufficiently. Vitrification is another approach to prevent ice crystal formation. Intracytoplasmic sperm injection is used to overcome possible zona hardening from the release of cortical granules. The microtubules of meiotic spindles are vulnerable to the thermal changes and would depolymerize. Cryopreserved oocytes exhibited serious disturbances of the microtubules immediately after thawing. Fertilization of oocytes with disorganized spindles could lead to chromosomal aneuploidy, digyny, and arrest of cleavage. After incubation, the microtubules would repolymerize in a time-dependent way. Normal fertilization and development of cryopreserved oocytes improved after appropriate incubation and timing of insemination, compatible with recovery of the spindles. With the improvement of survival, fertilization, and cleavage, oocyte cryopreservation would gain an imperative role.

  16. Rex Rabbit Somatic Cell Nuclear Transfer with In Vitro-Matured Oocytes.

    Science.gov (United States)

    Liu, Yong; Wang, Huili; Lu, Jinhua; Miao, Yiliang; Cao, Xinyan; Zhang, Ling; Wu, Xiaoqing; Wu, Fengrui; Ding, Biao; Wang, Rong; Luo, Mingjiu; Li, Wenyong; Tan, Jinghe

    2016-06-01

    Somatic cell nuclear transfer (SCNT) requires large numbers of matured oocytes. In vitro-matured (IVM) oocytes have been used in SCNT in many animals. We investigated the use of IVM oocytes in Rex rabbit SCNT using Rex rabbit ovaries obtained from a local abattoir. The meiotic ability of oocytes isolated from follicles of different diameters was studied. Rex rabbit SCNT was optimized for denucleation, activation, and donor cell synchronization. Rex rabbit oocytes grew to the largest diameter (110 μm) when the follicle diameter was 1.0 mm. Oocytes isolated from 0.7-mm follicles acquired maturation ability. More than 90% of these oocytes matured after IVC for 18 h. The developmental potential of oocytes isolated from >1-mm follicles was greater than that of oocytes isolated from 0.7- to 1.0-mm follicles. The highest activation rates for IVM Rex rabbit oocytes were seen after treatment with 2.5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) and 5 μg/mL cycloheximide (CHX) for 1 h. Ionomycin induced the chromatin of IVM oocytes to protrude from the oocyte surface, promoting denucleation. Fetal fibroblast cells (FFCs) and cumulus cells (CCs) were more suitable for Rex rabbit SCNT than skin fibroblast cells (SFCs) (blastocyst rate was 35.6 ± 2.2% and 38.0 ± 6.0% vs. 19.7 ± 3.1%). The best fusion condition was a 2DC interval for 1 sec, 1.6 kV/cm voltages, and 40 μsec duration in 0.28 M mannitol. In conclusion, the in vitro maturation of Rex rabbit oocytes and SCNT procedures were studied systematically and optimized in this study.

  17. Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage.

    Science.gov (United States)

    Biase, Fernando Henrique; Everts, Robin Edward; Oliveira, Rosane; Santos-Biase, Weruska Karyna Freitas; Fonseca Merighe, Giovana Krempel; Smith, Lawrence Charles; Martelli, Lúcia; Lewin, Harris; Meirelles, Flávio Vieira

    2014-02-01

    The mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8-16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: 'RNA processing', 'translation' and 'mRNA metabolic process'. Genes that are important to the molecular functions of 'RNA binding' and 'translation factor activity, RNA binding' were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

  18. The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum

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    Sánchez Mercedes

    2003-02-01

    Full Text Available Abstract A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1 when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE, 2 after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE, and 3 after oocyte activation (surrounded by the fertilization envelope, (FE. The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP shows proteolytic activity on gp75 of the VE.

  19. Effect of guaianolides in the meiosis reinitiation of amphibian oocytes.

    Science.gov (United States)

    Zapata-Martínez, J; Sánchez-Toranzo, G; Chaín, F; Catalán, C A N; Bühler, M I

    2017-02-01

    Sesquiterpene lactones (STLs) are a large and structurally diverse group of plant metabolites generally found in the Asteraceae family. STLs exhibit a wide spectrum of biological activities and it is generally accepted that their major mechanism of action is the alkylation of the thiol groups of biological molecules. The guaianolides is one of various groups of STLs. Anti-tumour and anti-migraine effects, an allergenic agent, an inhibitor of smooth muscle cells and of meristematic cell proliferation are only a few of the most commonly reported activities of STLs. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under stimulus with progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. There are previous records of the inhibitory effect of dehydroleucodin (DhL), a guaianolide lactone, on the progression of meiosis. It has been also shown that DhL and its 11,13-dihydroderivative (2H-DhL; a mixture of epimers at C-11) act as blockers of the resumption of meiosis in fully grown ovarian oocytes from the amphibian Rhinella arenarum (formerly classified as Bufo arenarum). The aim of this study was to analyze the effect of four closely related guaianolides, i.e., DhL, achillin, desacetoxymatricarin and estafietin as possible inhibitors of meiosis in oocytes of amphibians in vitro and discuss some structure-activity relationships. It was found that the inhibitory effect on meiosis resumption is greater when the lactone has two potentially reactive centres, either a α,β-α',β'-diunsaturated cyclopentanone moiety or an epoxide group plus an exo-methylene-γ-lactone function.

  20. Acentrosomal Spindle Assembly & Chromosome Segregation During Oocyte Meiosis

    OpenAIRE

    Dumont, Julien; Desai, Arshad

    2012-01-01

    The ability to reproduce relies in most eukaryotes on specialized cells called gametes. Gametes are formed by the process of meiosis in which, after a single round of replication, two successive cell divisions reduce the ploidy of the genome. Fusion of gametes at fertilization reconstitutes diploidy. In most animal species, chromosome segregation during female meiosis occurs on spindles assembled in the absence of the major microtubule-organizing center, the centrosome. In mammals, oocyte mei...

  1. Multiple requirements of PLK1 during mouse oocyte maturation.

    Directory of Open Access Journals (Sweden)

    Petr Solc

    Full Text Available Polo-like kinase 1 (PLK1 orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC. Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.

  2. Calcium and actin in the saga of awakening oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Santella, Luigia, E-mail: santella@szn.it; Limatola, Nunzia; Chun, Jong T.

    2015-04-24

    The interaction of the spermatozoon with the egg at fertilization remains one of the most fascinating mysteries of life. Much of our scientific knowledge on fertilization comes from studies on sea urchin and starfish, which provide plenty of gametes. Large and transparent, these eggs have served as excellent model systems for studying egg activation and embryo development in seawater, a plain natural medium. Starfish oocytes allow the study of the cortical, cytoplasmic and nuclear changes during the meiotic maturation process, which can also be triggered in vitro by hormonal stimulation. These morphological and biochemical changes ensure successful fertilization of the eggs at the first metaphase. On the other hand, sea urchin eggs are fertilized after the completion of meiosis, and are particularly suitable for the study of sperm–egg interaction, early events of egg activation, and embryonic development, as a large number of mature eggs can be fertilized synchronously. Starfish and sea urchin eggs undergo abrupt changes in the cytoskeleton and ion fluxes in response to the fertilizing spermatozoon. The plasma membrane and cortex of an egg thus represent “excitable media” that quickly respond to the stimulus with the Ca{sup 2+} swings and structural changes. In this article, we review some of the key findings on the rapid dynamic rearrangements of the actin cytoskeleton in the oocyte/egg cortex upon hormonal or sperm stimulation and their roles in the modulation of the Ca{sup 2+} signals and in the control of monospermic fertilization. - Highlights: • Besides microtubules, microfilaments may anchor the nucleus to oocyte surface. • The cortical Ca{sup 2+} flash and wave at fertilization mirror electrical membrane change. • Artificial egg activation lacks microvilli extension in the perivitelline space. • Calcium is necessary but not sufficient for cortical granules exocytosis. • Actin cytoskeleton modulates Ca{sup 2+} release at oocyte maturation

  3. Sohlh2 affects differentiation of KIT positive oocytes and spermatogonia.

    Science.gov (United States)

    Toyoda, Shuichi; Miyazaki, Tatsushi; Miyazaki, Satsuki; Yoshimura, Takuji; Yamamoto, Mayu; Tashiro, Fumi; Yamato, Eiji; Miyazaki, Jun-ichi

    2009-01-01

    The differentiation programs of spermatogenesis and oogenesis are largely independent. In the early stages, however, the mechanisms partly overlap. Here we demonstrated that a germ-cell-specific basic helix-loop-helix (bHLH) transcription factor gene, Sohlh2, is required for early spermatogenesis and oogenesis. SOHLH2 was expressed in mouse spermatogonia from the undifferentiated stage through differentiation and in primordial-to-primary oocytes. Sohlh2-null mice, produced by gene targeting, showed both male and female sterility, owing to the disrupted differentiation of mature (KIT(+)) spermatogonia and oocytes. The Sohlh2-null mice also showed the downregulation of genes involved in spermatogenesis and oogenesis, including the Sohlh1 gene, which is essential for these processes. Furthermore, we showed that SOHLH2 and SOHLH1 could form heterodimers. These observations suggested that SOHLH2 might coordinate with SOHLH1 to control spermatogonial and oocyte genes, including Sohlh1, to promote the differentiation of KIT(+) germ cells in vivo. This study lays the foundation for further dissection of the bHLH network that regulates early spermatogenesis and oogenesis.

  4. Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

    Science.gov (United States)

    Mitalipov, S M; Nusser, K D; Wolf, D P

    2001-07-01

    This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.

  5. Pharmaceutical Options for Triggering of Final Oocyte Maturation in ART

    Directory of Open Access Journals (Sweden)

    Juan Carlos Castillo

    2014-01-01

    Full Text Available Since the pioneering days of in vitro fertilization, hCG has been the gold standard to induce final follicular maturation. We herein reviewed different pharmaceutical options for triggering of final oocyte maturation in ART. The new upcoming agent seems to be GnRHa with its potential advantages over hCG trigger. GnRHa triggering elicits a surge of gonadotropins resembling the natural midcycle surge of gonadotropins, without the prolonged action of hCG, resulting in the retrieval of more mature oocytes and a significant reduction in or elimination of OHSS as compared to hCG triggering. The induction of final follicular maturation using GnRHa represents a paradigm shift in the ovulation triggering concept in ART and, thus, a way to develop a safer IVF procedure. Kisspeptins are key central regulators of the neuroendocrine mechanisms of human reproduction, who have been shown to effectively elicit an LH surge and to induce final oocyte maturation in IVF cycles. This new trigger concept may, therefore, offer a completely new, “natural” pharmacological option for ovulation induction. Whether kisspeptins will be the future agent to trigger ovulation remains to be further explored.

  6. Targeting oocyte maturation to improve fertility in older women.

    Science.gov (United States)

    Liu, X Johné

    2016-01-01

    Reproductive aging is an increasingly pressing problem facing women in modern society, due to delay in child bearing. According to Statistics Canada, 52% of all Canadian births in 2011 were by women aged 30 years and older, up from 24% in 1981 ( http://www.statcan.gc.ca/pub/91-209-x/2013001/article/11784-eng.htm ). Women older than 35 years of age experience significantly increased risks of infertility, miscarriage and congenital birth defects, mostly due to poor quality of the eggs. Increasingly sophisticated, and often invasive, assisted reproductive technologies (ARTs) have helped millions of women to achieve reproductive success. However, by and large, ARTs do not address the fundamental issue of reproductive aging in women: age-related decline in egg quality. More importantly, ARTs are not, and will never be, the main solution for the general population. Here, I attempt to review the scientific literature on age-related egg quality decline, based mostly on studies in mice and in humans. Emphasis is given to the brief period of time called oocyte maturation, which occurs just prior to ovulation. The rationale for this emphasis is that oocyte maturation represents a critical window where unfavorable ovarian conditions in older females contribute significantly to the decline of egg quality, and that science-based intervention during oocyte maturation represents the best chance of improving egg quality in older women. Finally, I summarize our own work in recent years on peri-ovulatory putrescine supplementation as a possible remedy for reproductive aging.

  7. Diploid oocyte formation and tetraploid embryo development induced by cytochalasin B in bovine.

    Science.gov (United States)

    Bai, Chunling; Liu, Hui; Liu, Ying; Wu, Xia; Cheng, Lei; Bou, Shorgan; Li, Guang-Peng

    2011-02-01

    Tetraploid embryos are a useful model for postimplantation development of polyploidy cells, and tetraploid cells are an advantage in studies for chimeras yielding offspring completely derived from embryo stem cells or induced pluripotent cells. This study was designed to investigate the effects of cytochalasin B (CB) on bovine oocyte meiosis, and to induce the formation of diploid oocytes and tetraploid embryos. The results showed that: (1) incubation of oocytes in CB at ≥2.0 μg/mL concentrations for 24 h significantly decreased oocyte maturation and the matured oocytes' haploid composition. Over 50% of the CB-treated oocytes did not expel PB1 (non-PB1), and most of the non-PB1 oocytes contained 2n (60) chromosomes. (2) Pretreatment of oocytes with CB at concentrations of 7.5 and 15 μg/mL for 10 h significantly decreased oocyte maturation. Posttreatment of oocytes with CB resulted in most of the oocytes containing 2n chromosomes. (3) The parthenogenetic blastocysts (25-28%) derived from the non-PB1 oocytes of posttreatment group was significantly higher than that from pretreatment, whole period treatment, and the control oocytes (12-16%). (4) Cytogenetic analysis of the embryos derived from CB-treated non-PB1 oocytes resulted in 74% of the one-cell stage embryos being 4n = 120 chromosomes, 82% of two-cell stage embryos contained 4n chromosomes in each blastomere, and 75% of the blastocysts were tetraploidy (4n = 120). (6) The stopped uncleaved one-cell embryos showed an amazing phenomenon of over 15% of them containing extra chromosomes, which suggested multiple DNA duplication occurred within 40 h after activation. In conclusion, CB inhibits PB1 extrusion, disfigures spindle structure, decreases oocyte maturation, and results in formation of diploid (2n or 4c) oocytes. The diploid oocytes resulted in a higher development of tetraploid embryos, which would be a unique approach for the production of tetraploid embryos in bovine.

  8. EFFECT OF CYSTEAMINE ON THE RATE OF IN VITRO MATURATION OF OOCYTES IN TWO MEDIA

    Directory of Open Access Journals (Sweden)

    A. Mohammadi-Rousheh

    2006-07-01

    Full Text Available Rate of in vitro maturation of oocytes is one of the challenges of assisted reproductive techniques. In this study we investigated the effects of supplementation of cysteamine on the rate of in vitro maturation of oocytes in two different media. Germinal vesicle oocytes were collected from mouse ovary and cultured in two media (TCM199 and MEME with 0, 50, 100, 200, 500 µM/ml cysteamine. Number of germinal vesicle breakdowns and metaphase II oocytes were recorded. The results showed that the rate of in vitro maturation in 100 µM/ml cysteamine was significantly higher compared to control (P < 0.05. Evaluation of two media in this study showed that TCM199 improved the rate of in vitro maturation and oocyte maturation better than MEME; however, this difference was not statistically significant. These findings indicate that TCM199 as compared to MEME was better in rate of in vitro maturation of oocytes.

  9. Effect of the meiotic inhibitor cilostamide on resumption of meiosis and cytoskeletal distribution in buffalo oocytes.

    Science.gov (United States)

    Li, Qing-Yang; Lou, Juan; Yang, Xiao-Gan; Lu, Yang-Qing; Lu, Sheng-Sheng; Lu, Ke-Huan

    2016-11-01

    Improving the quality of in vitro maturated buffalo oocytes is essential for embryo production. We report here the effects on microtubules and microfilaments in oocytes and embryo development that result from treating buffalo oocytes with the phosphodiesterase 3 (PDE3) inhibitor cilostamide. Addition of 20μM or 50μM cilostamide for 24h during in vitro maturation showed no differences in the percentage of oocytes arrested at the germinal vesicle (GV) stage. When 20μM cilostamide was added to the pre-maturation culture for 6h, 12h or 24h and continued for another 24h without cilostamide, oocytes resumed meiosis, but with significantly lower (P0.05). In summary, cilostamide reversibly arrested the resumption of meiosis without any adverse impact on the dynamic changes in microtubules and microfilaments in buffalo oocytes and their in vitro developmental capacity. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K;

    2002-01-01

    will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells...... and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual......-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels....

  11. Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes.

    Science.gov (United States)

    Tsuji, Ai; Nakamura, Toshinobu; Shibata, Katsumi

    2015-01-01

    Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.

  12. The ultrastructural aspects of vitellogenesis or oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Serrasalminae).

    Science.gov (United States)

    Guimarães, A C D; Quagio-Grassiotto, I

    2002-04-01

    Oocyte secondary growth in S. spiloleura corresponds to the period in which different vesicular structures are formed, including the cortical alveoli and the yolk granules. The oocytes with cortical alveolus formation show vesicular structures with filamentous content in the cortical cytoplasmic region, which are the cortical alveolus precursors. In these oocytes, electron-dense vesicles of heterogenous content are dispersed in the inner cytoplasmic region and their nuclei are irregular, showing many nucleoli of different sizes. The oocytes in vitellogenesis are filled with many vesicles. The cortical alveolus precursors are in the peripheral region, and electron-dense granules are seen near to the nucleus. These fuse and form yolk granules. The oocytes in vitellogenesis show a very irregular nucleus that has nucleoli of different sizes. In the oocytes in final vitellogenesis, the yolk granules are scattered throughout the cytoplasm, displacing the cortical alveoli toward cell periphery. The nucleus is similar to the other stages.

  13. Observations Regardin Oocyte in Vitro Maturation after Recovery from Slaughter House Females

    Directory of Open Access Journals (Sweden)

    Valeriu Carabă

    2011-05-01

    Full Text Available The oocytes viability must be taken as an important selection parameter for successful in vitro cultivation. The ovaries were collected from the slaughterhouse and maintained at 4°C for 7 days. Fallowing cumulus -oocytes complexes recovery the viability was tested using two staining methods. For the first experiment we used 27 cumulus - oocytes complexes, stained with Neutral red and for the second experiment we used 11 cumulus - oocytes complexes stained with Trypan blue. Fallowing staining with Neutral red 23 cumulus - oocytes complexes were assessed as viable (were stained in red – enzymatic activity within the cells and for the Trypan blue staining 11 cumulus - oocytes complexes were assessed as viable (remained unstained – integers cellular membranes.

  14. Revisiting oocyte–somatic cell interactions: in search of novel intrafollicular predictors and regulators of oocyte developmental competence

    Science.gov (United States)

    Li, Qinglei; McKenzie, Laurie J.; Matzuk, Martin M.

    2008-01-01

    Prediction and improvement of oocyte competence are two critical issues in assisted reproductive technology to improve infertility therapy. The lack of reliable and objective predictors of oocyte developmental competence for oocyte/embryo selection during in vitro fertilization hampers the effectiveness of this technology. Likewise, the low pregnancy rate resulting from in vitro maturation of human oocytes represents a major obstacle for its clinical application. Oocyte competence is progressively acquired during follicular development, and the oocyte plays a dominant role in regulating granulosa cell functions and maintaining the microenvironment appropriate for the development of its competence. Hence, granulosa cell functions are reflective of oocyte competence, and molecular markers of granulosa cells are potentially reliable predictors of oocyte quality. With the advent of the functional genomics era, the transcriptome of granulosa cells has been extensively characterized. Experimental data supporting granulosa cell markers as predictors of oocyte competence are now emerging in both animal models and humans. Future efforts should focus on integrating granulosa cell genetic markers as parameters for oocyte/embryo selection. Moreover, novel in vitro evidence highlights the effectiveness of exogenous oocyte-secreted factors in promoting oocyte developmental competence in animal models. The challenge in evaluating the effect of oocyte-secreted factors on oocyte quality in a clinical setting is to standardize the various preparations of these recombinant proteins and decipher their complex interactions/cooperativity within the germline-somatic cell regulatory loop. PMID:18996952

  15. p27(Kip1) negatively regulates the activation of murine primordial oocytes.

    Science.gov (United States)

    Hirashima, Yumiko; Moniruzzaman, Mohammad; Miyano, Takashi

    2011-04-01

    In mice, small oocytes (primordial oocytes) are enclosed within flattened granulosa cells to form primordial follicles around birth. A small number of primordial oocytes enter the growth phase, whereas others are quiescent. The mechanism regulating this selection of primordial oocytes is not well understood. The objective of the present study was to understand the role of p27(Kip1), which regulates cell cycle progression in somatic cells, in the growth initiation of primordial oocytes in neonatal mice. We studied the localization of p27(Kip1) in 0-, 3-, 5-, 7- and 21-day-old mouse ovaries by immunohistochemistry. Ovaries from 3-day-old mice were treated with p27(Kip1) siRNAs (small interfering RNAs), and knockdown of p27(Kip1) was determined by immunohistochemistry and Western blotting. Ovaries treated with siRNAs were organ-cultured for 6 days, and oocyte growth was estimated histologically. Expression of p27(Kip1) was undetectable in the primordial oocytes of newborn mice. In the 3-day-old ovaries (n=3), p27(Kip1) was demonstrated in the nucleus of 36 ± 6% primordial oocytes. The percentage of p27(Kip1)-positive primordial oocytes increased to 72 ± 8 (n=3), 85 ± 7 (n=3) and 93 ± 5 (n=3) in the 5-, 7- and 21-day-old mouse ovaries, respectively. After knockdown of the p27(Kip1) protein by siRNAs, a higher proportion of oocytes entered the growth phase in cultured ovaries than those in the control. These results suggest that p27(Kip1) negatively regulates primordial oocyte growth and that knockdown of p27(Kip1) leads primordial oocytes to enter the growth phase in vitro.

  16. Proteomics-based systems biology modeling of bovine germinal vesicle stage oocyte and cumulus cell interaction.

    Directory of Open Access Journals (Sweden)

    Divyaswetha Peddinti

    Full Text Available BACKGROUND: Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at germinal vesicle (GV stage are considered essential for proper maturation or 'programming' of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication. METHODOLOGY/PRINCIPAL FINDINGS: We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation. CONCLUSIONS/SIGNIFICANCE: Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level.

  17. Grading of Mammalian Cumulus Oocyte Complexes using Machine Learning for in Vitro Embryo Culture

    OpenAIRE

    Sudarshan, Viswanath P; Weiser, Tobias; Chintala, Phalgun; Mandal, Subhamoy; Dutta, Rahul

    2016-01-01

    Visual observation of Cumulus Oocyte Complexes provides only limited information about its functional competence, whereas the molecular evaluations methods are cumbersome or costly. Image analysis of mammalian oocytes can provide attractive alternative to address this challenge. However, it is complex, given the huge number of oocytes under inspection and the subjective nature of the features inspected for identification. Supervised machine learning methods like random forest with annotations...

  18. Vitrification affects nuclear maturation and gene expression of immature human oocytes

    Directory of Open Access Journals (Sweden)

    Abbas Shahedi

    2017-02-01

    Full Text Available Background: Vitrification of oocytes is a fast-freezing technique, which may affect the quality of the human oocyte, and consequently affects the embryo development, pregnancy and birth. The aim of the current study was to investigate the consequence of in-vitro vitrification on maturation status of immature human oocytes, additionally, expression levels of stress, and apoptosis related genes. Materials and Methods: The total of 213 human immature oocytes which routinely discarded from assisted reproduction clinics were collected and divided into two groups including: (I fresh germinal vesicle (GV oocytes (n=106 (matured in-vitro  (fIVM , and  (II GV oocytes (n=107 that initially vitrified, then matured in  in-vitro (vIVM. After 36 hours of incubation, the oocytes were evaluated for nuclear maturation and expression level of DNA methyltransferase (DNMT1, stress related genes (Sod1 and Hsp70, and apoptotic related genes (Bax and Bcl-2 by quantitative Real-Time PCR. Results: Oocyte maturation rates were reduced in vIVM compared to fIVM oocytes (P=0.001. The expression of stress (Sod1 and Hsp70, and apoptotic-related genes (Bax and Bcl-2 in vIVM were significantly higher compared to the fIVM group. Additionally, pro-apoptotic gene up-regulated 4.3 times more than anti-apoptotic gene in vIVM oocyte. However, DNMT1 gene expression was reduced in vIVM oocyte (P = 0.047. Conclusions: The low survival rate of vitrified In-vitro matured GV oocytes could definitely be explained by the alterations of their gene expression profile. 

  19. Pregnancy and fertilization potential of immature oocytes retrieved in intracytoplasmic sperm injection cycles.

    Science.gov (United States)

    Ko, Duck Sung; Lee, Sun-Hee; Park, Dong-Wook; Yang, Kwang Moon; Lim, Chun Kyu

    2015-09-01

    The goal of this study was to evaluate the pregnancy potential of immature (metaphase I or germinal vesicle stage) oocytes retrieved in intracytoplasmic sperm injection (ICSI) cycles. A total of 1,871 couples with infertility underwent 2,984 ICSI cycles. Cycles in which three or fewer oocytes were retrieved were included in this study in order to evaluate the pregnancy potential of immature oocytes. Cycles were divided into five groups (group I-V), according to the maturation status of the oocytes at the time of cumulus cell removal and ICSI. The fertilization and pregnancy rates after ICSI were analyzed and compared among the study groups based on the maturation status of the retrieved oocytes. The retrieval of only immature oocytes was associated with a significant decrease in the fertilization rate (76.1%±37.3% vs. 49.0%±49.1%, 66.7%±48.7%; group I vs. group II, group III, respectively) and the average number of transferred embryos (1.5±0.7 vs. 1.1±0.4, 1.1±0.6). The cycle cancellation rate was significantly higher when only immature oocytes were retrieved. The clinical pregnancy rate decreased significantly when the transferred embryos had originated from immature oocytes (16.9% vs. 10.3%, 1.2%). In ICSI cycles, the fertilization potential and pregnancy potential of the immature oocytes retrieved in ICSI cycles were inferior to those of mature oocytes. Therefore, increasing the number of injectable oocytes and transferrable embryos by using immature oocytes after their spontaneous in vitro maturation does not necessarily improve pregnancy outcomes.

  20. The potential significance of binovular follicles and binucleate giant oocytes for the development of genetic abnormalities

    Indian Academy of Sciences (India)

    Bernd Rosenbusch

    2012-12-01

    Normal development of a fertilizable female gamete emanates from a follicle containing only one oocyte that becomes haploid after first meiotic division. Binovular follicles including two oocytes and binucleate giant oocytes that are diploid after first meiosis constitute notable exceptions from this rule. Data provided by programmes of human-assisted reproduction on the occurrence of both phenomena have been reviewed to evaluate possible implications for the formation of genetic abnormalities. To exclude confusion with oocytes aspirated from two adjacent individual follicles, true binovularity has been defined as inclusion of two oocytes within a common zona pellucida or their fusion in the zonal region. A total of 18 conjoined oocytes have been reported and one of the oocyte was normally fertilized in seven cases. Simultaneous fertilization of both female gametes occurred only once. No pregnancy was achieved after transfer of an embryo from a binovular follicle. Binucleate giant oocytes have been observed sporadically but a few reports suggest an incidence of up to 0.3% of all gametes retrieved. Extensive studies performed by two independent centres demonstrated that giant oocytes are diploid at metaphase II, can undergo fertilization in vitro with formation of two or three pronuclei and develop into triploid zygotes and triploid or triploid/mosaic embryos. In summary, giant binucleate oocytes may be responsible for the development of digynic triploidy whereas the currently available data do not support a role of conjoined oocytes in producing dizygotic twins, mosaicism, chimaeras or tetraploidy. However, more information on the maturity and fertilizability of oocytes from binovular follicles is needed. Future studies should also evaluate a possible impact of pharmaceutical and environmental oestrogens on the formation of multiovular follicles.

  1. Vitrification of human germinal vesicle oocytes; before or after in vitro maturation?

    Directory of Open Access Journals (Sweden)

    Evangelia Kasapi

    2017-03-01

    Full Text Available Background The use of immature oocytes derived from stimulated cycles could be of great importance, particularly for urgent fertility preservation cases. The current study aimed to determine whether in vitro maturation (IVM was more successful before or after vitrification of these oocytes. Materials and Methods This prospective study was performed in a private in vitro fertilization (IVF center. We collected 318 germinal vesicle (GV oocytes from 104 stimulated oocyte donation cycles. Oocytes were divided into two groups according to whether vitrification was applied at the GV stage (group 1 or in vitro matured to the metaphase II (MII stage and then vitrified (group 2. In the control group (group 3, oocytes were in vitro matured without vitrification. In all three groups, we assessed survival rate after warming, maturation rate, and MII-spindle/chromosome configurations. The chi-square test was used to compare rates between the three groups. Statistical significance was defined at P<0.05 and we used Bonferroni criterion to assess statistical significance regarding the various pairs of groups. The Statistical Package for the Social Sciences version 17.0 was used to perform statistical analysis. Results There was no significant difference in the survival rate after vitrification and warming of GV (93.5% and MII oocytes (90.8%. A significantly higher maturation rate occurred when IVM was performed before vitrification (82.9% compared to after vitrification (51%. There was no significant difference in the incidence of normal spindle/ chromosome configurations among warmed oocytes matured in vitro before (50.0% or after (41.2% vitrification. However, a higher incidence of normal spindle/chromosome configurations existed in the in vitro matured oocytes which were not subjected to vitrification (fresh oocytes, 77.9%. Conclusion In stimulated cycles, vitrification of in vitro matured MII oocytes rather than GV oocytes seems to be more efficient. This

  2. VHA-19 is essential in Caenorhabditis elegans oocytes for embryogenesis and is involved in trafficking in oocytes.

    Science.gov (United States)

    Knight, Alison J; Johnson, Nicholas M; Behm, Carolyn A

    2012-01-01

    There is an urgent need to develop new drugs against parasitic nematodes, which are a significant burden on human health and agriculture. Information about the function of essential nematode-specific genes provides insight to key nematode-specific processes that could be targeted with drugs. We have characterized the function of a novel, nematode-specific Caenorhabditis elegans protein, VHA-19, and show that VHA-19 is essential in the germline and, specifically, the oocytes, for the completion of embryogenesis. VHA-19 is also involved in trafficking the oocyte receptor RME-2 to the oocyte plasma membrane and is essential for osmoregulation in the embryo, probably because VHA-19 is required for proper eggshell formation via exocytosis of cortical granules or other essential components of the eggshell. VHA-19 may also have a role in cytokinesis, either directly or as an indirect effect of its role in osmoregulation. Critically, VHA-19 is expressed in the excretory cell in both larvae and adults, suggesting that it may have a role in osmoregulation in C. elegans more generally, probably in trafficking or secretion pathways. This is the first time a role for VHA-19 has been described.

  3. Melatonin delivery by nanocapsules during in vitro bovine oocyte maturation decreased the reactive oxygen species of oocytes and embryos.

    Science.gov (United States)

    Remião, Mariana Härter; Lucas, Caroline Gomes; Domingues, William Borges; Silveira, Tony; Barther, Nathaniele Nebel; Komninou, Eliza Rossi; Basso, Andrea Cristina; Jornada, Denise Soledade; Beck, Ruy Carlos Ruver; Pohlmann, Adriana Raffin; Junior, Antonio Sérgio Varela; Seixas, Fabiana Kömmling; Campos, Vinicius Farias; Guterres, Silvia Stanisçuaski; Collares, Tiago

    2016-08-01

    In this work, a promising approach to increase the advantageous properties of melatonin through its encapsulation into lipid-core nanocapsules (LNC) was examined. Oocytes were treated during in vitro maturation with non-encapsulated melatonin (Mel), melatonin-loaded lipid-core nanocapsules (Mel-LNC), and unloaded LNC. Cytotoxicity, meiotic maturation rate, development to the blastocyst stage, reactive oxygen species (ROS) and glutathione levels, mean cell number and apoptotic cell/blastocyst, and mRNA quantification were evaluated. Both Mel and Mel-LNC enhanced in vitro embryo production, however, Mel-LNC proved to be more effective at decreasing ROS levels and the apoptotic cell number/blastocyst, increasing the cleavage and blastocyst rates, up-regulating the GPX1 and SOD2 genes, and down-regulating the CASP3 and BAX genes. Mel-LNC could penetrate into oocytes and remain inside the cells until they reach the blastocyst stage. In conclusion, when melatonin was encapsulated in LNC and applied during in vitro oocyte maturation, some quality aspects of the blastocysts were improved.

  4. VHA-19 is essential in Caenorhabditis elegans oocytes for embryogenesis and is involved in trafficking in oocytes.

    Directory of Open Access Journals (Sweden)

    Alison J Knight

    Full Text Available There is an urgent need to develop new drugs against parasitic nematodes, which are a significant burden on human health and agriculture. Information about the function of essential nematode-specific genes provides insight to key nematode-specific processes that could be targeted with drugs. We have characterized the function of a novel, nematode-specific Caenorhabditis elegans protein, VHA-19, and show that VHA-19 is essential in the germline and, specifically, the oocytes, for the completion of embryogenesis. VHA-19 is also involved in trafficking the oocyte receptor RME-2 to the oocyte plasma membrane and is essential for osmoregulation in the embryo, probably because VHA-19 is required for proper eggshell formation via exocytosis of cortical granules or other essential components of the eggshell. VHA-19 may also have a role in cytokinesis, either directly or as an indirect effect of its role in osmoregulation. Critically, VHA-19 is expressed in the excretory cell in both larvae and adults, suggesting that it may have a role in osmoregulation in C. elegans more generally, probably in trafficking or secretion pathways. This is the first time a role for VHA-19 has been described.

  5. Identification of some unknown transcripts from SSH cDNA library of buffalo follicular oocytes.

    Science.gov (United States)

    Rajput, S K; Kumar, P; Roy, B; Verma, A; Pandey, H P; Singh, D; De, S; Datta, T K

    2013-03-01

    A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.

  6. Casein kinase G may be the target of spermine during progesterone-induced oocyte maturation

    Institute of Scientific and Technical Information of China (English)

    LIRUNSHENG; JIAKETSO

    1993-01-01

    Casein kinase G (CKG) with more than 2500-fold enrichraent was purified from Bufo bufo gargarizans ovaries. The catalytic activity of the enzyme was found to be associated with its 42 kD subunit, and its 26 kD subunit was found to be the major tsrget for the enzyme autophos phorylation. Each fuU-grown oocyte contained 1.9 units of CKG corresponding to an intracellular concentration of 93 nM. After injecting an amount of 0,38 units of the enzyme into the oocyte, approximately 50% of the progesterone-induoed maturation was inhibited. The inhibitory effect was enhanced in oocytes pretreated with spermine, which was consistent with the results that the enzyme was activated in vitro in the presence of spermine, The MPF-induced oocyte maturation was delayed and even prohibited in the kinase-microinjected oocytes. A 55 kD oocyte protein was identified as an suhstrate of CKG both in vivo and in vitro, and the enhancement of the 55 kD protein phosphory[ation was associated with kinase inhibition on maturation and on protein synthesis in kinase-microinjected oocytes. As the endogenous spermine level decreased in the course of progesteroneinduced oocyte maturation. 55 kD protein was dephosphorylated, Heparin, a specific inhibitor of CKG, potentiated the progesterone-induced oocyte maturation. Altogether the experimental reSults indicated Strongly that CKG may be the physiological target of spermine.

  7. Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency.

    Science.gov (United States)

    Cagnone, Gael L M; Tsai, Te-Sha; Makanji, Yogeshwar; Matthews, Pamela; Gould, Jodee; Bonkowski, Michael S; Elgass, Kirstin D; Wong, Ashley S A; Wu, Lindsay E; McKenzie, Matthew; Sinclair, David A; St John, Justin C

    2016-03-18

    An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.

  8. Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro

    Science.gov (United States)

    Liu, Shuzhen; Jiang, Ligang; Zhong, Tao; Kong, Shuhui; Zheng, Rongbin; Kong, Fengyun; Zhang, Cong; Zhang, Lei; An, Liguo

    2015-01-01

    Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro. PMID:26275143

  9. Possible participation of mitochondria in lipid yolk formation in oocytes of paddlefish and sturgeon.

    Science.gov (United States)

    Zelazowska, Monika; Kilarski, Wincenty

    2009-03-01

    The ovary of paddlefish and sturgeons (Acipenseriformes) is composed of discrete units: the ovarian nests and ovarian follicles. The ovarian nests comprise oogonia and numerous early dictyotene oocytes surrounded by somatic prefollicular cells. Each ovarian follicle consists of a spherical oocyte and a layer of follicular cells situated on a thick basal lamina, encompassed by thecal cells. The cytoplasm of previtellogenic oocytes is differentiated into two distinct zones: the homogeneous and granular zones. The homogeneous cytoplasm is organelle-free, whereas the granular cytoplasm contains numerous organelles, including mitochondria and lipid droplets. We have analyzed the cytoplasm of early dictyotene and previtellogenic oocytes ultrastructurally and histologically. In the cytoplasm of early dictyotene oocytes, two morphologically different types of mitochondria can be distinguished: (1) with well-developed cristae and (2) with distorted and fused cristae. In previtellogenic oocytes, the mitochondria of the second type show various stages of cristae distortion; they contain and release material morphologically similar to that of lipid droplets and eventually degenerate. This process of mitochondrial transformation is accompanied by an accumulation of lipid droplets that form a single large accumulation (lipid body) located in the vicinity of the oocyte nucleus (germinal vesicle). The lipid body eventually disperses in the oocyte center. The possible participation of these mitochondria in the formation of oocyte lipid droplets is discussed.

  10. Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro.

    Directory of Open Access Journals (Sweden)

    Shuzhen Liu

    Full Text Available Acrylamide (ACR is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus-oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus-oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.

  11. Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes

    Science.gov (United States)

    Miledi, Ricardo; Eusebi, Fabrizio; Martínez-Torres, Ataúlfo; Palma, Eleonora; Trettel, Flavia

    2002-01-01

    The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl− channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to γ-aminobutyric acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders. PMID:12237406

  12. Does Cryopreservation of Ovarian Tissue Affect the Distribution and Function of Germinal Vesicle Oocytes Mitochondria?

    Directory of Open Access Journals (Sweden)

    Mojdeh Salehnia

    2013-01-01

    Full Text Available The aim of this study was to evaluate mitochondrial alteration and ATP content of germinal vesicle (GV oocytes isolated from fresh and vitrified ovaries. After superovulation, the ovaries from adult mice were collected and divided into control and vitrified groups. GV oocytes were isolated mechanically from each group. Half were cultured for 24 hours and their maturation was assessed. Metaphase II oocytes were collected and submitted to in vitro fertilization and their fertilization rates and development to the blastocyst stage were evaluated. In the remaining GV oocytes, ATP levels were quantified, and mitochondrial distribution, mitochondrial membrane potential, and intracellular free calcium were detected with rhodamine 123, JC-1 and Flou-4 AM staining, using laser-scanning confocal microscopy. Maturation and fertilization rates of GV oocytes and the developmental rates of subsequent embryos were significantly lower in vitrified samples (P<0.05. The ATP content and Ca2+ levels differed significantly in fresh and vitrified GV oocytes (P<0.05. Most mitochondria were seen as large and homogenous aggregates (66.6% in fresh GV oocytes compared to vitrified oocytes (50%. No significant differences in mitochondrial membrane potential were found between the groups. The lower maturation and fertilization rates of GV oocytes from vitrified ovaries may be due to changes in their mitochondrial function and distribution.

  13. The role of phosphatidylinositol signaling pathway in regulating serotonin-induced oocyte maturation in Mercenaria mercenaria

    Institute of Scientific and Technical Information of China (English)

    WANG Qing; ZHANG Tao

    2011-01-01

    Serotonin (5-HT) has been found to stimulate meiotic maturation of oocytes in many molluscs. During maturation, several signaling pathways are involved, especially the phosphatidylinositol and cAMP pathways. In order to examine the possible role of the phosphatidylinositol signaling pathway in regulating oocyte maturation in Mercenaria mercenaria, the effects of the activator/inhibitor of phospholipase (PLC) and protein kinase C (PKC) on serotonin-induced maturation were studied. Results show that high-concentrations of neomycin (inhibitor of PLC) blocked oocyte maturation, while 9, 10-dimethyl- 1, 2-benzanthracene (DMBA, activator of PLC) promoted oocyte maturation in the presence of serotonin. It was also found that in the presence of serotonin, phorbol 12-myristate 13-acetate (PMA,activator of PKC) inhibited oocyte maturation, while sphingosine (inhibitor of PKC) stimulated oocyte maturation. These results indicate that serotonin-induced oocyte maturation requires the activation of the phosphatidylinositol pathway. Decrease of PLC inhibited serotonin-induced oocyte maturation, whereas a decrease of PKC stimulated the maturation. Thus, our study indicates that serotonin promotes maturation of M. mercenaria oocytes through PLC stimulated increase in calcium ion concentration via inositol-1,4, 5-trisphosphate (IP3) but not PKC.

  14. Generation of meiomaps of genome-wide recombination and chromosome segregation in human oocytes

    DEFF Research Database (Denmark)

    Ottolini, Christian S; Capalbo, Antonio; Newnham, Louise

    2016-01-01

    We have developed a protocol for the generation of genome-wide maps (meiomaps) of recombination and chromosome segregation for the three products of human female meiosis: the first and second polar bodies (PB1 and PB2) and the corresponding oocyte. PB1 is biopsied and the oocyte is artificially......-nucleotide polymorphisms (SNPs) genome-wide by microarray. Informative maternal heterozygous SNPs are phased using a haploid PB2 or oocyte as a reference. A simple algorithm is then used to identify the maternal haplotypes for each chromosome, in all of the products of meiosis for each oocyte. This allows mapping...

  15. Xenopus tropicalis oocytes as an advantageous model system for the study of intracellular Ca(2+) signalling.

    Science.gov (United States)

    Marchant, J S; Parker, I

    2001-04-01

    1. The purpose of this study was to compare oocytes from the pipid frogs Xenopus tropicalis and Xenopus laevis, with respect to their utility for studying Ca(2+) signalling mechanisms and for expression of heterologous proteins. 2. We show that X. tropicalis oocytes possess an intracellular Ca(2+) store that is mobilized by inositol (1,4,5) trisphosphate (IP(3)). Ca(2+) signalling is activated by endogenous lysophosphatidic acid receptors and cytosolic Ca(2+) activates a plasma membrane chloride conductance. The spatiotemporal organization of cytosolic Ca(2+) signals, from the microscopic architecture of elementary Ca(2+) 'puffs' to the macroscopic patterns of Ca(2+) spiking are closely similar to the local and global patterns of Ca(2+) release previously characterized in oocytes from X. laevis. 3. By injecting X. tropicalis oocytes with cDNA encoding an ER-targeted fluorescent protein construct, we demonstrate the capacity of the X. tropicalis oocyte to readily express heterologous proteins. The organization of ER is polarized across the oocyte, with the IP(3)-releaseable store targeted within an approximately 8 microm wide band that circumscribes the cell. 4. We conclude that the X. tropicalis oocyte shares many of the characteristics that have made oocytes of X. laevis a favoured system for studying Ca(2+) signalling mechanisms. Moreover, X. tropicalis oocytes display further practical advantages in terms of imaging depth, Ca(2+) signal magnitude and electrical properties. These further enhance the appeal of X. tropicalis as an experimental system, in addition to its greater amenability to transgenic approaches.

  16. Dimethyl Sulfoxide Perturbs Cell Cycle Progression and Spindle Organization in Porcine Meiotic Oocytes.

    Directory of Open Access Journals (Sweden)

    Xuan Li

    Full Text Available Meiotic maturation of mammalian oocytes is a precisely orchestrated and complex process. Dimethyl sulfoxide (DMSO, a widely used solvent, drug, and cryoprotectant, is capable of disturbing asymmetric cytokinesis of oocyte meiosis in mice. However, in pigs, DMSO's effect on oocyte meiosis still remains unknown. We aimed to evaluate if DMSO treatment will affect porcine oocyte meiosis and the underlying molecular changes as well. Interestingly, we did not observe the formation of the large first polar body and symmetric division for porcine oocytes treated with DMSO, contrary to findings reported in mice. 3% DMSO treatment could inhibit cumulus expansion, increase nuclear abnormality, disturb spindle organization, decrease reactive oxygen species level, and elevate mitochondrial membrane potential of porcine oocytes. There was no effect on germinal vesicle breakdown rate regardless of DMSO concentration. 3% DMSO treatment did not affect expression of genes involved in spindle organization (Bub1 and Mad2 and apoptosis (NF-κB, Pten, Bcl2, Caspase3 and Caspase9, however, it significantly decreased expression levels of pluripotency genes (Oct4, Sox2 and Lin28 in mature oocytes. Therefore, we demonstrated that disturbed cumulus expansion, chromosome alignment, spindle organization and pluripotency gene expression could be responsible for DMSO-induced porcine oocyte meiotic arrest and the lower capacity of subsequent embryo development. Our results provide new insights on DMSO's effect on porcine oocyte meiosis and raise safety concerns over DMSO's usage on female reproduction in both farm animals and humans.

  17. Imbalance between the expression dosages of X-chromosome and autosomal genes in mammalian oocytes.

    Science.gov (United States)

    Fukuda, Atsushi; Tanino, Motohiko; Matoba, Ryo; Umezawa, Akihiro; Akutsu, Hidenori

    2015-09-15

    Oocytes have unique characteristics compared with other cell types. In mouse and human oocytes, two X chromosomes are maintained in the active state. Previous microarray studies have shown that the balance of the expression state is maintained in haploid oocytes. Here, we investigated transcripts using RNA-sequence technology in mouse and human oocytes. The median expression ratio between X chromosome and autosomal genes (X:A) in immature mouse oocytes increased as the gene expression levels increased, reaching a value of 1. However, the ratio in mature oocytes was under 1 for all expression categories. Moreover, we observed a markedly low ratio resulting from the bimodal expression patterns of X-linked genes. The low X:A expression ratio in mature oocyte was independent of DNA methylation. While mature human oocytes exhibited a slightly low X:A expression ratio, this was the result of the skewed high frequency of lowly expressed X-linked genes rather than the bimodal state. We propose that this imbalance between the expression dosages of X-chromosome and autosomal genes is a feature of transcripts in mammalian oocytes lacking X-chromosome inactivation.

  18. Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes

    Institute of Scientific and Technical Information of China (English)

    Shen Xinghui; Zhou Dongjie; Gu Yanli; Zhang Na; Li Tong; Wu Xi; Lei Lei

    2015-01-01

    Objective: To observe the effect of DMSO on mouse oocyte meiotic maturation. Results: In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell-like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap forma-tion and spindle migration. These features are among the primary causes of abnormal symmetric division;however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each "blasto-mere" of the 2-cell-like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each "blastomere" and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection ( ICSI ) . Further-more, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division fail-ure. Conclusions:Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric di-vision. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI.

  19. [In vitro maturation of the oocytes of the common frog with and without follicular envelopes as affected by cholesterol].

    Science.gov (United States)

    Skoblina, M N; Kondrat'eva, O T

    1983-01-01

    Cholesterol was shown to induce in vitro maturation of the Rana temporaria oocytes both in the presence and in the absence of the follicle cells. The maturation of the denuded oocytes required their much shorter treatment with cholesterol. Ethidium bromide inhibited the cholesterol-induced maturation of the oocytes with follicle envelopes and either did not affect the oocyte maturation after the follicle envelope removal or stimulated it. Amino-gluthetimide inhibited the cholesterol-induced oocyte maturation irrespective of the presence of follicle cells. The mechanism inducing effect of cholesterol on follicle and oocyte is discussed.

  20. The Relationship Between Transcript Expression Levels of Nuclear Encoded (TFAM, NRF1 and Mitochondrial Encoded (MT-CO1 Genes in Single Human Oocytes During Oocyte Maturation

    Directory of Open Access Journals (Sweden)

    Ghaffari Novin M.

    2015-06-01

    Full Text Available In some cases of infertility in women, human oocytes fail to mature when they reach the metaphase II (MII stage. Mitochondria plays an important role in oocyte maturation. A large number of mitochondrial DNA (mtDNA, copied in oocytes, is essential for providing adenosine triphosphate (ATP during oocyte maturation. The purpose of this study was to identify the relationship between transcript expression levels of the mitochondrial encoded gene (MT-CO1 and two nuclear encoded genes, nuclear respiratory factor 1 (NRF1 and mitochondrial transcription factor A (TFAM in various stages of human oocyte maturation. Nine consenting patients, age 21-35 years old, with male factors were selected for ovarian stimulation and intracytoplasmic sperm injection (ICSI procedures. mRNA levels of mitochondrial- related genes were performed by singlecell TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR. There was no significant relationship between the relative expression levels in germinal vesicle (GV stage oocytes (p = 0.62. On the contrary, a significant relationship was seen between the relative expression levels of TFAM and NRF1 and the MT-CO1 genes at the stages of metaphase I (MI and MII (p = 0.03 and p = 0.002. A relationship exists between the transcript expression levels of TFAM and NRF1, and MT-CO1 genes in various stages of human oocyte maturation.

  1. Elevated intracellular pH appears in aged oocytes and causes oocyte aneuploidy associated with the loss of cohesion in mice.

    Science.gov (United States)

    Cheng, Jin-Mei; Li, Jian; Tang, Ji-Xin; Chen, Su-Ren; Deng, Shou-Long; Jin, Cheng; Zhang, Yan; Wang, Xiu-Xia; Zhou, Chen-Xi; Liu, Yi-Xun

    2016-09-16

    Increases in the aneuploidy rate caused by the deterioration of cohesion with increasing maternal age have been well documented. However, the molecular mechanism for the loss of cohesion in aged oocytes remains unknown. In this study, we found that intracellular pH (pHi) was elevated in aged oocytes, which might disturb the structure of the cohesin ring to induce aneuploidy. We observed for the first time that full-grown germinal vesicle (GV) oocytes displayed an increase in pHi with advancing age in CD1 mice. Furthermore, during the in vitro oocyte maturation process, the pHi was maintained at a high level, up to ∼7.6, in 12-month-old mice. Normal pHi is necessary to maintain protein localization and function. Thus, we put forward a hypothesis that the elevated oocyte pHi might be related to the loss of cohesion and the increased aneuploidy in aged mice. Through the in vitro alkalinization treatment of young oocytes, we observed that the increased pHi caused an increase in the aneuploidy rate and the sister inter-kinetochore (iKT) distance associated with the strength of cohesion and caused a decline in the cohesin subunit SMC3 protein level. Young oocytes with elevated pHi exhibited substantially the increase in chromosome misalignment.

  2. Tri-directional anaphases as a novel chromosome segregation defect in human oocytes.

    Science.gov (United States)

    Haverfield, Jenna; Dean, Nicola L; Nöel, Diana; Rémillard-Labrosse, Gaudeline; Paradis, Veronique; Kadoch, Isaac-Jacques; FitzHarris, Greg

    2017-06-01

    What are the chromosome segregation errors in human oocyte meiosis-I that may underlie oocyte aneuploidy? Multiple modes of chromosome segregation error were observed, including tri-directional anaphases, which we attribute to loss of bipolar spindle structure at anaphase-I. Oocyte aneuploidy is common and associated with infertility, but mechanistic information on the chromosome segregation errors underlying these defects is scarce. Lagging chromosomes were recently reported as a possible mechanism by which segregation errors occur. Long-term confocal imaging of chromosome dynamics in 50 human oocytes collected between January 2015 and May 2016. Germinal vesicle (GV) stage oocytes were collected from women undergoing intracytoplasmic sperm injection cycles and also CD1 mice. Oocytes were microinjected with complementary RNAs to label chromosomes, and in a subset of oocytes, the meiotic spindle. Oocytes were imaged live through meiosis-I using confocal microscopy. 3D image reconstruction was used to classify chromosome segregation phenotypes at anaphase-I. Segregation phenotypes were related to spindle dynamics and cell cycle timings. Most (87%) mouse oocytes segregated chromosomes with no obvious defects. We found that 20% of human oocytes segregated chromosomes bi-directionally with no lagging chromosomes. The rest were categorised as bi-directional anaphase with lagging chromosomes (20%), bi-directional anaphase with chromatin mass separation (34%) or tri-directional anaphase (26%). Segregation errors correlated with chromosome misalignment prior to anaphase. Spindles were tripolar when tri-directional anaphases occurred. Anaphase phenotypes did not correlate with meiosis-I duration (P = 0.73). Not applicable. Oocytes were recovered at GV stage after gonadotrophin-stimulation, and the usual oocyte quality caveats apply. Whilst the possibility that imaging may affect oocyte physiology cannot be formally excluded, detailed controls and justifications are presented

  3. Oocyte maturation in humans: the role of gonadotropins and growth factors.

    Science.gov (United States)

    Gómez, E; Tarín, J J; Pellicer, A

    1993-07-01

    To determine the effect of FSH/LH in vivo and epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in vitro on human oocyte maturation. Oocyte-cumulus complexes were harvested from three different groups of patients: [1] unstimulated ovaries from women undergoing surgery; [2] multifollicular development achieved with a combination of FSH and LH in the absence of an ovulatory dose of hCG; and [3] oocyte-cumulus complexes retrieved after appropriate ovarian stimulation with FSH/LH and hCG for IVF purposes. In vitro fertilization program and patients undergoing surgery for benign disorders at the Instituto Valenciano de Infertilidad, Valencia, Spain. Oocyte-cumulus complexes from unstimulated ovaries collected at surgery by follicular puncture and washing. Oocyte-cumulus complexes from stimulated cycles obtained by ultrasound-guided transvaginal aspiration. Oocyte-cumulus complexes cultured in vitro in the absence or presence of different concentrations of EGF and IGF-I. Germinal vesicle breakdown and metaphase-II stage after 24 and 48 hours. Comparison of the spontaneous resumption of meiosis and metaphase II oocytes among groups showed significant differences between unstimulated and stimulated ovaries after 24 and 48 hours in culture. Administration of hCG accelerated the percentage of maturation by 24 hours. Further incubation of unstimulated oocyte-cumulus complexes with EGF and IGF-I significantly increased the percentage of metaphase-II oocytes after 24 and 48 hours in culture. Epidermal growth factor and IGF-I are able to augment spontaneous maturaion in immature human oocytes. Because spontaneous maturation is mainly observed when follicles have been exposed to pharmacological doses of hMG, it is suggested that increasing FSH levels within the follicle is coincident with the generation of a positive signal necessary to complete oocyte maturation in humans. This signal may be linked to the dynamics of growth factors within the follicle itself.

  4. Different fates of oocytes with DNA double-strand breaks in vitro and in vivo.

    Science.gov (United States)

    Lin, Fei; Ma, Xue-Shan; Wang, Zhen-Bo; Wang, Zhong-Wei; Luo, Yi-Bo; Huang, Lin; Jiang, Zong-Zhe; Hu, Meng-Wen; Schatten, Heide; Sun, Qing-Yuan

    2014-01-01

    In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.

  5. Short-term preservation of porcine oocytes in ambient temperature: novel approaches.

    Directory of Open Access Journals (Sweden)

    Cai-Rong Yang

    Full Text Available The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs were preserved in TCM-199, porcine follicular fluid (pFF and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10% with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.

  6. G beta gamma signaling reduces intracellular cAMP to promote meiotic progression in mouse oocytes.

    Science.gov (United States)

    Gill, Arvind; Hammes, Stephen R

    2007-02-01

    In nearly every vertebrate species, elevated intracellular cAMP maintains oocytes in prophase I of meiosis. Prior to ovulation, gonadotropins trigger various intra-ovarian processes, including the breakdown of gap junctions, the activation of EGF receptors, and the secretion of steroids. These events in turn decrease intracellular cAMP levels in select oocytes to allow meiotic progression, or maturation, to resume. Studies suggest that cAMP levels are kept elevated in resting oocytes by constitutive G protein signaling, and that the drop in intracellular cAMP that accompanies maturation may be due in part to attenuation of this inhibitory G protein-mediated signaling. Interestingly, one of these G protein regulators of meiotic arrest is the Galpha(s) protein, which stimulates adenylyl cyclase to raise intracellular cAMP in two important animal models of oocyte development: Xenopus leavis frogs and mice. In addition to G(alpha)(s), constitutive Gbetagamma activity similarly stimulates adenylyl cyclase to raise cAMP and prevent maturation in Xenopus oocytes; however, the role of Gbetagamma in regulating meiosis in mouse oocytes has not been examined. Here we show that Gbetagamma does not contribute to the maintenance of murine oocyte meiotic arrest. In fact, contrary to observations in frog oocytes, Gbetagamma signaling in mouse oocytes reduces cAMP and promotes oocyte maturation, suggesting that Gbetagamma might in fact play a positive role in promoting oocyte maturation. These observations emphasize that, while many general concepts and components of meiotic regulation are conserved from frogs to mice, specific differences exist that may lead to important insights regarding ovarian development in vertebrates.

  7. Obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy.

    Science.gov (United States)

    Boudoures, Anna L; Saben, Jessica; Drury, Andrea; Scheaffer, Suzanne; Modi, Zeel; Zhang, Wendy; Moley, Kelle H

    2017-06-01

    Mitochondria are the most prominent organelle in the oocyte. Somatic cells maintain a healthy population of mitochondria by degrading damaged mitochondria via mitophagy, a specialized autophagy pathway. However, evidence from previous work investigating the more general macroautophagy pathway in oocytes suggests that mitophagy may not be active in the oocyte. This would leave the vast numbers of mitochondria - poised to be inherited by the offspring - vulnerable to damage. Here we test the hypothesis that inactive mitophagy in the oocyte underlies maternal transmission of dysfunctional mitochondria. To determine whether oocytes can complete mitophagy, we used either CCCP or AntimycinA to depolarize mitochondria and trigger mitophagy. After depolarization, we did not detect co-localization of mitochondria with autophagosomes and mitochondrial DNA copy number remained unchanged, indicating the non-functional mitochondrial population was not removed. To investigate the impact of an absence of mitophagy in oocytes with damaged mitochondria on offspring mitochondrial function, we utilized in vitro fertilization of high fat high sugar (HF/HS)-exposed oocytes, which have lower mitochondrial membrane potential and damaged mitochondria. Here, we demonstrate that blastocysts generated from HF/HS oocytes have decreased mitochondrial membrane potential, lower metabolites involved in ATP generation, and accumulation of PINK1, a mitophagy marker protein. This mitochondrial phenotype in the blastocyst mirrors the phenotype we show in HF/HS exposed oocytes. Taken together, these data suggest that the mechanisms governing oocyte mitophagy are fundamentally distinct from those governing somatic cell mitophagy and that the absence of mitophagy in the setting of HF/HS exposure contributes to the oocyte-to-blastocyst transmission of dysfunctional mitochondria. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Zona pellucida birefringence and meiotic spindle visualisation of human oocytes are not influenced by IVM technology.

    Science.gov (United States)

    Omidi, Marjan; Khalili, Mohammad Ali; Ashourzadeh, Sareh; Rahimipour, Marzieh

    2014-03-01

    The aim of the present study was to investigate the relationship between the presence of the meiotic spindle and zona pellucida (ZP) birefringence with morphology of in vivo- and in vitro-matured human oocytes. Germinal vesicles (n=47) and MI (n=38) oocytes obtained from stimulated ovaries of patients undergoing intracytoplasmic sperm injection (ICSI) underwent IVM. Using a PolScope (OCTAX PolarAID; Octax, Herbon, Germany), the presence of spindles and ZP birefringence was assessed in both in vivo-matured (n=56) and IVM (n=56) oocytes. In addition, the morphology of each matured oocyte was evaluated microscopically. There were insignificant differences for ZP birefringence and meiotic spindle between the in vivo-matured and IVM MII oocytes. Subanalysis revealed that the rates of morphologically abnormal oocytes did not differ significantly between the two groups, except in the case of irregular shape (P=0.001), refractile body (P=0.001) and fragmented polar body (P=0.03), which were higher in IVM oocytes. In the case of in vivo-matured oocytes, a significantly higher percentage of oocytes with intracytoplasmic and both intra- and extracytoplasmic abnormalities have a low birefringent ZP (P=0.007 and P=0.02, respectively). There was no relationship between morphological abnormalities and spindle detection. The findings suggest that clinical IVM is a safe technology that maintains the high maturation rate and integrity of oocytes. In addition, the use of the non-invasive PolScope is recommended for the detection of oocytes most suitable for ICSI.

  9. The generation of live offspring from vitrified oocytes.

    Directory of Open Access Journals (Sweden)

    L Gabriel Sanchez-Partida

    Full Text Available Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P<0.05. As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P<0.05. Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10. When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and

  10. Refractory ceramic fibers (RCFs) induce germline aneuploidy in Drosophila oocytes.

    Science.gov (United States)

    Osgood, C J

    1994-06-01

    Mineral fibers are ubiquitous in the natural environment and are widely used in industry in such applications as insulators. We have previously shown that asbestos fibers induce aneuploidy in oocytes of Drosophila melanogaster; here we extend those studies by testing refractory ceramic fibers (RCFs) for their mutagenicity to germ cells. The results establish that the tested RCFs are inducers of aneuploidy following feeding of adult females. A subset of the RCFs were also effective following larval feeding. Our results suggest that RCFs, like certain asbestos fibers, may pose a hazard to germ cells.

  11. Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

    Science.gov (United States)

    Li, H J; Sutton-McDowall, M L; Wang, X; Sugimura, S; Thompson, J G; Gilchrist, R B

    2016-04-01

    Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P benefits of c

  12. A western blot protocol for detection of proteins heterologously expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins...

  13. Cyclic AMP in oocytes controls meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary.

    Science.gov (United States)

    Wang, Yijing; Teng, Zhen; Li, Ge; Mu, Xinyi; Wang, Zhengpin; Feng, Lizhao; Niu, Wanbao; Huang, Kun; Xiang, Xi; Wang, Chao; Zhang, Hua; Xia, Guoliang

    2015-01-15

    In mammalian ovaries, a fixed population of primordial follicles forms during the perinatal stage and the oocytes contained within are arrested at the dictyate stage of meiotic prophase I. In the current study, we provide evidence that the level of cyclic AMP (cAMP) in oocytes regulates oocyte meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary. Our results show that the early meiotic development of oocytes is closely correlated with increased levels of intra-oocyte cAMP. Inhibiting cAMP synthesis in fetal ovaries delayed oocyte meiotic progression and inhibited the disassembly and degradation of synaptonemal complex protein 1. In addition, inhibiting cAMP synthesis in in vitro cultured fetal ovaries prevented primordial follicle formation. Finally, using an in situ oocyte chromosome analysis approach, we found that the dictyate arrest of oocytes is essential for primordial follicle formation under physiological conditions. Taken together, these results suggest a role for cAMP in early meiotic development and primordial follicle formation in the mouse ovary. © 2015. Published by The Company of Biologists Ltd.

  14. Fundamental aspects of bovine oocyte maturation : the role of estradiol, VIP and GHRH

    NARCIS (Netherlands)

    Beker van Woudenberg, A.R.C.L. (Anna Rita Costa Lage)

    2004-01-01

    Chapter 1 presents an overview on the aspects of oocyte maturation. Growth hormone (GH), released from the pituitary by the stimulus of GHRH, increases cumulus expansion and improves cytoplasmic maturation in bovine oocytes. GHRH is also expressed in extraneural tissues suggesting that GHRH also

  15. Evaluation of ribonucleic acid amplification protocols for human oocyte transcriptome analysis

    NARCIS (Netherlands)

    E. Mantikou; O. Bruning; S. Mastenbroek; S. Repping; T.M. Breit; M. de Jong

    2015-01-01

    OBJECTIVE: To develop a reliable, reproducible, and sensitive method for investigating gene-expression profiles from individual human oocytes. DESIGN: Five commercially available protocols were investigated for their efficiency to amplify messenger RNA (mRNA) from 54 single human oocytes. Protocols

  16. Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility.

    Science.gov (United States)

    Nicholas, Cory R; Haston, Kelly M; Grewall, Amarjeet K; Longacre, Teri A; Reijo Pera, Renee A

    2009-11-15

    Ten to 15% of couples are infertile, with the most common causes being linked to the production of few or no oocytes or sperm. Yet, our understanding of human germ cell development is poor, at least in part due to the inaccessibility of early stages to genetic and developmental studies. Embryonic stem cells (ESCs) provide an in vitro system to study oocyte development and potentially treat female infertility. However, most studies of ESC differentiation to oocytes have not documented fundamental properties of endogenous development, making it difficult to determine the physiologic relevance of differentiated germ cells. Here, we sought to establish fundamental parameters of oocyte development during ESC differentiation to explore suitability for basic developmental genetic applications using the mouse as a model prior to translating to the human system. We demonstrate a timeline of definitive germ cell differentiation from ESCs in vitro that initially parallels endogenous oocyte development in vivo by single-cell expression profiling and analysis of functional milestones including responsiveness to defined maturation media, shared genetic requirement of Dazl, and entry into meiosis. However, ESC-derived oocyte maturation ultimately fails in vitro. To overcome this obstacle, we transplant ESC-derived oocytes into an ovarian niche to direct their functional maturation and, thereby, present rigorous evidence of oocyte physiologic relevance and a potential therapeutic strategy for infertility.

  17. Dietary saccharides and sweet tastants have differential effects on colonization of Drosophila oocytes by Wolbachia endosymbionts

    Directory of Open Access Journals (Sweden)

    Moises Camacho

    2017-07-01

    Full Text Available Wolbachia bacteria are widespread, maternally transmitted endosymbionts of insects. Maintenance of sufficient Wolbachia titer in maternal germline cells is required for transmission efficacy. The mechanisms that regulate Wolbachia titer are not well understood; however, dietary sucrose was reported to elevate oocyte Wolbachia titer in Drosophila melanogaster whereas dietary yeast decreased oocyte titer. To further investigate how oocyte Wolbachia titer is controlled, this study analyzed the response of wMel Wolbachia to diets enriched in an array of natural sugars and other sweet tastants. Confocal imaging of D. melanogaster oocytes showed that food enriched in dietary galactose, lactose, maltose and trehalose elevated Wolbachia titer. However, oocyte Wolbachia titers were unaffected by exposure to the sweet tastants lactulose, erythritol, xylitol, aspartame and saccharin as compared to the control. Oocyte size was generally non-responsive to the nutrient-altered diets. Ovary size, however, was consistently smaller in response to all sugar- and sweetener-enriched diets. Furthermore, most dietary sugars administered in tandem with dietary yeast conferred complete rescue of oocyte titer suppression by yeast. All diets dually enriched in yeast and sugar also rescued yeast-associated ovary volume changes. This indicates oocyte colonization by Wolbachia to be a nutritionally sensitive process regulated by multiple mechanistic inputs.

  18. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes.

    Science.gov (United States)

    Pfender, Sybille; Kuznetsov, Vitaliy; Pasternak, Michał; Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-08-13

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals.

  19. Random Genetic Drift Determines the Level of Mutant mtDNA in Human Primary Oocytes

    OpenAIRE

    Brown, D T; Samuels, D C; Michael, E. M.; Turnbull, D.M.; Chinnery, P F

    2000-01-01

    We measured the proportion of mutant mtDNA (mutation load) in 82 primary oocytes from a woman who harbored the A3243G mtDNA mutation. The frequency distribution of mutation load indicates that random drift is the principal mechanism that determines the level of mutant mtDNA within individual oocytes.

  20. MITOCHONDRIAL DYNAMICS IN PRE- AND POSTPUBERTAL PIG OOCYTES BEFORE AND AFTER IN VITRO MATURATION

    DEFF Research Database (Denmark)

    Pedersen, H. S.; Løvendahl, P.; Nikolaisen, N. K.;

    2013-01-01

    Oocytes from prepubertal (PRE) or postpubertal (POST) pigs are used in, for example, somatic cell nuclear transfer and in vitro fertilization. Here we describe mitochondrial dynamics in pig oocytes of different sizes before and after in vitro maturation (IVM), isolated from PRE or POST animals...

  1. Folic Acid Deficiency Does Not Adversely Affect Oocyte Meiosis in Mice.

    Science.gov (United States)

    Tsuji, Ai; Noguchi, Rina; Nakamura, Toshinobu; Shibata, Katsumi

    2016-01-01

    Spindle defect and chromosome misalignment occuring in oocyte meiosis induce nondisjunction. Nondisjunction causes Down syndrome, also known as trisomy 21. Folic acid (FA) is an essential nutrient composition for fetal growth and development. It has been reported that FA nutritional status is associated with the risk of Down syndrome. However, to our knowledge, little is known about the effect of FA deficiency on abnormal oocytes (spindle defects, chromosome misalignments and immature oocyte) in vivo. In the present study, we investigate the effects of FA deficiency on oocyte meiosis in female mice. In order to induce FA deficiency in mice, female Crl:CD1 mice were fed a FA-free diet for 58 d. The diet also contained an antibiotic which has functions on limiting FA formation by intestinal microorganisms. The level of FA deficiency was determined by measuring the concentration of FA in the liver, hemocyte, uterus, ovary, and urine. FA concentrations in these samples from the FA-deficient group were 50-90% lower. Despite this, the frequency of abnormal oocytes was no different between the FA-deficient and control groups (20.0% vs 14.6%). According to the past research, FA transporter was strongly expressed in oocytes. Hence, it is possible that FA-free diets may not affect the concentration of oocyte FA in mice. To sum up these data, our study concluded that FA deficiency did not adversely affect oocyte meiosis.

  2. Exposure to HT-2 toxin causes oxidative stress induced apoptosis/autophagy in porcine oocytes

    Science.gov (United States)

    Zhang, Yue; Han, Jun; Zhu, Cheng-Cheng; Tang, Feng; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2016-01-01

    T-2 toxin is a main type A trichothecene mycotoxin which is the most toxic trichothecence. T-2 toxin has posed various toxic effects on human and animals in vigorous cell proliferation tissues like lymphoid, hematopoietic and gastrointestinal tissues, while HT-2 toxin is the major metabolite which is deacetylated by T-2 toxin. In this study, we focused on the toxic effects of HT-2 on porcine oocyte maturation. We treated the porcine oocyte with HT-2 toxin in vitro, and we first found that HT-2 treatment inhibited porcine oocyte polar body extrusion and cumulus cell expansion. We observed the disrupted meiotic spindle morphology after treatment, which might be due to the reduced p-MAPK protein level. Actin distribution was also disturbed, indicating that HT-2 affects cytoskeleton of porcine oocytes. We next explored the causes for the failure of oocyte maturation after HT-2 treatment. We found that HT-2 treated oocytes showed the increased ROS level, which indicated that oxidative stress had occurred. We also detected autophagy as well as early apoptosis in the treatment oocytes. Due to the fact that oxidative stress could induced apoptosis, our results indicated that HT-2 toxin caused oxidative stress induced apoptosis and autophagy, which further affected porcine oocyte maturation. PMID:27658477

  3. Why is chromosome segregation error in oocytes increased with maternal aging?

    Science.gov (United States)

    Wang, Zhen-Bo; Schatten, Heide; Sun, Qing-Yuan

    2011-10-01

    It is well documented that female fertility is decreased with advanced maternal age due to chromosome abnormality in oocytes. Increased chromosome missegregation is mainly caused by centromeric cohesion reduction. Other factors such as weakened homologous recombination, improper spindle organization, spindle assembly checkpoint (SAC) malfunction, chromatin epigenetic changes, and extra-oocyte factors may also cause chromosome errors.

  4. High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival

    DEFF Research Database (Denmark)

    Dupont, Yoko; Lin, Lin; Schmidt, Mette

    2008-01-01

    and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups...

  5. Application of oocyte cryopreservation technology in TALEN-mediated mouse genome editing.

    Science.gov (United States)

    Nakagawa, Yoshiko; Sakuma, Tetsushi; Nakagata, Naomi; Yamasaki, Sho; Takeda, Naoki; Ohmuraya, Masaki; Yamamoto, Takashi

    2014-01-01

    Reproductive engineering techniques, such as in vitro fertilization (IVF) and cryopreservation of embryos or spermatozoa, are essential for preservation, reproduction, and transportation of genetically engineered mice. However, it has not yet been elucidated whether these techniques can be applied for the generation of genome-edited mice using engineered nucleases such as transcription activator-like effector nucleases (TALENs). Here, we demonstrate the usefulness of frozen oocytes fertilized in vitro using frozen sperm for TALEN-mediated genome editing in mice. We examined side-by-side comparisons concerning sperm (fresh vs. frozen), fertilization method (mating vs. IVF), and fertilized oocytes (fresh vs. frozen) for the source of oocytes used for TALEN injection; we found that fertilized oocytes created under all tested conditions were applicable for TALEN-mediated mutagenesis. In addition, we investigated whether the ages in weeks of parental female mice can affect the efficiency of gene modification, by comparing 5-week-old and 8-12-week-old mice as the source of oocytes used for TALEN injection. The genome editing efficiency of an endogenous gene was consistently 95-100% when either 5-week-old or 8-12-week-old mice were used with or without freezing the oocytes. Thus, our report describes the availability of freeze-thawed oocytes and oocytes from female mice at various weeks of age for TALEN-mediated genome editing, thus boosting the convenience of such innovative gene targeting strategies.

  6. Expression of rat liver cell membrane transporters for thyroid hormone in Xenopus laevis oocytes

    NARCIS (Netherlands)

    R. Docter (Roel); E.C.H. Friesema (Edith); P.G.J. van Stralen (Paul); E.P. Krenning (Eric); M.E. Everts (Maria); T.J. Visser (Theo); G. Hennemann

    1997-01-01

    textabstractThe present study was conducted to explore the possible use of Xenopus laevis oocytes for the expression cloning of cell membrane transporters for iodothyronines. Injection of stage V-VI X. laevis oocytes with 23 ng Wistar rat liver polyadenylated RNA (mRNA)

  7. Obesity modulates inflammation and lipid metabolism oocyte gene expression: A single cell transcriptome perspective

    Science.gov (United States)

    This study aimed to compare oocyte gene expression profiles and follicular fluid (FF) content from overweight/obese (OW) women and normal weight (NW) women who were undergoing fertility treatments. Using single cell transcriptomic analyses, we investigated oocyte gene expression using RNA-seq. Serum...

  8. TACC3 Is Important for Correct Progression of Meiosis in Bovine Oocytes

    NARCIS (Netherlands)

    Mahdipour, Mahdi; Leitoguinho, Ana Rita Canhoto; Zacarias Silva, Ricardo A; van Tol, Helena T A; Stout, Tom A E; Rodrigues, Gabriela; Roelen, Bernard A J

    2015-01-01

    Transforming acidic coiled-coil (TACC) proteins are key players during mitosis via stabilization of the spindle. The roles of TACCs during meiosis are however less clear. We used bovine oocytes to study the expression and function of TACC3 during meiosis. TACC3 mRNA was detected in bovine oocytes du

  9. Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes.

    Science.gov (United States)

    Sun, Xing-Shen; Liu, Yong; Yue, Kui-Zhong; Ma, Suo-Feng; Tan, Jing-He

    2004-10-01

    Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes were studied using a new method that allows a clearer visualization of both nucleolus and chromatin after Hoechst staining. The GV chromatin of porcine oocytes was classified into five configurations, based on the degree of chromatin condensation, and on nucleolus and nuclear membrane disappearance. While the GV1 to 4 configurations were similar to those reported by previous studies, the GV0 configuration was distinct by the diffuse, filamentous pattern of chromatin in the whole nuclear area. Most of the oocytes were at the GV0 stage in the layers of cumulus cells and those with less than one layer or no cumulus cells. Overall, our results suggested that (i) the GV0 configuration in porcine oocytes corresponded to the "nonsurrounded nucleolus" pattern in mice and other species; (ii) all the oocytes were synchronized at the GV1 stage before GVBD and this pattern might, therefore, represent a nonatretic state; (iii) the GV3 and GV4 configurations might represent stages toward atresia, or transient events prior to GVBD that could be switched toward either ovulation or atresia, depending upon circumstances; (iv) the in vitro systems currently used were not favorable for oocytes to switch toward ovulation (or final maturation); (v) the number of cumulus cells was not correlated with the chromatin configuration of oocytes, indicating that the beneficial effect of cumulus cells on oocyte maturation and development may simply be attributed to their presence during in vitro culture.

  10. Maternal death after oocyte donation at high maternal age: Case report

    NARCIS (Netherlands)

    J.M. Schutte (Joke); N.W.E. Schuitemaker (Nico); E.A.P. Steegers (Eric); J. van Roosmalen (Jos)

    2008-01-01

    textabstractBackground. The percentage of women giving birth after the age of 35 increased in many western countries. The number of women remaining childless also increased, mostly due to aging oocytes. The method of oocyte donation offers the possibility for infertile older women to become

  11. Maternal death after oocyte donation at high maternal age: Case report

    NARCIS (Netherlands)

    J.M. Schutte (Joke); N.W.E. Schuitemaker (Nico); E.A.P. Steegers (Eric); J. van Roosmalen (Jos)

    2008-01-01

    textabstractBackground. The percentage of women giving birth after the age of 35 increased in many western countries. The number of women remaining childless also increased, mostly due to aging oocytes. The method of oocyte donation offers the possibility for infertile older women to become pregnant

  12. A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

    Science.gov (United States)

    Santiquet, Nicolas W; Greene, Alison F; Becker, John; Barfield, Jennifer P; Schoolcraft, William B; Krisher, Rebecca L

    2017-09-01

    Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following

  13. Supplementation of l-carnitine during in vitro maturation improves embryo development from less competent bovine oocytes.

    Science.gov (United States)

    Knitlova, Drahomira; Hulinska, Pavlina; Jeseta, Michal; Hanzalova, Katerina; Kempisty, Bartosz; Machatkova, Marie

    2017-10-15

    The present study was designed to define the impact of l-carnitine, supplemented during maturation, on bovine oocytes with different meiotic competence in terms of their IVF outcomes. Meiotically more competent (MMC) and less competent (MLC) oocytes were obtained separately from differently sized follicles at selected phases of folliculogenesis. The oocytes were matured with or without l-carnitine, fertilized and cultured to the blastocyst stage. The oocytes were examined for nuclear maturation, mitochondrial cluster formation, lipid consumption, fertilization and embryo development. The proportion of oocytes at metaphase II was significantly higher in the l-carnitine-treated MMC than that in the l-carnitine-treated MLC oocytes. However in comparison with the untreated controls, the proportion of MII oocytes with mitochondrial clusters was significantly higher only in the l-carnitine-treated MLC oocytes, which also showed a significantly lower mean lipid content. The l-carnitine-treated MLC oocytes showed significantly higher fertilization and syngamy rates than the untreated MLC oocytes. On the other hand, in the l-carnitine-treated MMC oocytes, the fertilization rate was similar to that of the untreated controls and the syngamy rate was significantly delayed. Although no significant differences in cleavage on Day 2 were found among all oocyte categories, l-carnitine treatment resulted in a significantly higher blastocyst yield in the MLC oocytes on Day 7 and Day 8 and a significantly higher proportion of expanded blastocysts in relation to the total number of blastocysts in MMC oocytes on Day 8. It can be concluded that l-carnitine supplementation during maturation improves the development of bovine embryos from meiotically less competent oocytes and accelerates blastocyst formation from more competent oocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Sequential Analysis of Global Gene Expression Profiles in Immature and In vitro Matured Bovine Oocytes: Potential Molecular Markers of Oocyte Maturation

    LENUS (Irish Health Repository)

    Mamo, Solomon

    2011-03-16

    Abstract Background Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results 8489 transcripts were detected across the two oocyte groups, of which ~25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of α-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource

  15. Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrification

    DEFF Research Database (Denmark)

    Morató, Roser; Mogas, Teresa; Maddox-Hyttel, Poul

    2008-01-01

    Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups...... for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 microM Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 microM Taxol to the vitrification solution. All...... in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well...

  16. The role of Fyn kinase in the release from metaphase in mammalian oocytes.

    Science.gov (United States)

    Levi, M; Shalgi, R

    2010-01-27

    Meiosis in mammalian oocytes starts during embryonic life and arrests for the first time before birth, at prophase of the first meiotic division. The second meiotic arrest occurs after spindle formation at metaphase of the second meiotic division (MII) in selected oocytes designated for ovulation. The fertilizing spermatozoon induces the release from MII arrest only after the oocyte's spindle assembly checkpoint (SAC) was deactivated. Src family kinases (SFKs) are nine non-receptor protein tyrosine kinases that regulate many key cellular functions. Fyn is an SFK expressed in many cell types, including oocytes. Recent studies, including ours, imply a role for Fyn in exit from meiotic and mitotic metaphases. Other studies demonstrate that SFKs, particularly Fyn, are required for regulation of microtubules polymerization and spindle stabilization. Altogether, Fyn is suggested to play an essential role in signaling events that implicate SAC pathway and hence in regulating the exit from metaphase in oocytes and zygote. 2009 Elsevier Ireland Ltd. All rights reserved.

  17. Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling.

    Science.gov (United States)

    Navarro-Costa, Paulo; McCarthy, Alicia; Prudêncio, Pedro; Greer, Christina; Guilgur, Leonardo G; Becker, Jörg D; Secombe, Julie; Rangan, Prashanth; Martinho, Rui G

    2016-08-10

    Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I.

  18. Collection of superovulated mouse oocytes continuously by surgery and their development after activation

    Institute of Scientific and Technical Information of China (English)

    Wang Min-kang; Zhang Tian

    2005-01-01

    Objective: To establish a new way to collect superovulated oocytes or zygotes repeatedly from an individual mouse. Methods: Superovulations were induced by injection PMSG and hCG in Kunming strain mice. The ampullaes of oviduct of all anaesthetised mouse were put in a specially designed "U" sink and released. The second and third times of PMSG injection were made on the sixth day and eleventh day after the first superovulation injection. The capacity of development was examined by in vitro culture of parthenogenesis activation oocytes. Results: Development to blastocyst stage was not significantly different between the first and second time collection. The percentage of blastocyst stage in CD and Sr++ treatment was significantly higher (P<0.05) than the oocytes treated in CB and Sr++. Conclusion: This method enables us to collect oocytes or zygotes repeatedly from one individual mouse in an interval as short as 5 days and without influence on the quality of oocytes.

  19. The potential use of maturation in vitro of human oocytes in low responder patients.

    Science.gov (United States)

    Requena, A; Neuspiller, F; Cobo, A C; Aragonés, M; Remohí, J; Simón, C; Pellicer, A

    2000-05-01

    To assess whether maturation in vitro of human oocytes (MIVHO) could be an alternative treatment in low responders to ovarian stimulation for in vitro fertilization (IVF). Prospective case-control study. Spontaneously ovulatory women who volunteered were included in our program of MIVHO at the Instituto Valenciano de Infertilidad. Rates of oocyte retrieval, in vitro maturation, fertilization, and development up to the blastocyst stage were studied. A significantly increased rate of oocyte retrieval was found when the pickup was performed before follicular selection. No differences were found when MIVHO was used in a low responder patient with an ovarian content of early antral follicles > 5 as compared to normal responders. MIVHO could be a successful choice in low responder patients with an acceptable number of early antral follicles. Oocyte retrieval should be performed before follicular selection in order to obtain more oocytes.

  20. Structure of the insect oocyte nucleus with special reference to interchromatin granule clusters and cajal bodies.

    Science.gov (United States)

    Bogolyubov, Dmitry; Parfenov, Vladimir

    2008-01-01

    The nuclear structure of insect oocyte is described with reference to interchromatin granule clusters and Cajal bodies. It is suggested that the intranuclear structure is determined by the ovary organization and reflects the sources of oocyte RNA. Inactivated oocyte chromosomes in meroistic ovaries form a karyosphere, and their features are discussed. In insects with panoistic ovaries, the organization and molecular composition of complicated oocyte Cajal bodies are discussed, and the homology between a structure located inside Cajal bodies and interchromatin granule clusters is proved. Along with revealing some essential Cajal body components and factors involved in RNA polymerase II transcription, we analyze the absence of RNAP II itself from oocyte Cajal bodies of Acheta. In insects with meroistic ovaries, Cajal bodies of Drosophila and homology of Panorpa nuclear bodies to Cajal bodies and their peculiarities are described, because the nuclear bodies may share some features of both Cajal bodies and interchromatin granule clusters to represent a single nuclear domain.

  1. Localization of RNA transcription sites in insect oocytes using microinjections of 5-bromouridine 5'-triphosphate.

    Directory of Open Access Journals (Sweden)

    Dmitry Bogolyubov

    2007-06-01

    Full Text Available In the present study we used 5-bromouridine 5'-triphosphate (BrUTP microinjections to localize the transcription sites in oocytes of insects with different types of the ovarium structure: panoistic, meroistic polytrophic, and meroistic telotrophic. We found that in an insect with panoistic ovaries (Acheta domesticus, oocyte nuclei maintain their transcription activity during the long period of oocyte growth. In insects with meroistic ovaries (Tenebrio molitor and Panorpa communis, early oocyte chromosomes were found to be transcriptionally active, and some transcription activity still persist while the karyosphere, a compact structure formed by all condensed oocyte chromosomes, begins to develop. At the latest stages of karyosphere development, no anti-Br-RNA signal was registered in the karyosphere.

  2. Neural image analysis in the process of quality assessment: domestic pig oocytes

    Science.gov (United States)

    Boniecki, P.; Przybył, J.; Kuzimska, T.; Mueller, W.; Raba, B.; Lewicki, A.; Przybył, K.; Zaborowicz, M.; Koszela, K.

    2014-04-01

    The questions related to quality classification of animal oocytes are explored by numerous scientific and research centres. This research is important, particularly in the context of improving the breeding value of farm animals. The methods leading to the stimulation of normal development of a larger number of fertilised animal oocytes in extracorporeal conditions are of special importance. Growing interest in the techniques of supported reproduction resulted in searching for new, increasingly effective methods for quality assessment of mammalian gametes and embryos. Progress in the production of in vitro animal embryos in fact depends on proper classification of obtained oocytes. The aim of this paper was the development of an original method for quality assessment of oocytes, performed on the basis of their graphical presentation in the form of microscopic digital images. The classification process was implemented on the basis of the information coded in the form of microphotographic pictures of the oocytes of domestic pig, using the modern methods of neural image analysis.

  3. Low temperature induces oocytes p34cdc2 synthesis and accumulation—the acquisition of competence to resume meiosis in toad oocytes

    Institute of Scientific and Technical Information of China (English)

    LUJINING; ZHENGGU; 等

    1996-01-01

    Full grown oocytes derived from Bufo Bufo gargarizans rearing at high temperature environment (24℃), never underwent GVBD after progesterone treatment.No p34cdc2 Hl kinase activity was detected in the oocytes after progesterone stimulation or OA microinjection;Western blotting analysis showed that the level of p34cdc2 and p33 in the oocytes are significantly lower than those in the oocytes derived from the hibernating toads (below 10℃).35S-Met incorporation analysis showed that when the oocytes were incubated at 6℃,synthesis of about thirty defferent polypeptides was promoted or induced,including p34cdc2 and some other p13suc1-binding proteins.All these results indicated that a low temperature environment is essential for the oocytes of Bufo Bufo gargarizans to express and stord some cell cycle drivers and its regulators,and to gain the maturation competence.These results will also provide a nwe clue for explaining the molecular mechanisms why gametogenesis of some organisms depends on a relative low temperature and how to maintain the geographical distribution of some animals.

  4. Laser researches on livestock semen and oocytes: A brief review

    Directory of Open Access Journals (Sweden)

    Z. Abdel-Salam

    2015-05-01

    Full Text Available This article presents a brief review of the past and present literature pertinent to laser effects on sperm motility parameters, improvement of oocyte maturation and characterization of semen in livestock. The aim was, on one hand, to make the readers aware of such knowledge and on the other hand to trigger the interest of the animal reproduction scientific community in attempting some laser techniques that have not yet been fully exploited in the field of artificial insemination. With respect to the conventional methods, laser is a more sensitive and less costly technology that can be used for improving artificial insemination and embryo production system. Since 1980s, laser treatment came on the biological samples scene; its applications have continuously been developed thereafter. Exploitation of laser light by various researchers for improving the reproductive efficiency of sperm cells and the maturation rate in different livestock is demonstrated herein. Laser irradiation, in principal, can increase the production of adenosine triphosphate (ATP and consequently increases the energy provided to the cell. Since sperm motility and oocyte maturation depend on the energy consumption, an increase in the energy supply to the cells will be of great importance. In addition, the authors also discuss the use of laser spectrochemical analytical techniques, such as laser induced breakdown spectroscopy (LIBS and laser induced fluorescence (LIF, in characterization of semen samples.

  5. Zoroastrians Support Oocyte and Embryo Donation Program for Infertile Couples

    Science.gov (United States)

    Halvaei, Iman; Khalili, Mohammad Ali; Ghasemi-Esmailabad, Saeed; Nabi, Ali; Shamsi, Farimah

    2014-01-01

    Background The main goal was to evaluate the attitudes and knowledge of Zoroastrians living in Iran towards oocyte donation (OD) and embryo donation (ED) program. Methods This cross sectional study consisted of 318 Zoroastrians (n=175 for OD and n=143 for ED) of both sexes. The questionnaire form comprised two parts of general demographic characteristics of the participants and twenty multiple-choice questions about attitude and knowledge of participants towards OD and ED. For statistical analysis, the chi-square test was applied for comparison of data generated from ED and OD groups. Results Majority of the participants supported OD (69.7%) and ED (71.3%) for infertile patients. In addition, 40% and 42% preferred donation program (OD and ED, respectively), compared to adoption. About 60% of the respondents believed that the donors have no right to find the child and claim it as their own. In addition, more than half of the respondents thought that the recipients of oocyte/embryo should never know the name and address of the donors. More than half of the participants did not know whether their religion accepts donation program or not. Approximately, 80% of respondents supported psychological counseling for both donors and recipients. Moreover, about 56% of the participants necessitated the advertisement on OD/ED program in the mass media. Conclusion Our preliminary data showed that Zoroastrians supported both OD and ED program equally for infertile couples. PMID:25473631

  6. Donor type and parental disclosure following oocyte donation

    Institute of Scientific and Technical Information of China (English)

    John Stephenson; Eric Blyth; Wendy Kramer; Jennifer Schneider

    2012-01-01

    Objective:To ascertain the perspectives of parents of children conceived via oocyte donation regarding donor anonymity and disclosure of the nature of their conception to their children. Methods:Information was gathered by means of an anonymous online survey initiated by theDonorSiblingRegistry, in which108 parents with143 children conceived following oocyte donation and aged between one year and15 yearsparticipated.Results:Parental use of an anonymous or open-identity donor-and regardless of parental choice of donor-makes very little difference to the timing of parental disclosure to their donor-conceived child about their conception.The median age of children at disclosure is about3.5years;UK/Australian parents seem more ready to tell their children at an early stage(median age around two years) thanNorth American parents(median age around4.5 years), although about three quarters of all children have been told by the age of six years.Considerable ambiguity among parents who intend to disclose to their children as to the optimal age of disclosure is evidenced.Conclusions:Parents’ experiences of disclosure to children at different ages need to be more thoroughly examined in order to establisha coherent body of knowledge that may facilitate improved evidence-based parental decision making.

  7. Biotin-binding proteins and biotin transport to oocytes.

    Science.gov (United States)

    White, H B

    1985-01-01

    The eggs of chickens and other birds contain two proteins that bind biotin. Both are homotetrameric proteins of similar size. In contrast to the well-characterized egg white avidin, egg yolk biotin-binding protein has a very acidic isoelectric point, binds biotin with lower affinity, and is usually saturated with biotin. Like other egg yolk proteins, biotin-binding protein appears to be synthesized in the liver, transported by the blood stream to the ovary and deposited in the developing oocyte. Since the yolk of a chicken egg contains over 90% of the biotin in an egg and all of the biotin is bound to biotin-binding protein, the function of biotin-binding protein is undoubtedly to transport biotin to the egg for future use by the developing embryo. Avidin is produced by the oviduct and in the egg it is presumed to deter microbial growth around the oocyte by sequestering biotin. Among the eggs examined, those from turkeys have the lowest amount of biotin-binding protein and the highest amount of avidin. Furthermore, the majority of the biotin in turkey eggs can be bound to avidin in the egg white, suggesting a nutritional role for avidin in turkeys. An assay has been developed to conveniently measure apo- and holobiotin-binding proteins.

  8. Somatic cells initiate primordial follicle activation and govern the development of dormant oocytes in mice.

    Science.gov (United States)

    Zhang, Hua; Risal, Sanjiv; Gorre, Nagaraju; Busayavalasa, Kiran; Li, Xin; Shen, Yan; Bosbach, Benedikt; Brännström, Mats; Liu, Kui

    2014-11-03

    The majority of oocytes in the mammalian ovary are dormant oocytes that are enclosed in primordial follicles by several somatic cells, which we refer to as primordial follicle granulosa cells (pfGCs). Very little is known, however, about how the pfGCs control the activation of primordial follicles and the developmental fates of dormant oocytes. By targeting molecules in pfGCs with several mutant mouse models, we demonstrate that the somatic pfGCs initiate the activation of primordial follicles and govern the quiescence or awakening of dormant oocytes. Inhibition of mTORC1 signaling in pfGCs prevents the differentiation of pfGCs into granulosa cells, and this arrests the dormant oocytes in their quiescent states, leading to oocyte death. Overactivation of mTORC1 signaling in pfGCs accelerates the differentiation of pfGCs into granulosa cells and causes premature activation of all dormant oocytes and primordial follicles. We further show that pfGCs trigger the awakening of dormant oocytes through KIT ligand (KITL), and we present an essential communication network between the somatic cells and germ cells that is based on signaling between the mTORC1-KITL cascade in pfGCs and KIT-PI3K signaling in oocytes. Our findings provide a relatively complete picture of how mammalian primordial follicles are activated. The microenvironment surrounding primordial follicles can activate mTORC1-KITL signaling in pfGCs, and these cells trigger the awakening of dormant oocytes and complete the process of follicular activation. Such communication between the microenvironment, somatic cells, and germ cells is essential to maintaining the proper reproductive lifespan in mammals. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Age-associated metabolic and morphologic changes in mitochondria of individual mouse and hamster oocytes.

    Directory of Open Access Journals (Sweden)

    Fatma Simsek-Duran

    Full Text Available BACKGROUND: In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus and mouse (Mus musculus ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. METHODOLOGY/PRINCIPAL FINDINGS: Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. CONCLUSIONS/SIGNIFICANCE: In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae.

  10. Molecular and Cellular Mechanisms of Sperm-Oocyte Interactions Opinions Relative to in Vitro Fertilization (IVF

    Directory of Open Access Journals (Sweden)

    George Anifandis

    2014-07-01

    Full Text Available One of the biggest prerequisites for pregnancy is the fertilization step, where a human haploid spermatozoon interacts and penetrates one haploid oocyte in order to produce the diploid zygote. Although fertilization is defined by the presence of two pronuclei and the extraction of the second polar body the process itself requires preparation of both gametes for fertilization to take place at a specific time. These preparations include a number of consecutive biochemical and molecular events with the help of specific molecules and with the consequential interaction between the two gametes. These events take place at three different levels and in a precise order, where the moving spermatozoon penetrates (a the outer vestments of the oocyte, known as the cumulus cell layer; (b the zona pellucida (ZP; where exocytosis of the acrosome contents take place and (c direct interaction of the spermatozoon with the plasma membrane of the oocyte, which involves a firm adhesion of the head of the spermatozoon with the oocyte plasma membrane that culminates with the fusion of both sperm and oocyte membranes (Part I. After the above interactions, a cascade of molecular signal transductions is initiated which results in oocyte activation. Soon after the entry of the first spermatozoon into the oocyte and oocyte activation, the oocyte’s coat (the ZP and the oocyte’s plasma membrane seem to change quickly in order to initiate a fast block to a second spermatozoon (Part II. Sometimes, two spermatozoa fuse with one oocyte, an incidence of 1%–2%, resulting in polyploid fetuses that account for up to 10%–20% of spontaneously aborted human conceptuses. The present review aims to focus on the first part of the human sperm and oocyte interactions, emphasizing the latest molecular and cellular mechanisms controlling this process.

  11. IVF for premature ovarian failure: first reported births using oocytes donated from a twin sister

    Directory of Open Access Journals (Sweden)

    Sills Eric

    2010-03-01

    Full Text Available Abstract Background Premature ovarian failure (POF remains a clinically challenging entity because in vitro fertilisation (IVF with donor oocytes is currently the only treatment known to be effective. Methods A 33 year-old nulligravid patient with a normal karyotype was diagnosed with POF; she had a history of failed fertility treatments and had an elevated serum FSH (42 mIU/ml. Oocytes donated by her dizygotic twin sister were used for IVF. The donor had already completed a successful pregnancy herself and subsequently produced a total of 10 oocytes after a combined FSH/LH superovulation regime. These eggs were fertilised with sperm from the recipient's husband via intracytoplasmic injection and two fresh embryos were transferred to the recipient on day three. Results A healthy twin pregnancy resulted from IVF; two boys were delivered by caesarean section at 39 weeks' gestation. Additionally, four embryos were cryopreserved for the recipient's future use. The sister-donor achieved another natural pregnancy six months after oocyte retrieval, resulting in a healthy singleton delivery. Conclusion POF is believed to affect approximately 1% of reproductive age females, and POF patients with a sister who can be an oocyte donor for IVF are rare. Most such IVF patients will conceive from treatment using oocytes from an anonymous oocyte donor. This is the first report of births following sister-donor oocyte IVF in Ireland. Indeed, while sister-donor IVF has been successfully undertaken by IVF units elsewhere, this is the only known case where oocyte donation involved twin sisters. As with all types of donor gamete therapy, pre-treatment counselling is important in the circumstance of sister oocyte donation.

  12. Effect of high fat diet on artificial oocyte activation following superovulation in mice.

    Science.gov (United States)

    Yamamoto, Daisuke; Yasui, Toshiyuki; Kobayashi, Chika; Kitazato, Takane; Iwasa, Takeshi; Irahara, Minoru

    2016-04-01

    The aim of the present study was to determine the effects of increased dietary intake and high fat diet (HFD) in mice on artificial oocyte activation by using puromycin or roscovitine. Six-week-old mice were fed as either a control diet group, an increased dietary intake group or an HFD group for 4 weeks. Oocytes were obtained following superovulation and were divided into three treatment groups (no activation treatment, calcium ionophore and puromycin treatment, and calcium ionophore and roscovitine treatment) and were incubated for 4 h. Retrieved oocytes and numbers of oocytes activated as assessed by morphological changes were compared among the three treatment groups. The proportion of degenerated oocytes in HFD mice was significantly higher than that in control diet mice. The rates of activation in oocytes treated with roscovitine were 90.3% in control diet mice, 89.8% in increased dietary intake mice and 67.9% in HFD mice. The rate of activation in oocytes treated with roscovitine in HFD mice was significantly lower than the rates in control diet mice and increased dietary intake mice. The rates of activation in oocytes treated with puromycin were 90.6% in control diet mice, 94.0% in increased dietary intake mice and 71.4% in HFD mice, and the rate of activation in oocytes treated with puromycin in HFD mice was significantly lower than the rates in control diet mice and increased dietary intake mice. HFD-induced obesity deteriorated induction of oocyte activation by roscovitine or puromycin in mice.

  13. Effects of mineral supplements on ovulation and maturation of dog oocytes.

    Science.gov (United States)

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Park, Eun Jung; Jang, Goo; Lee, Byeong Chun

    2012-07-01

    The aim of this study was to assess the effects of trace mineral supplements near the time of ovulation on the number of ovulated oocytes, in vivo oocyte maturation and pregnancy for dog cloning. Sixteen oocyte donor dogs were used in each control and mineral supplement group, and 136 and 166 corpora lutea were counted from each group. No significant difference was observed between oocyte recovery rates in the control (91.2 ± 2.7%) and mineral (89.9 ± 2.7) groups. Proportions of mature (86.2 ± 7.2 and 88.4 ± 6.8%) and aged (13.8 ± 7.2 and 11.6 ± 6.8%) oocytes were not different in the control and mineral groups, respectively. Oocytes with fair (91.5 ± 3.6 and 93.6 ± 2.1%) and poor (8.5 ± 3.6 and 6.4 ± 2.1%) quality also showed no difference between the control and mineral groups. The concentrations of manganese and ferrous iron were higher and lower on the day of ovulation, respectively, in both groups, but trace element concentrations in peripheral blood were not affected by mineral treatment. Oocytes were used to make cloned embryos; after embryo transfer, four and two pups were delivered from the control and mineral group, respectively, but there was no difference in the delivery rate (4.6 and 2.7%). In conclusion, intravenous mineral supplements administered once close to the LH surge in oocyte donor dogs and recipients had no effect on the number of ovulated oocytes, in vivo oocyte maturation or pregnancy in dog cloning in this study. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. IVF for premature ovarian failure: first reported births using oocytes donated from a twin sister.

    LENUS (Irish Health Repository)

    Sills, Eric Scott

    2010-01-01

    BACKGROUND: Premature ovarian failure (POF) remains a clinically challenging entity because in vitro fertilisation (IVF) with donor oocytes is currently the only treatment known to be effective. METHODS: A 33 year-old nulligravid patient with a normal karyotype was diagnosed with POF; she had a history of failed fertility treatments and had an elevated serum FSH (42 mIU\\/ml). Oocytes donated by her dizygotic twin sister were used for IVF. The donor had already completed a successful pregnancy herself and subsequently produced a total of 10 oocytes after a combined FSH\\/LH superovulation regime. These eggs were fertilised with sperm from the recipient\\'s husband via intracytoplasmic injection and two fresh embryos were transferred to the recipient on day three. RESULTS: A healthy twin pregnancy resulted from IVF; two boys were delivered by caesarean section at 39 weeks\\' gestation. Additionally, four embryos were cryopreserved for the recipient\\'s future use. The sister-donor achieved another natural pregnancy six months after oocyte retrieval, resulting in a healthy singleton delivery. CONCLUSION: POF is believed to affect approximately 1% of reproductive age females, and POF patients with a sister who can be an oocyte donor for IVF are rare. Most such IVF patients will conceive from treatment using oocytes from an anonymous oocyte donor. This is the first report of births following sister-donor oocyte IVF in Ireland. Indeed, while sister-donor IVF has been successfully undertaken by IVF units elsewhere, this is the only known case where oocyte donation involved twin sisters. As with all types of donor gamete therapy, pre-treatment counselling is important in the circumstance of sister oocyte donation.

  15. Perioperative and post-operative complications of transvaginal ultrasound-guided oocyte retrieval: prospective study of >1000 oocyte retrievals.

    Science.gov (United States)

    Ludwig, A K; Glawatz, M; Griesinger, G; Diedrich, K; Ludwig, M

    2006-12-01

    Although transvaginal ultrasound-guided oocyte retrievals (OR) are performed routinely worldwide, there is very little systematic data about its complications. We performed a prospective cohort study following the perioperative and post-operative complications of over 1058 ORs. Additionally, we assessed the pain experienced during the OR. A total of 1166 OR were performed during the study period, of which 1058 (90.7%) ORs were included prospectively. Incomplete data meant that the remaining 9.3% were excluded. No complications were caused by sedation or general anaesthesia. Vaginal bleeding was observed in 2.8% of procedures, without any cases of intra-abdominal bleeding. An injury of pelvic structures (a ureteral lesion) occurred in one case. No case of pelvic infection, but one case of unexplained fever, was observed. A severe ovarian hyperstimulation syndrome (OHSS) occurred in 2.7% of cases. Although most patients tolerated the OR well, 3% of patients experienced severe to very severe pain after the OR and 2% of patients were still suffering from severe pain 2 days after the procedure. The pain level increased with the number of oocytes retrieved. About 0.7% of patients required hospitalization for pain treatment. Patients can be reassured that overall OR is a safe procedure. However, patients have to be counselled about the minor risks of the OR. The literature on complications is reviewed in the article.

  16. Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model

    Institute of Scientific and Technical Information of China (English)

    Yan-Guang Wu; Yong Liu; Ping Zhou; Guo-Cheng Lan; Dong Han; De-Qiang Miao; Jing-He Tan

    2007-01-01

    Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue (BCB) staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained (BCB+) and those unstained (BCB-) according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB+ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotro-pin stimulation on the competence of BCB+ oocytes were also analyzed. In the large- and medium-size groups, BCB+ oocytes were larger and showed more surrounded nucleoli (SN) chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity (determined as the intracellular GSH level and pattern of mitochondrial distribution) and higher developmental potential after in vitro maturation (IVM) than the BCB- oocytes. Adult mice produced more BCB+ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB+ oocytes. The BCB+ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB+ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.

  17. [Comparison of the frequency of chromosomal disorders in populations of in vitro-matured and ovulating rat oocytes].

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    Kitaev, E M; Pimenova, M N

    1980-12-01

    The rat oocytes extracted from the rat ovaries and cultivated for 42-46 hours were compared with ovulated oocytes by the chromosomal aberration rate. The chromosomal aberration rate in the population of "follicular" oocytes was 8.2% on the average whereas in ovulated oocytes, it did not exceed 1.8%. Analysis of the chromosomal aberrations depending on the phase of the estral cycle suggests that the main portion of chromosomal aberrations in cultivated oocytes occurs during the physiological process of follicular atresia.

  18. Muscarinic receptor heterogeneity in follicle-enclosed Xenopus oocytes

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    Arellano, Rogelio O; Garay, Edith; Miledi, Ricardo

    1999-01-01

    Ionic current responses elicited by acetylcholine (ACh) in follicle-enclosed Xenopus oocytes (follicles) were studied using the two-electrode voltage-clamp technique. ACh generated a fast chloride current (Fin) and inhibited K+ currents gated by cAMP (IK,cAMP) following receptor activation by adenosine, follicle-stimulating hormone or noradrenaline. These previously described cholinergic responses were confirmed to be of the muscarinic type, and were independently generated among follicles from different frogs.Inhibition of IK,cAMP was about 100 times more sensitive to ACh than Fin activation; the half-maximal effective concentrations (EC50) were 6.6 ± 0.4 and 784 ± 4 nm, respectively.Both responses were blocked by several muscarinic receptor antagonists. Using the respective EC50 concentrations of ACh as standard, the antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the two effects with very different potencies. Fin was blocked with a half-maximal inhibitory concentration (IC50) of 2.4 ± 0.07 nm, whilst the IC50 for IK,cAMP inhibition was 5.9 ± 0.2 μm.Oxotremorine, a muscarinic agonist, preferentially stimulated IK,cAMP inhibition (EC50= 15.8 ± 1.4 μm), whilst Fin was only weakly activated. In contrast, oxotremorine inhibited Fin generated by ACh with an IC50 of 2.3 ± 0.7 μm.Fin elicited via purinergic receptor stimulation was not affected by oxotremorine, indicating that the inhibition produced was specific to the muscarinic receptor, and suggesting that muscarinic actions do not exert a strong effect on follicular cell-oocyte coupling.Using reverse transcription-PCR, transcripts of a previously cloned muscarinic receptor from Xenopus (XlmR) were amplified from the RNA of both the isolated follicular cells and the oocyte. The pharmacological and molecular characteristics suggest that XlmR is involved in IK,cAMP inhibition.In conclusion, follicular cells possess two different muscarinic receptors, one resembling the M2 (or M4) subtype

  19. Development of porcine tetraploid somatic cell nuclear transfer embryos is influenced by oocyte nuclei.

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    Fu, Bo; Liu, Di; Ma, Hong; Guo, Zhen-Hua; Wang, Liang; Li, Zhong-Qiu; Peng, Fu-Gang; Bai, Jing

    2016-02-01

    Cloning efficiency in mammalian systems remains low because reprogramming of donor cells is frequently incomplete. Nuclear factors in the oocyte are removed by enucleation, and this removal may adversely affect reprogramming efficiency. Here, we investigated the role of porcine oocyte nuclear factors during reprogramming. We introduced somatic cell nuclei into intact MII oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. We then examined the influence of the oocyte nucleus on tetraploid SCNT embryo development by assessing characteristics including pronucleus formation, cleavage rate, and blastocyst formation. Overall, tetraploid SCNT embryos have a higher developmental competence than do standard diploid SCNT embryos. Therefore, we have established an embryonic model in which a fetal fibroblast nucleus and an oocyte metaphase II plate coexist. Tetraploid SCNT represents a new research platform that is potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.

  20. Influence of Insulin-like Growth Factor 1 on Nuclear Maturation of Germinal Vesicle Mouse Oocytes

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    R mahmoudi

    2014-09-01

    Full Text Available Background & aim: In vitro maturation and fertilization of oocytes play an important role in reproductive biotechnology. The aim of this study is to define the IGF-1 effect on in vitro maturation, fertilization and development of mice immature oocytes to 2-cells in TCM199 medium cultures. Methods: In this study 4 week old NMRI mice were used. Ovaries stimulation carried out using PMSG. GV oocytes with or without cumulus cells were isolated from ovaries and cultured in TCM199 in presence of 100 ng IGF-1 for 24hr.The oocytes (MII were inseminated with sperm in T6 medium for fertilization and development of 2-cells stage and they were investigated under inverted microscope. Data analysis was performed by using Chi- 2 test. Results: In cumulus cell group and in the presence of insulin-like growth factor fertilization of oocytes, forming embryos and the formation of 2-cells compared to the group without cumulus cells significantly increased (p < 0.05. Conclusion: As the results showed oocytes with cumulus cells in the presence of insulin-like growth factor enhances maturation, fertilization and embryonic development in 2-cells oocytes compared to group without cumulus cells TCM199.

  1. Isolation and Identification of Transforming Growth Factor β from In Vitro Matured Cumulus Oocyte Complexes

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    AULANI’AM

    2012-03-01

    Full Text Available Transforming growth factor-β (TGF-β is a two-chain polypeptide with molecular weight of 25 kDa which takes significant role in the steroidogenesis process. In the ovarian oocyte in particular, TGF-β has an important role in regulating reproductive function. TGF-β represents a key intrafollicular protein that regulates follicle development and aromatization process. The purpose of this research was to characterize and identify a protein fraction of TGF-β from the bovine isolated oocytes, which is synthesized during in vitro oocyte maturation process. Oocytes were collected from follicles with diameter of 3-8 mm. Oocytes were then matured in TCM 199 media supplemented with 5 μg/mg LH, 3% BSA, and 50 μg/ml gentamicin sulfate, and cultured in CO2 incubator (5%, 38.5 oC for 20 hours. TGF-β receptors were identified immunohistochemically. Characteristics of the TGF-β protein were determined using SDS PAGE and TGF-β specification was tested using Western Blotting. The results showed that TGF-β receptors were identified and found in cumulus oocyte complexes (COCs. TGF-β protein was isolated from bovine oocytes with molecular weight 25 kDa and it was identified by Western blotting methods in the same molecular weight.

  2. Effects of M Ⅱ stage oocytes zona pellucida birefringence on pregnancy outcome

    Institute of Scientific and Technical Information of China (English)

    Jia Luo; Yan-Wen Xu; Ming-Fang Zhang; Ling Gao; Cong Fang; Can-Quan Zhou

    2013-01-01

    Objective: To explore the effects of different MⅡ stage oocytes zona pellucida birefringence on pregnancy outcome. Methods: A total of 46 couples with infertile which induced by single cause received in-vitro fertilization treatment were analyzed retrospectively, and randomly divided into the high zona birefringence (HZB)/HZB group, HZB/low zona birefringence (LZB) group and LZB/LZB group according to different oocytes zona pellucida birefringence. Intracytoplasmic sperm injection outcome was analyzed and compared. Results: The proportion of HZB oocytes, implantation rate and the pregnancy rate were decreased in three groups (HZB/HZB group>HZB/LZB group>LZB/LZB group) (P0.05). Logistic regression analysis showed that factors affect M Ⅱ stage oocytes zona pellucida birefringence were age, basal FSH level and the LH level on the day of HCG injection. Age and FSH levels were negatively correlated with the single oocyte zona pellucida birefringence; While the LH level on the day of hCG injection was positively correlated with the single oocyte zona pellucida birefringence. Conclusions: The primary influence factors on M Ⅱ stage oocytes zona pellucida are age, basal FSH level and the LH level on the day of hCG injection. The birefringence value of zona pellucida can affect the pregnancy outcome.

  3. Perceptions of oocyte banking from women intending to circumvent age-related fertility decline.

    Science.gov (United States)

    de Groot, Marije; Dancet, Eline; Repping, Sjoerd; Goddijn, Mariette; Stoop, Dominic; van der Veen, Fulco; Gerrits, Trudie

    2016-12-01

    Women can now opt to bank their oocytes with the intention of increasing their chances of achieving a pregnancy after their fertility has declined. This exploratory study aimed to gain insight into how women, considering oocyte banking to circumvent age-related fertility decline, perceive this intervention. We conducted a qualitative study in a Dutch university medical center and held in-depth interviews with women on the waiting list for oocyte banking. We recorded the interviews, transcribed them verbatim and used thematic analysis. All women were financially independent and lived in single-person urban households. They opted for oocyte banking because they wished to share parenthood with a future partner rather than becoming a single parent. This strong desire was key in their interpretation of all aspects of the intervention. Women set aside information about the limited success rates and potential risks, as they were optimistic about their own prognosis, thought that the chances for success were equally likely as the chances it would fail, and because of "anticipatory regret". They perceived oocyte banking as a "helping hand" to achieve shared parenthood. Although women found the costs of the intervention high, they were willing to invest their money to increase their chances for shared parenthood. Oocyte banking allows women to circumvent age-related fertility decline. The prospect of potential shared parenthood overrules the perceived health risks and burden. Health professionals should take this into account when informing potential users of oocyte banking. © 2016 Nordic Federation of Societies of Obstetrics and Gynecology.

  4. Zearalenone exposure affects mouse oocyte meiotic maturation and granulosa cell proliferation.

    Science.gov (United States)

    Hou, Yan-Jun; Zhu, Cheng-Cheng; Xu, Yin-Xue; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2015-09-01

    Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN-treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule-free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose-dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. © 2014 Wiley Periodicals, Inc.

  5. Effects of Aroclor 1254 on in vivo oocyte maturation in the mouse.

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    ShuZhen Liu

    Full Text Available Polychlorinated biphenyls (PCBs are stable, lipophilic compounds that accumulate in the environment and in the food chain. Though some studies provided evidence that PCBs had adverse effects on reproductive function, most of these results were from in vitro models. Therefore we investigated the effect of Aroclor 1254 (a commercial PCBs mixture treatments on in vivo maturation and developmental potential of mouse oocytes. In the present study, female ICR mice were treated with different doses (12.5, 25 and 50 mg/kg of Aroclor 1254 (a commercial PCB mixture once every 72 hours by intraperitoneal injection for 9 days. After three treatments of Aroclor 1254, the mice were superovulated to collect oocytes one day after the last exposure. The effects of Aroclor 1254 on oocyte maturation, fertilization, and preimplantation embryonic development were investigated. Immunofluorescence-stained oocytes were observed under a confocal microscope to assess the effects of Aroclor 1254 on spindle morphology. Parthenogenic activation and the incidence of cumulus apoptosis in cumulus-oocyte complexes were observed as well. Oocytes exposed to different doses of Aroclor 1254 in vivo were associated with a significant decrease in outgrowth potential, abnormal spindle configurations, and the inhibition of parthenogenetic activation of ovulated oocytes. Furthermore, the incidence of apoptosis in cumulus cells was increased after exposed to Aroclor 1254. These results may provide reference for the treatment of reproductive diseases such as infertility or miscarriage caused by environmental contaminants.

  6. Mitochondrial fission factor Drp1 maintains oocyte quality via dynamic rearrangement of multiple organelles.

    Science.gov (United States)

    Udagawa, Osamu; Ishihara, Takaya; Maeda, Maki; Matsunaga, Yui; Tsukamoto, Satoshi; Kawano, Natsuko; Miyado, Kenji; Shitara, Hiroshi; Yokota, Sadaki; Nomura, Masatoshi; Mihara, Katsuyoshi; Mizushima, Noboru; Ishihara, Naotada

    2014-10-20

    Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1, as well as analyzing a human patient harboring a point mutation in Drp1, showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically, and their organelle aggregation is related to germ cell formation and epigenetic regulation. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an age-dependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca(2+) signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement.

  7. PP2A regulates kinetochore-microtubule attachment during meiosis I in oocyte.

    Science.gov (United States)

    Tang, An; Shi, Peiliang; Song, Anying; Zou, Dayuan; Zhou, Yue; Gu, Pengyu; Huang, Zan; Wang, Qinghua; Lin, Zhaoyu; Gao, Xiang

    2016-06-02

    Studies using in vitro cultured oocytes have indicated that the protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, participates in multiple steps of meiosis. Details of oocyte maturation regulation by PP2A remain unclear and an in vivo model can provide more convincing information. Here, we inactivated PP2A by mutating genes encoding for its catalytic subunits (PP2Acs) in mouse oocytes. We found that eliminating both PP2Acs caused female infertility. Oocytes lacking PP2Acs failed to complete 1(st) meiotic division due to chromosome misalignment and abnormal spindle assembly. In mitosis, PP2A counteracts Aurora kinase B/C (AurkB/C) to facilitate correct kinetochore-microtubule (KT-MT) attachment. In meiosis I in oocyte, we found that PP2Ac deficiency destabilized KT-MT attachments. Chemical inhibition of AurkB/C in PP2Ac-null oocytes partly restored the formation of lateral/merotelic KT-MT attachments but not correct KT-MT attachments. Taken together, our findings demonstrate that PP2Acs are essential for chromosome alignments and regulate the formation of correct KT-MT attachments in meiosis I in oocytes.

  8. Prediction of Oocyte Number for Take-Home Baby Rate in Fresh ART Cycles

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    Spitzer D

    2015-01-01

    Full Text Available To guarantee the IVF success, the retrieval of several oocytes is mandatory to compensate those which reveal fertilization failure or growth arrest and in order to transfer a viable embryo. Thus, the aim of controlled ovarian hyperstimulation (COH is the growth of multiple follicles and to obtain a high number of mature (metaphase II [MII] oocytes. Various factors such as the etiology of infertility, the stimulation protocol, or female age can influence the quality as well as quantity of oocytes. However, the pivotal question is the optimal oocyte number for a successful IVF therapy. Therefore, the aim of this retrospective study was to investigate a putative correlation between the oocyte number obtained per fresh cycle for IVF success and the baby take-home rate. In the period 2006–2009, 1345 fresh cycles using the long GnRH-agonist protocol were evaluated. Patients were grouped according to their age and COH response. The number of oocytes obtained per ovum pick-up (OPU and pregnancy outcome were found to be related to the age of patients. Pregnancy and birth rates were significantly lower in patients when oocyte number was below the expected median of the age group.

  9. Negative energy balance affects imprint stability in oocytes recovered from postpartum dairy cows.

    Science.gov (United States)

    O'Doherty, Alan M; O'Gorman, Aoife; al Naib, Abdullah; Brennan, Lorraine; Daly, Edward; Duffy, Pat; Fair, Trudee

    2014-09-01

    Ovarian follicle development in post-partum, high-producing dairy cows, occurs in a compromised endogenous metabolic environment (referred to as negative energy balance, NEB). Key events that occur during oocyte/follicle growth, such as the vital process of genomic imprinting, may be detrimentally affected by this altered ovarian environment. Imprinting is crucial for placental function and regulation of fetal growth, therefore failure to establish and maintain imprints during oocyte growth may contribute to early embryonic loss. Using ovum pick-up (OPU), oocytes and follicular fluid samples were recovered from cows between days 20 and 115 post-calving, encompassing the NEB period. In a complimentary study, cumulus oocyte complexes were in vitro matured under high non-esterified fatty acid (NEFA) concentrations and in the presence of the methyl-donor S-adenosylmethionine (SAM). Pyrosequencing revealed the loss of methylation at several imprinted loci in the OPU derived oocytes. The loss of DNA methylation was observed at the PLAGL1 locus in oocytes, following in vitro maturation (IVM) in the presence of elevated NEFAs and SAM. Finally, metabolomic analysis of postpartum follicular fluid samples revealed significant differences in several branched chain amino acids, with fatty acid profiles bearing similarities to those characteristic of lactating dairy cows. These results provide the first evidence that (1) the postpartum ovarian environment may affect maternal imprint acquisition and (2) elevated NEFAs during IVM can lead to the loss of imprinted gene methylation in bovine oocytes.

  10. Ultrastructural Interactions and Genotoxicity Assay of Cerium Dioxide Nanoparticles on Mouse Oocytes

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    Blandine Courbiere

    2013-10-01

    Full Text Available Cerium dioxide nanoparticles (CeO2 ENPs are on the priority list of nanomaterials requiring evaluation. We performed in vitro assays on mature mouse oocytes incubated with CeO2 ENPs to study (1 physicochemical biotransformation of ENPs in culture medium; (2 ultrastructural interactions with follicular cells and oocytes using Transmission Electron Microscopy (TEM; (3 genotoxicity of CeO2 ENPs on follicular cells and oocytes using a comet assay. DNA damage was quantified as Olive Tail Moment. We show that ENPs aggregated, but their crystal structure remained stable in culture medium. TEM showed endocytosis of CeO2 ENP aggregates in follicular cells. In oocytes, CeO2 ENP aggregates were only observed around the zona pellucida (ZP. The comet assay revealed significant DNA damage in follicular cells. In oocytes, the comet assay showed a dose-related increase in DNA damage and a significant increase only at the highest concentrations. DNA damage decreased significantly both in follicular cells and in oocytes when an anti-oxidant agent was added in the culture medium. We hypothesise that at low concentrations of CeO2 ENPs oocytes could be protected against indirect oxidative stress due to a double defence system composed of follicular cells and ZP.

  11. Oocyte quality in mice is affected by a mycotoxin-contaminated diet.

    Science.gov (United States)

    Hou, Yan-Jun; Xiong, Bo; Zheng, Wei-Jiang; Duan, Xing; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Qiang; Xu, Yin-Xue; Sun, Shao-Chen

    2014-05-01

    Mycotoxins, such as deoxynivalenol (DON), zearalenone (ZEN), and aflatoxin (AF), are commonly found in many food commodities and may impair the growth and reproductive efficiency of animals and humans. We investigated the effects of a mycotoxin-contaminated diet on mouse oocyte quality. Maize contaminated with DON (3.875 mg/kg), ZEN (1,897 μg/kg), and AF (806 μg/kg) was incorporated into a mouse diet at three different levels (0, 15, and 30% w/w). After 4 weeks, ovarian and germinal vesicle oocyte indices decreased in mycotoxin-fed mice. Oocytes from these mice exhibited low developmental competence with reduced germinal vesicle breakdown and polar body extrusion rates. Embryo developmental competence also showed a similar pattern, and the majority of embryos could not develop to the morula stage. Actin expression was also reduced in both the oocyte cortex and cytoplasm, which was accompanied by decreased expression of the actin nucleation factors profilin-1 and mDia1. Moreover, a large percentage of oocytes derived from mice that were fed a mycotoxin-contaminated diet exhibited aberrant spindle morphology, a loss of the cortical granule-free domain, and abnormal mitochondrial distributions, which further supported the decreased oocyte quality. Thus, our results demonstrate that mycotoxins are toxic to the mouse reproductive system by affecting oocyte quality.

  12. Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography.

    Science.gov (United States)

    Mio, Yasuyuki; Iwata, Kyoko; Yumoto, Keitaro; Kai, Yoshiteru; Sargant, Haruka C; Mizoguchi, Chizuru; Ueda, Minako; Tsuchie, Yuka; Imajo, Akifumi; Iba, Yumiko; Nishikori, Kyoko

    2012-09-01

    To analyze the fertilization process related to polyspermy block in human oocytes using an in vitro culturing system for time-lapse cinematography. We had 122 oocytes donated for this study from couples that provided informed consent. We recorded human oocytes at 2,000 to 2,800 frames every 10 s during the fertilization process and thereafter every 2 min using a new in vitro culture system originally developed by the authors for time-lapse cinematography. We displayed 30 frames per second for analysis of the polyspermy block during fertilization. Three oocytes showed the leading and following sperm within the zona pellucida in the same microscopic field. The dynamic images obtained during the fertilization process using this new system revealed that once a leading sperm penetrated the zona pellucida and attached to the oocyte membrane, a following sperm was arrested from further penetration into the zona pellucida within 10 s. The present results strongly suggest the existence of a novel mechanism of polyspermy block that takes place at the zona pellucida immediately after fertilization. These findings are clearly different from previous mechanisms describing polyspermy block as the oocyte membrane block to sperm penetration and the zona reaction. The finding presented herein thus represents a novel discovery about the highly complicated polyspermy block mechanism occurring in human oocytes.

  13. Localization of interchromatin granule cluster and Cajal body components in oocyte nuclear bodies of the hemipterans.

    Science.gov (United States)

    Bogolyubov, D S; Batalova, F M; Ogorzałek, A

    2007-10-01

    An oocyte nucleus contains different extrachromosomal nuclear domains collectively called nuclear bodies (NBs). In the present work we revealed, using immunogold labeling electron microscopy, some marker components of interchromatin granule clusters (IGCs) and Cajal bodies (CBs) in morphologically heterogeneous oocyte NBs studied in three hemipteran species: Notostira elongata, Capsodes gothicus (Miridae) and Velia caprai (Veliidae). Both IGC and CB counterparts were revealed in oocyte nuclei of the studied species but morphological and biochemical criteria were found to be not sufficient to determine carefully the define type of oocyte NBs. We found that the molecular markers of the CBs (coilin and non-phosphorylated RNA polymerase II) and IGCs (SC35 protein) may be localized in the same NB. Anti-SC35 antibody may decorate not only a granular material representing "true" interchromatin granules but also masks some fibrillar parts of complex NBs. Our first observations on the hemipteran oocyte NBs confirm the high complexity and heterogeneity of insect oocyte IGCs and CBs in comparison with those in mammalian somatic cells and amphibian oocytes.

  14. Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish.

    Science.gov (United States)

    Toraya, Tetsuo; Kida, Tetsuo; Kuyama, Atsushi; Matsuda, Shinjiro; Tanaka, Seiichi; Komatsu, Yo; Tsurukai, Taro

    2017-02-04

    Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N(6)-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised against a 1-MeAde hapten, a single band with Mr of 47.5 K was detected. The band was lost when extract was heated at 100 °C. A similar 47.5 K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors.

  15. The Effects of Polyadenylation Status on MPFs During In Vitro Porcine Oocyte Maturation

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    Huiyu Liu

    2016-10-01

    Full Text Available Aims: This study aims to clarify the effects of polyadenylation status on M-phase promoting factors (MPFs during in vitro porcine oocyte maturation. Methods: In this study, porcine follicular oocytes from large follicles (> 5 millimeter (mm and small follicles (Cyclin B1 and cell division cycle 2 (Cdc2 were determined by real-time quantitative PCR. Immunofluorescence was used to assess spindle formation and chromosome alignment in the examined oocytes. Results: In large-follicle oocytes, the effects of inhibiting polyadenylation caused the percentage of mature to be significantly lower for the treated group than for the untreated group (p in vitro and inhibits Cdc2 polyadenylation. Cyclin B1 plays a significant role in promoting the maturation of large-follicle oocytes. Polyadenylation contributes to the formation of dominant follicles and facilitates the selection of dominant follicles. However, the inhibition of adenylation affected spindle formation-related propulsion and chromosome alignment in both large- and small-follicle oocytes. The first polar body could not be extruded in certain large follicles. Conclusions: 3'-da can significantly improve the rate of small oocyte maturation in vitro, but it can also affect spindle formation-related propulsion and chromosome alignment.

  16. Oocytes with a dark zona pellucida demonstrate lower fertilization, implantation and clinical pregnancy rates in IVF/ICSI cycles.

    Science.gov (United States)

    Shi, Wei; Xu, Bo; Wu, Li-Min; Jin, Ren-Tao; Luan, Hong-Bing; Luo, Li-Hua; Zhu, Qing; Johansson, Lars; Liu, Yu-Sheng; Tong, Xian-Hong

    2014-01-01

    The morphological assessment of oocytes is important for embryologists to identify and select MII oocytes in IVF/ICSI cycles. Dysmorphism of oocytes decreases viability and the developmental potential of oocytes as well as the clinical pregnancy rate. Several reports have suggested that oocytes with a dark zona pellucida (DZP) correlate with the outcome of IVF treatment. However, the effect of DZP on oocyte quality, fertilization, implantation, and pregnancy outcome were not investigated in detail. In this study, a retrospective analysis was performed in 268 infertile patients with fallopian tube obstruction and/or male factor infertility. In 204 of these patients, all oocytes were surrounded by a normal zona pellucida (NZP, control group), whereas 46 patients were found to have part of their retrieved oocytes enclosed by NZP and the other by DZP (Group A). In addition, all oocytes enclosed by DZP were retrieved from 18 patients (Group B). No differences were detected between the control and group A. Compared to the control group, the rates of fertilization, good quality embryos, implantation and clinical pregnancy were significantly decreased in group B. Furthermore, mitochondria in oocytes with a DZP in both of the two study groups (A and B) were severely damaged with several ultrastructural alterations, which were associated with an increased density of the zona pellucida and vacuolization. Briefly, oocytes with a DZP affected the clinical outcome in IVF/ICSI cycles and appeared to contain more ultrastructural alterations. Thus, DZP could be used as a potential selective marker for embryologists during daily laboratory work.

  17. Effects of vitrification procedures on subsequent development and ultrastructure of in vitro-matured swamp buffalo (Bubalus bubalis) oocytes.

    Science.gov (United States)

    Boonkusol, Duangjai; Faisaikarm, Tassanee; Dinnyes, Andras; Kitiyanant, Yindee

    2007-01-01

    The purpose of the present study was to investigate the effects of two vitrification procedures on developmental capacity and ultrastructural changes of matured swamp buffalo oocytes. In vitro-matured oocytes were vitrified by using 35 and 40% ethylene glycol as vitrification solution for solid surface vitrification (SSV) and in-straw vitrification (ISV), respectively. Survival rate of vitrified-warmed oocytes, evaluated on the basis of ooplasm homogeneity, oolemma integrity and zona pellucida intactness, as well as parthenogenetic blastocyst rates of vitrified-warmed oocytes were significantly higher with SSV (89.3 and 13.6%, respectively) than ISV (81.8 and 5.5%, respectively). However, they were still significantly lower than that of control oocytes (100 and 34.2%, respectively). For examining the ultrastructural changes, fresh, VS-exposed (ISV and SSV), and vitrified-warmed (ISV and SSV) oocytes were processed for transmission electron microscopy. In VS-exposed oocytes, reduction of microvilli abundance and damage of mitochondrial membrane were found only in the ISV group. In vitrified-warmed oocytes, however, it was clear that both methods of vitrification induced profound ultrastructural modifications to microvilli, mitochondria, oolemma and cortical granules as well as to the size and position of vesicles. Damaged mitochondria were, however, more abundant in ISV vitrified oocytes than in SSV vitrified oocytes, which correlated with the developmental data, showing the superiority of the SSV method. The present study demonstrated the feasibility of vitrification of in vitro-matured swamp buffalo oocytes.

  18. Effect of the volume of medium and number of oocytes during in vitro fertilization on embryo development in pigs.

    Science.gov (United States)

    Gil, Maria Antonia; Abeydeera, Lalantha R; Day, Billy N; Vazquez, Juan M; Roca, Jordi; Martinez, Emilio A

    2003-09-01

    The present study was designed to determine the effect of the volume of medium (VM) and the number of oocytes (NOOC) during in vitro fertilization (IVF) on embryo development in pigs. Groups of 15, 30 and 50 in vitro matured oocytes were transferred to 2, 1 and 0.1 ml of modified Tris-buffered medium (mTBM) and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) in a 3 x 3 factorial experiment. A total of 2739 oocytes from four replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The efficiency of fertilization (EF: number of monospermic oocytes/total number of inseminated oocytes) and BF decreased (PIVF medium (0.1 ml) and the number of oocytes during IVF (30-50) can improve the in vitro embryo production in pigs.

  19. In vitro and in vivo Development of Cloned Ovine Embryos using in vitro and in vivo Matured Oocytes

    DEFF Research Database (Denmark)

    Holm, P; Nagashima, H; Sun, F-J

    1995-01-01

    Cloning of sheep embryos by nucleus transplantation can be achieved by using in vivo matured (oviductal) oocytes and in vivo culture. However, these steps involve cumbersome procedures. Therefore, the effects of in vivo vs. the equivalent in vitro procedures on the pre-implantation development...... of cloned embryos were compared using: l. In vivo oocytes and in vivo culture; 2. In vivo oocytes and in vitro culture; and 3. In vitro oocytes and in vitro culture. Selected embryos were transferred to recipients. Donor embryos and oviductal oocytes were collected from superovulated Merino ewes. In vivo...... matured oocytes were enucleated and fused with inserted blastomeres from donor embryos. In vitro matured oocytes were enucleated and allowed to age prior to blastomere insertion and electrofusion. Fused embryos were cultured for approximately 132 h either in vivo in ligated sheep oviducts or in vitro...

  20. The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality.

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    Faten Shaeib

    Full Text Available Myeloperoxidase (MPO, an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl, a damaging reactive oxygen species (ROS utilizing hydrogen peroxide (H2O2 and chloride (Cl-. Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1 Incubation with a catalytic amount of MPO (40 nM for different incubation periods in the presence of 100 mM Cl- with and without H2O2 and with and without melatonin (100 μM, at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells; 2 Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 106 cells/ml in the absence and presence of melatonin (200 μM, an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200; 3 Untreated oocytes incubated for 4 hrs as controls (n = 73/64. Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes.

  1. Loss of glycogen synthase kinase 3 isoforms during murine oocyte growth induces offspring cardiac dysfunction.

    Science.gov (United States)

    Monteiro da Rocha, André; Ding, Jun; Slawny, Nicole; Wolf, Amber M; Smith, Gary D

    2015-05-01

    Glycogen synthase kinase-3 (GSK3) is a constitutively active serine threonine kinase with 1) two isoforms (GSK3A and GSK3B) that have unique and overlapping functions, 2) multiple molecular intracellular mechanisms that involve phosphorylation of diverse substrates, and 3) implications in pathogenesis of many diseases. Insulin causes phosphorylation and inactivation of GSK3 and mammalian oocytes have a functional insulin-signaling pathway whereby prolonged elevated insulin during follicle/oocyte development causes GSK3 hyperphosphorylation, reduced GSK3 activity, and altered oocyte chromatin remodeling. Periconceptional diabetes and chronic hyperinsulinemia are associated with congenital malformations and onset of adult diseases of cardiovascular origin. Objectives were to produce transgenic mice with individual or concomitant loss of GSK3A and/or GSK3B and investigate the in vivo role of oocyte GSK3 on fertility, fetal development, and offspring health. Wild-type males bred to females with individual or concomitant loss of oocyte GSK3 isoforms did not have reduced fertility. However, concomitant loss of GSK3A and GSK3B in the oocyte significantly increased neonatal death rate due to congestive heart failure secondary to ventricular hyperplasia. Individual loss of oocyte GSK3A or GSK3B did not induce this lethal phenotype. In conclusion, absence of oocyte GSK3 in the periconceptional period does not alter fertility yet causes offspring cardiac hyperplasia, cardiovascular defects, and significant neonatal death. These results support a developmental mechanism by which periconceptional hyperinsulinemia associated with maternal metabolic syndrome, obesity, and/or diabetes can act on the oocyte and affect offspring cardiovascular development, function, and congenital heart malformation.

  2. Number and Quality of Oocytes Collected from Heterotopic Autografted Mice Ovary after PMSG Induction

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    NURBARIAH

    2011-12-01

    Full Text Available Heterotopic grafting sites can be useful in producing oocytes for in vitro Fertilization, therefore, maximising the oocyte yield from the graft by gonadotrophin stimulation would be advantageous. The aim of this study was to investigate the number and quality of oocytes collected from heterotopic autografted ovary after Pregnant Mare Serum Gonadothropin (PMSG induction. Graft recipients were treated either with or without PMSG stimulation 48 hours prior to graft collection. Ovarian tissue from four weeks old mice (DDY strain were autotransplanted under the kidney capsule of the same ovariectomized mice and the oocytes were collected 21 days after autotransplantation. The results showed that the average number of oocytes collected from autografted ovaries without PMSG induction were 9.0. + 2.8 not significantly different with those received PMSG induction, 10.9 + 5.1. The percentage of matured and fertilized oocytes and the developed embryos from the autografted ovaries without PMSG induction were 52.4, 33.4, and 26.0%, respectively not significantly different with those received PMSG induction, 53.2, 35.1, and 29.9%, respectively. The number of oocytes and the capacity to matured, fertilized and developed were significantly lower (P < 0.05 compared to the superovulated nongrafted (control ovaries. In conclusion, PMSG induction on the graft recipients did not significantly increase oocytes yield from grafted heterotopic ovaries. The number and quality of oocytes produced from the autografted ovaries were lower than the superovulated nongrafted ovaries, but still can be used for in vitro embryo production after sequential in vitro maturation and fertilization.

  3. The effects of roscovitine on cumulus cell apoptosis and the developmental competence of domestic cat oocytes.

    Science.gov (United States)

    Sananmuang, T; Techakumphu, M; Tharasanit, T

    2010-01-15

    The developmental competence of cat oocytes matured in vitro is relatively poor when compared with that of in vivo oocytes. The study aimed to investigate the effect of roscovitine on the developmental competence of cat Felis catus oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were classified as Grade I and II to III. Groups of COCs were cultured in 0, 12.5, 25, 50, 100, and 200 microM roscovitine for 24h and were either fixed to assess the stages of nuclear maturation (Experiment 1) or additionally matured in vitro for 24h before fixation (Experiment 2). In Experiment 3, cumulus cells from the COCs treated with roscovitine were examined for apoptosis. Experiment 4 examined the developmental competence of cat oocytes after roscovitine treatment and in vitro fertilization in terms of cleavage and morula and blastocyst formation rates. Roscovitine reversibly arrested cat oocytes at an immature stage in a dose-dependent manner. Roscovitine at 12.5 and 25 microM demonstrated less efficiency compared with that of other doses. However, higher doses of roscovitine induced cumulus cell apoptosis and resulted in a high number of degenerated oocytes after in vitro maturation. Roscovitine at 12.5 and 25 microM were therefore used to evaluate their effect on embryo development. Pretreatment with 12.5 and 25 microM roscovitine prior to in vitro maturation decreased the developmental competence of cat oocytes compared with that of non-roscovitine-treated controls. In conclusion, roscovitine reversibly maintained cat oocytes at the germinal vesicle stage without detrimental effect on nuclear maturation. However, it negatively affected cumulus cell viability and developmental competence.

  4. Maturation of pig oocytes in vitro in a medium with pyruvate

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    H. Gonzales-Figueroa

    2005-06-01

    Full Text Available The aim of in vitro maturation oocyte systems is to produce oocytes of comparable quality to those derived in vivo. The present study was designed to examine the surface morphological changes of the cumulus-oocyte complex (COC and nuclear maturation in a culture system containing pyruvate. Ovaries were obtained from a slaughterhouseand transported to the laboratory within 2 h at 35-39ºC,and rinsed three times in 0.9% NaCl. The COCs were harvested from the ovaries and in vitro maturation was evaluated in San Marcos (SM medium, a chemically defined culture system containing 22.3 mM sodium pyruvate. Oocytes were cultured in SM, SM + porcine follicular fluid (pFF and in SM + pFF + gonadotropins (eCG and hCG for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Oocytes matured in SM (40.9% and SM + pFF (42.9% showed moderate cumulus expansion, whereas oocytes matured in SM + pFF + gonadotropins (54.6% showed high cumulus expansion. The maturation rate of cultured oocytes, measured in function of the presence of the polar corpuscle, did not differ significantly between SM (40.9 ± 3.6% and SM + pFF (42.9 ± 3.7%. These results indicate that pig oocytes can be successfully matured in a chemically definedmedium and suggest a possible bifunctional role of pyruvate as an energy substrate and as an antioxidant protecting oocytes against the stress of the in vitro environment.

  5. Does the number of oocytes retrieved influence pregnancy after fresh embryo transfer?

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    Qianfang Cai

    Full Text Available BACKGROUND: The nature of the association between the number of oocytes retrieved and in vitro fertilization (IVF outcomes after fresh embryo transfer remains unclear because of conflicting results reported in the studies on this subject. In addition, the influence of the quality of the embryos transferred is usually neglected. The objective of this study is to assess the relationships of the number of oocytes retrieved, the number and quality of embryos transferred, and the prospects of pregnancy after fresh embryo transfer. METHODS: The data on 3131 infertile women undergoing their first IVF treatment cycle between January 2009 and December 2010 were collected retrospectively. Restricted cubic splines and stratified analyses were used to explore the relationships between the number of oocytes retrieved, the number and quality of embryos transferred, and the IVF outcomes. RESULTS: When stratified by the number and quality of transferred embryos, no significant differences in the chances for clinical pregnancy and live birth were found in three groups of oocytes yielded (≤6, 7-14, or ≥15. The relationship between the number of oocytes retrieved and pregnancy is nearly a reflection of the pattern of the relationship between the number of oocytes retrieved and the probability of having two good-quality embryos transferred. The patients with the "optimal" number of oocytes were not only younger but also had the highest probability of having two good-quality embryos replaced. CONCLUSIONS: Similarly aged patients have similar pregnancy prospects after fresh embryo transfer when the same number and quality of embryos are replaced, irrespective of their number of oocytes. Selecting the desired number of good-quality embryos for transfer is the key to IVF success. Thus, aiming at retrieving an optimal number of oocytes to maximize IVF outcomes in a fresh cycle could place undue stress on the patients and may not be the best medical decision.

  6. Analysis of oocyte-like cells differentiated from porcine fetal skin-derived stem cells.

    Science.gov (United States)

    Dyce, Paul W; Shen, Wei; Huynh, Evanna; Shao, Hua; Villagómez, Daniel A F; Kidder, Gerald M; King, W Allan; Li, Julang

    2011-05-01

    We previously reported the differentiation of cells derived from porcine female fetal skin into cells resembling germ cells and oocytes. A subpopulation of these cells expressed germ cell markers and formed aggregates resembling cumulus-oocyte complexes. Some of these aggregates extruded large oocyte-like cells (OLCs) that expressed markers consistent with those of oocytes. The objective of the current study was to further characterize OLCs differentiated from porcine skin-derived stem cells. Reverse transcriptase (RT)-polymerase chain reaction and Western blot revealed the expression of connexin37 and connexin43, both of which are characteristic of ovarian follicles. The expression of meiosis markers DMC1 and synaptonemal complex protein, but not STRA8 and REC8, was detected in the OLC cultures. Immunofluorescence with an antibody against synaptonemal complex protein on chromosome spreads revealed a very small subpopulation of stained OLCs that had a similar pattern to leptotene, zytotene, or pachytene nuclei during prophase I of meiosis. Sodium bisulfite sequencing of the differentially methylated region of H19 indicated that this region is almost completely demethylated in OLCs, similar to in vivo-derived oocytes. We also investigated the differentiation potential of male skin-derived stem cells in the same differentiation medium. Large cells with oocyte morphology were generated in the male stem cell differentiation cultures. These OLCs expressed oocyte genes such as octamer-binding transcription factor 4 (OCT4), growth differentiation factor-9b (GDF9B), deleted in azoospermia-like (DAZL), VASA, zona pellucida B (ZPB), and zona pellucida C (ZPC). It was concluded that skin-derived stem cells from both male and female porcine fetuses are capable of entering an oocyte differentiation pathway, but the culture system currently in place is inadequate to support the complete development of competent oocytes.

  7. Improvement on in vitro maturation, fertilization and development of minke whale (Balaenoptera acutorostrata) oocytes.

    Science.gov (United States)

    Asada, M; Tetsuka, M; Ishikawa, H; Ohsumi, S; Fukui, Y

    2001-09-01

    The aims of the present study were to improve in vitro maturation, fertilization and subsequent development of minke whale oocytes. We investigated the effects of different concentrations (0, 10 and 20%) of fetal whale serum (FWS) in maturation medium on nuclear maturation, morphological grade (A or B) of cumulus-oocyte complexes (COC) obtained from prepubertal and adult minke whales. Grade A (> or = 5 layers of cumulus cells) COC collected from the adult whales and cultured in the medium with 20% FWS had a higher (P whales and COC grades were not significantly affected by M-II oocytes. When in vitro fertilization of matured oocytes was performed in the presence of 20% FWS or 0.6% BSA in the fertilization medium, the proportions of sperm penetration and two-pronuclei formation in matured oocytes were not significantly different. Grade A COC cultured in a culture medium supplemented with 10% FWS cleaved at a higher rate (15.4%, P develop to morula (4.2%) compared with that of the oocytes from Grade B COC (2.5% and 0%). Coculture with granulosa cells during in vitro culture did not significantly affect cleavage and development to the morula stage. These results indicate that FWS addition in the maturation medium improved the rate of in vitro maturation and cleavage after insemination of minke whale oocytes. The BSA supplementation in fertilization medium was as effective as FWS supplementation for in vitro fertilization of matured oocytes. In vitro embryo production beyond the morula stage of minke whale oocytes could be possible, if Grade A COC was selected and cultured in the maturation medium supplemented with 10% or 20% FWS.

  8. Identification of maturation and protein synthesis related proteins from porcine oocytes during in vitro maturation

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    Seo Kang

    2011-06-01

    Full Text Available Abstract Background In vitro maturation (IVM of mammalian oocytes is divided into the GV (germinal vesicle stage, MI (metaphase I stage and MII (metaphase II stage stages, and only fully mature oocytes have acquired the ability to be fertilized and initiate zygotic development. These observations have been mostly based on morphological evaluations, but the molecular events governing these processes are not fully understood. The aim of the present study was to better understand the processes involved in the molecular regulation of IVM using 2-DE analysis followed by mass spectrometry to identify proteins that are differentially expressed during oocyte IVM. Result A total of 16 up-regulated and 12 down-regulated proteins were identified. To investigate the IVM process, we specifically focused on the proteins that were up-regulated during the MII stage when compared with the GV stage, which included PRDX 2, GST, SPSY, myomegalin, PED4D, PRKAB 1, and DTNA. These up-regulated proteins were functionally involved in redox regulation and the cAMP-dependent pathway, which are essential for the intracellular signaling involved in oocyte maturation. Interestingly, the PDE4D and its partner, myomegalin, during the MII stage was consistently confirmed up-regulation by western blot analyses. Conclusion These results could be used to better understand some aspects of the molecular mechanisms underlying porcine oocyte maturation. This study identified some regulatory proteins that may have important roles in the molecular events involved in porcine oocyte maturation, particularly with respect to the regulation of oocyte meiotic resumption, MII arrest and oocyte activation. In addition, this study may have beneficial applications not only to basic science with respect to the improvement of oocyte culture conditions but also to mammalian reproductive biotechnology with potential implications.

  9. Obstetric and neonatal complications in pregnancies conceived after oocyte donation

    DEFF Research Database (Denmark)

    Storgaard, M.; Loft, A.; Bergh, C.

    2017-01-01

    by conventional in-vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) or spontaneously. Search Strategy: A systematic search was performed in the PubMed, Cochrane and Embase databases from 1982–2016. Primary outcomes were hypertensive disorders of pregnancy, pre-eclampsia (PE), gestational diabetes......Background: Approximately 50 000 oocyte donation (OD) treatment cycles are now performed annually in Europe and the US. Objectives: To ascertain whether the risk of adverse obstetric and perinatal/neonatal outcomes is higher in pregnancies conceived by OD than in pregnancies conceived...... mellitus, postpartum haemorrhage, caesarean section, preterm birth, low birthweight and small for gestational age. Selection criteria: Inclusion criteria were original studies including at least five OD pregnancies with a control group of pregnancies conceived by conventional IVF/ICSI or spontaneous...

  10. The cytochemistry of oocytes of Chinese shrimp Penaeus orientalis

    Science.gov (United States)

    Chen, Qiu

    1991-06-01

    In the growth of oocytes of Penaeus orientalis Kishinouye, five stages were distinguished. Histochemical tests showed the presence of DNA in the chromatin and nucleolus of the cell. The cytoplasm at the previtellogenetic stage and the nucleolus are rich in RNA and the proteins abounding with cysteine, tyrosine and tryptophan. The yolk consists mainly of proteins and phospholipids. The 1,2-glycol groups of carbohydrate occur in the cytoplasm at stage II, and aggregate mostly into cortical rods at stages IV and V. Neutral lipid droplets, and protein containing disulfides, appear in the cytoplasm at stages III and IV respectively. The proteins in the cortical rod differ from those in other components of the cell in the presence of cystine and absence of arginine.

  11. Vitrification of oocytes: from basic science to clinical application.

    Science.gov (United States)

    Arav, Amir; Natan, Yehudit

    2013-01-01

    Vitrification is a physical process by which a liquid is transformed into a solid of amorphous glass form. It was only at the end of the nineteenth century (1898) that Gustav Heinrich Johann Apollon Tammann pointed out that a large number of substances can be obtained as glasses and suggested that this property might be universal (Tammann, Zeitschrift for Physikalische Chemie; 25: 441-479, 1898). Basically, vitrification is the supercooling of a liquid to a temperature at which the viscosity is so high that it can be defined as being at a solid state. The understanding of the vitrification process has been deepened over the years and has been applied for cryopreservation and currently is the method of choice for preserving oocytes and embryos.

  12. Intact fetal ovarian cord formation promotes mouse oocyte survival and development

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    Pera Renee

    2010-01-01

    Full Text Available Abstract Background Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined. Results Here, we examine whether intact fetal ovarian germ and somatic cell cord structures are required for oocyte development using mouse gonad re-aggregation and transplantation to disrupt gonadal organization. We observed that germ cells from disrupted female gonad prior to embryonic day e13.5 completed prophase I of meiosis but did not survive following transplantation. Furthermore, re-aggregated ovaries from e13.5 to e15.5 developed with a reduced number of oocytes. Oocyte loss occurred before follicle formation and was associated with an absence of ovarian cord structure and ovary disorganization. However, disrupted ovaries from e16.5 or later were resistant to the re-aggregation impairment and supported robust oocyte survival and development in follicles. Conclusions Thus, we demonstrate a critical window of oocyte development from e13.5 to e16.5 in the intact fetal mouse ovary, corresponding to the establishment of ovarian cord structure, which promotes oocyte interaction with neighboring ovarian somatic granulosa cells before birth and imparts oocytes with competence to survive and develop in follicles. Because germline cyst and ovarian cord structures are conserved in the

  13. Towards a new understanding on the regulation of mammalian oocyte meiosis resumption.

    Science.gov (United States)

    Sun, Qing-Yuan; Miao, Yi-Liang; Schatten, Heide

    2009-09-01

    Mammalian oocytes reach prophase of first meiosis around the time of birth, and remain at this stage for months or years, depending on the species. Only after puberty will the fully-grown oocytes begin to resume meiosis which is stimulated by gonadotropin surge. It has long been known that a high level of intra-oocyte cyclic adenosine 3',5'-monophosphate (cAMP) prevents oocyte meiosis resumption as indicated by germinal vesicle breakdown (GVBD). Recently, guanosine triphosphate-binding (G) protein-coupled receptors/G proteins/adenyl cyclase pathway endogenous to the oocyte as well as cAMP diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that prevent oocytes from resuming meiosis. Another second messager molecule, guanosine 3',5'-cyclic monophosphate (cGMP), has also recently been found to play important roles in maintaining oocyte meiosis arrest. cGMP in the follicular somatic cells diffuses into the oocyte and causes an increase in oocyte cAMP, presumably by acting on phosphodiesterase 3 (PDE3). The cGMP level in the somatic compartment of the follicle decreases in response to luteinizing hormone (LH), and this change may be mediated through the epidermal growth factor (EGF)-like factors and specific cGMP-phosphodiesterase subtype activity. It is well known that gonadotropic stimulation of meiotic resumption depends on mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle; recent studies show that LH, through cAMP/protein kinase A (PKA) and protein kinase C (PKC) pathways, induces the synthesis of paracine factors such as EGF-like facors and meiosis activating sterol (MAS) to regulate oocyte GVBD via the MAPK pathway in follicle cells. A recent granulosa cell-specific knockout study has for the first time provided in vivo evidence for the important role of extracellular regulated kinase 1 and 2 (ERK1/2), two main forms of MAPK, and their downstream molecules in

  14. Induction of abnormal oocyte division under the constant light in the young adult rat

    Institute of Scientific and Technical Information of China (English)

    Wei Wang; Fangxiong Shi

    2012-01-01

    In order to study the effect of constant light on the ovary, 12 young adult female rats were exposed to constant light for 37 days and their estrous cycles were recorded by daily examination and the ovaries were examined histologically. The results showed that constant light induced in an abnormal and uncontrolled division of oocytes. In these divided-oocyte follicles (DOFs), two or more divided-oocytes shared one zona pellucida and usually floated freely in the follicular antrum. This fantastic phenomenon was discovered for the first time, and it was different from multioocyte follicles (MOFs) and polyovular follicles (POFs) founded in rats, humans, rabbits, mice and dogs.

  15. Adnexal Torsion during Pregnancy after Oocyte In Vitro Maturation and Intracytoplasmic Sperm Injection Cycle

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    Simone Giulini

    2010-01-01

    Full Text Available We report a case of right adnexal torsion during pregnancy after an oocyte in vitro maturation and intracitoplasmic sperm injection cycle in patient with polycystic ovary syndrome. A 31-year-old woman with a typical clinical disorder of polycystic ovarian syndrome was included in an oocyte in vitro maturation program. Right adnexal torsion occurred two days after embryo transfer, and laparoscopy detorsion was successfully performed with preservation of adnexa. The patient had a full-term pregnancy and delivered a healthy infant at 40 weeks of gestation. To our knowledge this is the first report of adnexal torsion after an oocyte in vitro maturation and intracitoplasmic sperm injection program.

  16. Reversible phosphorylation and regulation of mammalian oocyte meiotic chromatin remodeling and segregation.

    Science.gov (United States)

    Swain, J E; Smith, G D

    2007-01-01

    The mammalian oocyte is notorious for high rates of chromosomal abnormalities. This results in subsequent embryonic aneuploidy, resulting in infertility and congenital defects. Therefore, understanding regulatory mechanisms involved in chromatin remodeling and chromosome segregation during oocyte meiotic maturation is imperative to fully understand the complex process and establish potential therapies. This review will focus on major events occurring during oocyte meiosis, critical to ensure proper cellular ploidy. Mechanistic and cellular events such as chromosome condensation, meiotic spindle formation, as well as cohesion of homologues and sister chromatids will be discussed, focusing on the role of reversible phosphorylation in control of these processes.

  17. Cumulus Cell Role on Mouse Germinal Vesicle Oocyte Maturation, Fertilization, and Subsequent Embryo Development to Blastocyst Stage In Vitro

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    Reza Mahmodi

    2009-01-01

    Full Text Available Objective: The purpose of this study is to investigate the effect of cumulus cells on maturation,fertilization and subsequent development of mouse germinal vesicle oocytes.Materials and Methods: A total of 470 germinal vesicle (GV oocytes were obtained from26 ovaries of 3- 4 week old ICR female mice 48 hours after injection of 5 IU pregnant mareserum gonadotropin (PMSG. Collected oocytes were divided into two groups; group I: GVoocytes without cumulus cells (denuded oocyte, group II: GV oocytes with cumulus cells(cumulus-oocyte complex. The oocytes in both groups were cultured in TCM-199 mediumsupplemented with 10% fetal bovine serum (FBS for 22- 24 hours in a humidified atmosphereof 5% CO2 in air at 37°C. Oocyte maturation was scored under inverted microscope.To do in vitro fertilization, matured oocytes from each group were placed in T6 mediumand capacitated spermatozoa were added. Then the fertilized oocytes were cultured andassessed for cleavage to the 2-cell stage 24 hours and production of blastocyst 120 hoursafter fertilization. Data was analyzed by chi-square test and differences in the values wereconsiderable significant when p<0.05.Results: Maturation, fertilization, cleavage and blastocyst rates in denuded oocytes were:76.32%, 57.49%, 51.15% and 19.14% respectively. In the cumulus-oocyte complex rateswere: 89.41%, 80.76%, 75.58% and 45.62% respectively; all in the cumulus-oocyte complexwere significantly higher than those of denuded oocytes (p<0.05.Conclusion: The present study indicates that cumulus cells have important role duringmaturation, fertilization and subsequent embryo development to the blastocyst stage.

  18. Effect of Leptin on In Vitro Nuclear Maturation and Apoptosis of Buffalo (Bubalus bubalis Oocyte

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    Amir Khaki

    2014-03-01

    Full Text Available Background: Leptin, as a 16 kDa adipokine, is a pleiotropic cytokine-like hormone that primarily secreted from adipose tissue. It also involves in the regulation of energy homeostasis, neuroendocrine function, immunity, lipid and glucose homeostasis, fatty acid oxidation, angiogenesis, puberty and reproduction. The aim of this study was to investigate the effects of in vitro addition of leptin to in vitro maturation (IVM medium on buffalo oocyte maturation and apoptosis. Materials and Methods: In this experimental study, Ovaries from apparently normal reproductive organs of slaughtered adult buffaloes (Bubalus bubalis with unknown breeding history were collected from Urmia Abattoir, Urmia, Iran, and were transported immediately to the laboratory in a thermos flask containing sterile normal saline with added antibiotics. Oocytes were aspirated from 2-8 mm visible follicles of the ovaries using an 18-G needle attached to a 10 ml syringe. IVM medium included tissue culture medium-199 (TCM-199, 10% fetal bovine serum (FBS, 22 μg/ml sodium pyruvate, 0.5 IU/ml ovine follicle-stimulating hormone (oFSH, 0.5 IU/ml ovine luteinizing hormone (oLH, 1 μg/ml oestradiol, 50 μg/ml gentamycin, and leptin [0 (control, 10, 50, and 100 ng/ml]. The good quality buffalo oocytes (batches of 10 oocytes were placed in a culture plate containing six 50 μl droplets of maturation medium, covered with sterilized mineral oil, and then incubated at 38.5˚C with 5% CO2 in air for 24 hours. The maturation of oocytes was evaluated under a stereomicroscope by detecting the first polar body extrusion of oocytes. FITC-Annexin V - propidium iodide (PI staining method was used to detect oocyte apoptosis. Results: From a total of 115 collected ovaries, 1100 oocytes were recovered among which 283 oocyte were suitable for IVM. In the groups of leptin treated with 0 (control, 10, 50 and 100 ng/ml, the percentage of oocytes maturation was 74.65, 83.81, 77.85, and 75.40%, while the

  19. Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells

    Directory of Open Access Journals (Sweden)

    Looft C

    2011-01-01

    Full Text Available Abstract Background The bi-directional communication between the oocyte and its companion cumulus cells (CCs is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa. Results We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO + CCs and without (OO - CCs CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs + OO or without (CCs - OO their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs + OO and CCs - OO, respectively. While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18, translation (EIF2AK1, EIF4ENIF1 and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI, protein metabolic processes (IHH, APOA1, PLOD1, steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7. Similarly, while transcripts over expressed in OO + CCs

  20. Gene expression, oocyte nuclear maturation and developmental competence of bovine oocytes and embryos produced after in vivo and in vitro heat shock.

    Science.gov (United States)

    Pavani, Krishna C; Baron, Erica; Correia, Pedro; Lourenço, Joana; Bettencourt, Bruno Filipe; Sousa, Madalena; da Silva, Fernando Moreira

    2016-10-01

    Three assays were performed. In assay 1, oocytes harvested during the winter months were subjected to kinetic heat shock by stressing the oocytes at 39.5°C (HS1) or at 40.5°C (HS2) for either 6, 12, 18 or 24 h and then matured at control temperature (38.5°C). The nuclear maturation rates (NMR) of all oocytes were recorded after 24 h. In assay 2, oocytes collected year-round maturated, were implanted via in vitro fertilization (IVF) and developed for 9 days. Gene expression analysis was performed on target genes (Cx43, CDH1, DNMT1, HSPA14) with reference to the two housekeeping genes (GAPDH and SDHA) in embryos. Similarly, in assay 3, genetic analysis was performed on the embryos produced from heat-stressed oocytes (from HS1 and HS2). In assay 1, the duration of heat stress resulted in a significant decline in NMR (P CDH1 genes (P < 0.05). Targeted gene expression was aberrant in embryo development, which can provide evidence on early embryo arrest and slowed embryo development.

  1. Deletion of Mylk1 in oocytes causes delayed morula-to-blastocyst transition and reduced fertility without affecting folliculogenesis and oocyte maturation in mice.

    Science.gov (United States)

    Liang, Qiu-Xia; Zhang, Qing-Hua; Qi, Shu-Tao; Wang, Zhong-Wei; Hu, Meng-Wen; Ma, Xue-Shan; Zhu, Min-Sheng; Schatten, Heide; Wang, Zhen-Bo; Sun, Qing-Yuan

    2015-04-01

    The mammalian oocyte undergoes two rounds of asymmetric cell divisions during meiotic maturation and fertilization. Acentric spindle positioning and cortical polarity are two major factors involved in asymmetric cell division, both of which are thought to depend on the dynamic interaction between myosin II and actin filaments. Myosin light chain kinase (MLCK), encoded by the Mylk1 gene, could directly phosphorylate and activate myosin II. To determine whether MLCK was required for oocyte asymmetric division, we specifically disrupted the Mylk1 gene in oocytes by Cre-loxP conditional knockout system. We found that Mylk1 mutant female mice showed severe subfertility. Unexpectedly, contrary to previously reported in vitro findings, our data showed that oocyte meiotic maturation including spindle organization, polarity establishment, homologous chromosomes separation, and polar body extrusion were not affected in Mylk1(fl/fl);GCre(+) females. Follicular development, ovulation, and early embryonic development up to compact morula occurred normally in Mylk1(fl/fl);GCre(+) females, but deletion of MLCK caused delayed morula-to-blastocyst transition. More than a third of embryos were at morula stage at 3.5 Days Postcoitum in vivo. The delayed embryos could develop further to early blastocyst stage in vitro on Day 4 when most control embryos reached expanded blastocysts. Our findings provide evidence that MLCK is linked to timely blastocyst formation, though it is dispensable for oocyte meiotic maturation. © 2015 by the Society for the Study of Reproduction, Inc.

  2. Gene expression patterns in granulosa cells and oocytes at various stages of follicle development as well as in in vitro grown oocyte-and-granulosa cell complexes.

    Science.gov (United States)

    Munakata, Yasuhisa; Kawahara-Miki, Ryoka; Shiratsuki, Shogo; Tasaki, Hidetaka; Itami, Nobuhiko; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2016-08-25

    Follicle development is accompanied by proliferation of granulosa cells and increasing oocyte size. To obtain high-quality oocytes in vitro, it is important to understand the processes that occur in oocytes and granulosa cells during follicle development and the differences between in vivo and in vitro follicle development. In the present study, oocytes and granulosa cells were collected from early antral follicles (EAFs, 0.5-0.7 mm in diameter), small antral follicles (SAFs, 1-3 mm in diameter), large antral follicles (LAFs, 3-7 mm in diameter), and in vitro grown oocyte-and-granulosa cell complexes (OGCs), which were cultured for 14 days after collection from EAFs. Gene expression was analyzed comprehensively using the next-generation sequencing technology. We found top upstream regulators during the in vivo follicle development and compared them with those in in vitro developed OGCs. The comparison revealed that HIF1 is among the top regulators during both in vivo and in vitro development of OGCs. In addition, we found that HIF1-mediated upregulation of glycolysis in granulosa cells is important for the growth of OGCs, but the cellular metabolism differs between in vitro and in vivo grown OGCs. Furthermore, on the basis of comparison of upstream regulators between in vivo and in vitro development of OGCs, we believe that low expression levels of FLT1 (VEGFA receptor), SPP1, and PCSK6 can be considered causal factors of the suboptimal development under in vitro culture conditions.

  3. Risk and prevention of bovine viral diarrhea virus (BVDV) transmission through embryo production via somatic cell nuclear transfer (SCNT) using oocytes from persistently infected donors.

    Science.gov (United States)

    Gregg, K; Riddell, K P; Chen, S H; Galik, P K; Xiang, T; Guerra, T; Marley, M S; Polejaeva, I; Givens, M D

    2010-07-01

    The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 x 10(5). The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production.

  4. Grading of Mammalian Cumulus Oocyte Complexes using Machine Learning for in Vitro Embryo Culture

    CERN Document Server

    Sudarshan, Viswanath P; Chintala, Phalgun; Mandal, Subhamoy; Dutta, Rahul

    2016-01-01

    Visual observation of Cumulus Oocyte Complexes provides only limited information about its functional competence, whereas the molecular evaluations methods are cumbersome or costly. Image analysis of mammalian oocytes can provide attractive alternative to address this challenge. However, it is complex, given the huge number of oocytes under inspection and the subjective nature of the features inspected for identification. Supervised machine learning methods like random forest with annotations from expert biologists can make the analysis task standardized and reduces inter-subject variability. We present a semi-automatic framework for predicting the class an oocyte belongs to, based on multi-object parametric segmentation on the acquired microscopic image followed by a feature based classification using random forests.

  5. Using oocyte nuclei for studies on chromatin structure and gene expression.

    Science.gov (United States)

    Sommerville, John

    2010-05-01

    The giant nucleus of amphibian oocytes is generally referred to as the germinal vesicle (GV). Its size allows relatively easy manual isolation from the rest of the oocyte and also presents a large target in situ for microinjection of macromolecules including plasmid DNA, RNA species, antibodies and other proteins and even whole organelles, including somatic cell nuclei. Thus the use of GVs is excellent for two major types of study: the function of endogenous nuclear processes such as gene transcription, RNA processing and intra-nuclear dynamics; and the use of the nuclear components to effect processes such as chromatin assembly, expression of foreign genes and nucleocytoplasmic transport of injected biomolecules. This article outlines some basic techniques appropriate for GV studies, particularly the preparation of oocytes for microinjection and the isolation of germinal vesicles into an oil phase. As an aid to the targeting of the GV within the nucleus, descriptions are given of the use of oocytes from albino animals.

  6. Radiation- and drug-induced DNA repair in mammalian oocytes and embryos

    Energy Technology Data Exchange (ETDEWEB)

    Pedersen, R A; Brandriff, B

    1979-01-01

    A review of studies showing ultraviolet- or drug-induced unscheduled DNA synthesis in mammalian oocytes and embryos suggests that the female gamete has an excision repair capacity from the earliest stages of oocyte growth. The oocyte's demonstrable excision repair capacity decreases at the time of meiotic maturation for unknown reasons, but the fully mature oocyte maintans a repair capacity, in contrast to the mature sperm, and contributes this to the zygote. Early embryo cells maintain relatively constant levels of excision repair until late fetal stages, when they lose their capacity for excision repair. These apparent changes in excision repair capacity do not have a simple relationship to known differences in radiation sensitivity of germ cells and embryos.

  7. Nucleolus precursor body (NPB): a distinct structure in mammalian oocytes and zygotes.

    Science.gov (United States)

    Kyogoku, Hirohisa; Kitajima, Tomoya S; Miyano, Takashi

    2014-01-01

    Nucleoli in mammalian oocytes and zygotes, sometimes referred to as nucleolus precursor bodies (NPBs), are compact and morphologically different from nucleoli in somatic cells. We applied a unique NPB analyzing method "enucleolation" technique to zygotes to remove the NPBs. It has been reported that oocyte NPBs are essential for embryonic development; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygotes and embryos, leading to developmental failure. However, we found that when NPBs were removed from zygotes, the zygotes developed successfully to live-born pups. These results indicated that oocyte NPBs are essential for embryonic development, but zygote NPBs are not. In addition, the enucleolated zygotes formed somatic-type nucleoli during early embryonic development, demonstrating that somatic-type nucleoli do not originate from zygote NPBs. We summarize our recent investigation on NPBs, and provide additional comments and findings.

  8. Meiosis I in Xenopus oocytes is not error-prone despite lacking spindle assembly checkpoint.

    Science.gov (United States)

    Liu, Dandan; Shao, Hua; Wang, Hongmei; Liu, X Johné

    2014-01-01

    The spindle assembly checkpoint, SAC, is a surveillance mechanism to control the onset of anaphase during cell division. SAC prevents anaphase initiation until all chromosome pairs have achieved bipolar attachment and aligned at the metaphase plate of the spindle. In doing so, SAC is thought to be the key mechanism to prevent chromosome nondisjunction in mitosis and meiosis. We have recently demonstrated that Xenopus oocyte meiosis lacks SAC control. This prompted the question of whether Xenopus oocyte meiosis is particularly error-prone. In this study, we have karyotyped a total of 313 Xenopus eggs following in vitro oocyte maturation. We found no hyperploid egg, out of 204 metaphase II eggs with countable chromosome spreads. Therefore, chromosome nondisjunction is very rare during Xenopus oocyte meiosis I, despite the lack of SAC.

  9. Microtransplantation of acetylcholine receptors from normal or denervated rat skeletal muscles to frog oocytes

    Science.gov (United States)

    Bernareggi, Annalisa; Reyes-Ruiz, Jorge Mauricio; Lorenzon, Paola; Ruzzier, Fabio; Miledi, Ricardo

    2011-01-01

    Cell membranes, carrying neurotransmitter receptors and ion channels, can be ‘microtransplanted’ into frog oocytes. This technique allows a direct functional characterization of the original membrane proteins, together with any associated molecules they may have, still embedded in their natural lipid environment. This approach has been previously demonstrated to be very useful to study neurotransmitter receptors and ion channels contained in cell membranes isolated from human brains. Here, we examined the possibility of using the microtransplantation method to study acetylcholine receptors from normal and denervated rat skeletal muscles. We found that the muscle membranes, carrying their fetal or adult acetylcholine receptor isoforms, could be efficiently microtransplanted to the oocyte membrane, making the oocytes become sensitive to acetylcholine. These results show that oocytes injected with skeletal muscle membranes efficiently incorporate functional acetylcholine receptors, thus making the microtransplantation approach a valuable tool to further investigate receptors and ion channels of human muscle diseases. PMID:21224230

  10. Low oocyte yield during IVF treatment and the risk of a trisomic pregnancy

    DEFF Research Database (Denmark)

    Honorato, Talita; Hoek, Annemieke; Henningsen, Anna-Karina

    2017-01-01

    .2% (22/357) controls. Low oocyte yield in IVF treatment was not associated with a higher risk of trisomic pregnancy (OR 1.43, 95% CI 0.64 to 3.19). Stratification for female age, adjustment for history of ovarian surgery, and gonadotrophin-releasing hormone protocol used did not change the results.......A low number of antral follicles may result in the selection of suboptimal oocytes that are prone to meiotic errors. The aim of this case-control study was to evaluate women receiving IVF treatment with low oocyte yield (defined as three or fewer oocytes retrieved after ovarian stimulation) who...... are at an increased risk of a trisomic pregnancy. Data were obtained from Danish and Dutch medical registries between 1983 and 2011. Analyses were carried out in 105 cases and 442 controls matched by age and year of IVF treatment. Cases were women with a trisomic pregnancy (trisomies 13, 18 or 21) resulting from...

  11. Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes

    National Research Council Canada - National Science Library

    Preller, Ana; Wilson, Christian A.M; Quiroga-Roger, Diego; Ureta, Tito

    2013-01-01

    .... Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase...

  12. Increased stress tolerance of matured pig oocytes after high hydrostatic presure

    DEFF Research Database (Denmark)

    Pribenszky, Cs; Du, Y; Molnár, M;

    2008-01-01

    treated with different pressure impulses in the range of 20-80 MPa (200-800 times greater than atmospheric pressure) for 30-120 min at 24 °C. For parthenogenetic activation a single dc of 12.5 kV/cm was used, to test shock tolerance of the treated vs. control oocytes and also compare their developmental...... electric (single dc of 1.25 kV/cm) and chemical treatment after warming. According to our investigations performed with a total of 1980 oocytes and 3-5 replicates, pressure treatment increased cleavage and blastocyst rates after activation and cleavage rates after vitrification followed by activation. Our...... results indicate that the sublethal pressure treatment may induce specific responses in oocytes increasing their resistance and developmental competence. The mechanism that may lie behind the observations is the sublethal stress-induced post-transcriptional activation of shock proteins in the oocytes...

  13. Pain relief for women undergoing oocyte retrieval for assisted reproduction.

    Science.gov (United States)

    Kwan, Irene; Bhattacharya, Siladitya; Knox, Fiona; McNeil, Alex

    2013-01-31

    Various methods of conscious sedation and analgesia have been used for pain relief during oocyte recovery in in vitro fertilisation (IVF) and intra-cytoplasmic sperm injection (ICSI) procedures. The choice of agent has also been influenced by the quality of sedation and analgesia as well as by concerns about possible detrimental effects on reproductive outcomes. To assess the effectiveness and safety of different methods of conscious sedation and analgesia on pain relief and pregnancy outcomes in women undergoing transvaginal oocyte retrieval. We searched the Menstrual Disorders and Subfertility Group Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL) on The Cochrane Library, MEDLINE, EMBASE, PsycINFO, CINAHL (from their inception to present); the National Research Register and Current Controlled Trials. We searched reference lists of included studies for relevant studies and contacted authors for information on unpublished and ongoing trials. There was no language restriction. The search was updated in July 2012. Only randomised controlled trials comparing different methods of conscious sedation and analgesia for pain relief during oocyte recovery were included. Quality assessment and data extraction were performed independently by two review authors. Interventions were classified and analysed under broad categories or strategies of sedation and pain relief to compare different methods and administrative protocols of conscious sedation and analgesia. Outcomes were extracted and the data were pooled when appropriate. With this update, nine new studies were identified resulting in a total of 21 trials including 2974 women undergoing oocyte retrieval. These trials compared five different categories of conscious sedation and analgesia: 1) conscious sedation and analgesia versus placebo; 2) conscious sedation and analgesia versus other active interventions such as general and acupuncture anaesthesia; 3) conscious sedation and analgesia plus

  14. Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

    Science.gov (United States)

    McGinnis, Lynda K; Luo, Jinping; Kinsey, William H

    2013-04-01

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

  15. Resveratrol-induced mitochondrial synthesis and autophagy in oocytes derived from early antral follicles of aged cows.

    Science.gov (United States)

    Sugiyama, Miyako; Kawahara-Miki, Ryoka; Kawana, Hirosuke; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2015-01-01

    Mitochondrial numbers increase during oocyte growth. In this study, we collected oocytes and granulosa cell complexes (OGCs) from early antral follicles (EAFs) of aged cows (> 120 months of age) and examined the effects of resveratrol on mitochondrial generation, degradation, and quality in oocytes grown in vitro. We also examined the effects of resveratrol on gene expression of the granulosa cells. Resveratrol (20 µM) enhanced the expression of SIRT1 and induced autophagy in both granulosa cells and oocytes derived from aged cows. Culturing the OGCs with resveratrol increased mitochondrial DNA copy numbers in oocytes grown in vitro. Furthermore, resveratrol increased the ATP content in oocytes and improved the developmental ability of the oocytes to the blastocyst stage. Gene expression profiles in granulosa cells, as evaluated by next-generation sequencing technology, revealed that resveratrol enhanced the expression of EIF2-related genes but downregulated the expression of mammalian target of rapamycin (mTOR)-, inflammation-, and cholesterol homeostasis-related genes in granulosa cells. In conclusion, resveratrol affected both oocytes and granulosa cells derived from aged cows and improved the quality of oocytes grown in vitro through upregulation of mitochondrial biogenesis and degradation in growing oocytes and conditioning of granulosa cells.

  16. Astaxanthin present in the maturation medium reduces negative effects of heat shock on the developmental competence of porcine oocytes.

    Science.gov (United States)

    Do, Lanh Thi Kim; Luu, Vien Viet; Morita, Yasuhiro; Taniguchi, Masayasu; Nii, Masahiro; Peter, Augustine T; Otoi, Takeshige

    2015-06-01

    Astaxanthin, one of the most common carotenoids, elicits antioxidant effects on cellular viability and embryonic development. This study was conducted to investigate the effects of astaxanthin on maturation, fertilization and development of porcine oocytes matured in vitro under heat stress conditions, and then fertilized and cultured under standard conditions. Porcine oocytes were cultured in maturation medium supplemented with different concentrations of astaxanthin (0, 0.25, 0.5 or 1 ppm) for 46 h at either 38.5 or 41 °C. In comparison to oocytes cultured at 38.5 °C, the exposure of porcine oocytes to 41.0 °C during in vitro maturation (IVM) significantly inhibited maturation and development of fertilized oocytes to the blastocyst stage. Supplementation of maturation medium with astaxanthin (0.5 ppm) significantly improved oocyte maturation, fertilization and development to the blastocysts stage in both oocyte groups. However, the total cell number and apoptosis index of blastocysts did not differ among groups. Moreover, astaxanthin (0.5 ppm) significantly increased the rate of oocytes that reached metaphase II and decreased proportion of apoptotic oocytes exposed to H2O2 (1.0mM) during IVM. In summary, we demonstrated that supplementation of maturation medium with astaxanthin (0.5 ppm) exerted antioxidative effects and improved the ability of maturation, fertilization, and development of porcine oocytes exposed to heat stress.

  17. Chemical activation of in vitro matured dromedary camel (Camelus dromedarius) oocytes: optimization of protocols.

    Science.gov (United States)

    Wani, N A

    2008-03-15

    Experiments were conducted to study the efficiency of sequential treatments of ionomycine and ethanol combined with phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine) in inducing artificial activation in dromedary M-II oocytes. Cumulus oocyte complexes (COCs), collected from slaughterhouse ovaries were cultured at 38.5 degrees C in an atmosphere of 5% CO2 in air for 24-48 h. In experiment 1, the COCs were either fertilized in vitro or activated with 5 microM ionomycine for 5 min or 7% ethanol for 7 min, both followed by exposure to 6-diethylaminopurine or roscovitine for 4h. After 14-15 h of in vitro culture, the oocytes were fixed and stained with 1% aceto-orcein to evaluate their nuclear status. In experiment 2, the oocytes were activated in the same manner as in experiment 1 but were cultured for 7 days to evaluate their post-parthenogenetic development. In experiment 3, oocytes were exposed to the ionomycine for 2, 3, 4 or 5 min to evaluate the better exposure time while as in experiment 4, the oocytes matured for 28-48 h were activated to see the effect of aging on post-parthenogenetic development. Higher proportion (P0.05) in the proportion of oocytes activated with ethanol when compared with in vitro fertilized group. No significant difference was seen on the proportion of morula on day 7 of culture, however the development to blastocyst stage was higher (P0.05). The proportion of blastocysts obtained was higher (Pdromedary camel oocytes with ionomycine/6-DMAP is demonstrated and optimized in the present study for further use in the development of assisted reproductive techniques in this species.

  18. Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

    Directory of Open Access Journals (Sweden)

    Paula Stein

    2015-02-01

    Full Text Available The RNase III enzyme DICER generates both microRNAs (miRNAs and endogenous short interfering RNAs (endo-siRNAs. Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO family of proteins. In mammals, there are four AGO proteins (AGO1-4, of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT, the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals.

  19. Lin28a is dormant, functional, and dispensable during mouse oocyte-to-embryo transition.

    Science.gov (United States)

    Flemr, Matyas; Moravec, Martin; Libova, Veronika; Sedlacek, Radislav; Svoboda, Petr

    2014-06-01

    The oocyte-to-embryo transition (OET) denotes transformation of a highly differentiated oocyte into totipotent blastomeres of the early mammalian embryo. OET depends exclusively on maternal RNAs and proteins accumulated during oocyte growth, which implies importance of post-transcriptional control of gene expression. OET includes replacement of abundant maternal microRNAs (miRNAs), enriched also in differentiated cells and exemplified by the Let-7 family, with embryonic miRNAs common in pluripotent stem cells (the miR-290 family in the mouse). Lin28a and its homolog Lin28b encode RNA-binding proteins, which interfere with Let-7 maturation and facilitate reprogramming of induced pluripotent stem cells. Both Lin28a and Lin28b transcripts are abundant in mouse oocytes. To test the role of maternal expression of Lin28a and Lin28b during oocyte-to-zygote reprogramming, we generated mice with oocyte-specific knockdown of both genes by using transgenic RNA interference. Lin28a and Lin28b are dispensable during oocyte growth because their knockdown has no effect on Let-7a levels in fully grown germinal vesicle (GV)-intact oocytes. Furthermore, transgenic females were fertile and produced healthy offspring, and their overall breeding performance was comparable to that of wild-type mice. At the same time, 2-cell embryos derived from transgenic females showed up-regulation of mature Let-7, suggesting that maternally provided LIN28A and LIN28B function during zygotic genome activation. Consistent with this conclusion is increased translation of Lin28a transcripts upon resumption of meiosis. Our data imply dual repression of Let-7 during OET in the mouse model, the selective suppression of Let-7 biogenesis by Lin28 homologs superimposed on previously reported global suppression of miRNA activity. © 2014 by the Society for the Study of Reproduction, Inc.

  20. Maternal death after oocyte donation at high maternal age: case report

    Directory of Open Access Journals (Sweden)

    Steegers Eric AP

    2008-12-01

    Full Text Available Abstract Background The percentage of women giving birth after the age of 35 increased in many western countries. The number of women remaining childless also increased, mostly due to aging oocytes. The method of oocyte donation offers the possibility for infertile older women to become pregnant. Gestation after oocyte-donation-IVF, however, is not without risks for the mother, especially at advanced age. Case presentation An infertile woman went abroad for oocyte-donation-IVF, since this treatment is not offered in The Netherlands after the age of 45. The first oocyte donation treatment resulted in multiple gestation, but was ended by induced abortion: the woman could not cope with the idea of being pregnant with twins. During the second pregnancy after oocyte donation, at the age of 50, she was mentally more stable. The pregnancy, again a multiple gestation, was uneventful until delivery. Immediately after delivery the woman had hypertension with nausea and vomiting. A few hours later she had an eclamptic fit. HELLP-syndrome was diagnosed. She died due to cerebral haemorrhage. Conclusion In The Netherlands, the age limit for women receiving donor oocytes is 45 years and commercial oocyte donation is forbidden by law. In other countries there is no age limit, the reason why some women are going abroad to receive the treatment of their choice. Advanced age, IVF and twin pregnancy are all risk factors for pre-eclampsia, the leading cause of maternal death in The Netherlands. Patient autonomy is an important ethical principle, but doctors are also bound to the principle of 'not doing harm', and do have the right to refuse medical treatment such as IVF-treatment. The discussion whether women above 50 should have children is still not closed. If the decision is made to offer this treatment to a woman at advanced age, the doctor should counsel her intensively about the risks before treatment is started.

  1. Permeability characteristics of human oocytes in the presence of the cryoprotectant dimethylsulphoxide.

    Science.gov (United States)

    Paynter, S J; Cooper, A; Gregory, L; Fuller, B J; Shaw, R W

    1999-09-01

    Equilibration of oocytes with cryoprotectants is a prerequisite of low temperature storage. However, cryoprotectant exposure may induce damage via osmotic stress. Knowledge of cell membrane permeability characteristics and their temperature dependence would facilitate the design of cryopreservation protocols in which osmotic stress is minimized and the incidence of intracellular freezing is reduced. To obtain such data, the volume change of donated human oocytes following exposure to cryoprotectant was measured at a variety of temperatures. After removal of cumulus cells, each oocyte was placed in a 5 microl droplet of phosphate-buffered medium. The oocyte was held in position by suction generated using a fine pipette and perfused with 1 ml 1.5 mol/l dimethylsulphoxide (DMSO) at 30, 24 or 10 degrees C. The volume of the oocyte before, during and after perfusion was recorded by videomicroscopy. Oocyte volume was calculated from radius measurements and the Kedem-Katchalsky (K-K) passive coupled transport coefficients, namely L(p) (hydraulic permeability), P(DMSO) (permeability to DMSO) and sigma (reflection coefficient) were derived. The resulting coefficients were L(p) = 1. 65 +/- 0.15, 0.70 +/- 0.06 and 0.28 +/- 0.04 microm/min.atm; P(DMSO) = 0.79 +/- 0.10, 0.25 +/- 0.04 and 0.06 +/- 0.01 microm/s and sigma = 0.97 +/- 0.01, 0.94 +/- 0.03 and 0.96 +/- 0.01 at 30, 24 and 10 degrees C respectively. The activation energy for L(p) was 14.70 and for P(DMSO) was 20.82 kcal/mol. The permeability parameters of human oocytes are higher than those of murine oocytes, suggesting that they require a shorter period of exposure to DMSO with concomitantly reduced toxic effects.

  2. In vitro maturation and embryo development of bovine oocytes after meiosis blockage with MPF inhibitors

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    Mariana Groke Marques

    2011-12-01

    Full Text Available This study evaluated the bovine oocyte maturation and embryo development after in vitro fertilization. The maturation of the oocytes was blocked using Butyrolactone I and Roscovitine using pre-maturation medium supplemented with fetal calf serum (FCS. The ocytes were divided in four groups: Control 0 hour, Control (24 hours of maturation, Roscovitine (maturation blockage with 50mM Roscovitine during 24 hours followed by 24 hours of maturation, and Butyrolactone I (maturation blockage with 150mM Butyrolactone I during 24 hours followed by 24 hours of maturation. The oocytes were fixed and stained with aceto orcein to evaluate the nuclear maturation. After the maturation period, the remaining oocytes of the Control group, Roscovitine, and Butyrolactone I were fertilized in vitro. Embryo development was assessed by the cleavage rate (D3 and blastocysts formation (D7. The Butyrolactone I group had similar rates of germinal vesical stage oocytes during blockage, and Metaphase 2 after maturation, comparing to Control group at 0 hour and Control group, respectively. On the other hand, the Roscovitine group had lower rates of vesical stage oocytes during blockage, and Metaphase 2 after maturation comparing to Control groups. After in vitro fertilization, higher rates of cleavage were observed in Control and Butyrolactone I groups. For the blastocyst formation rate, the Control group showed better results than Roscovitine group. In summary, Butyrolactone I group had similar results to the Control group, and for this reason, is suitable for pre-maturation of bovine oocytes using FCS. In contrast, Roscovitine group had lower oocyte maturation and embryo development.

  3. RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

    Science.gov (United States)

    Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

    2016-10-01

    Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

  4. Potential role for MATER in cytoplasmic lattice formation in murine oocytes.

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    Boram Kim

    Full Text Available BACKGROUND: Mater and Padi6 are maternal effect genes that are first expressed during oocyte growth and are required for embryonic development beyond the two-cell stage in the mouse. We have recently found that PADI6 localizes to, and is required for the formation of, abundant fibrillar Triton X-100 (Triton insoluble structures termed the oocyte cytoplasmic lattices (CPLs. Given their similar expression profiles and mutant mouse phenotypes, we have been testing the hypothesis that MATER also plays a role in CPL formation and/or function. METHODOLOGY/FINDINGS: Herein, we show that PADI6 and MATER co-localize throughout the oocyte cytoplasm following Triton extraction, suggesting that MATER co-localizes with PADI6 at the CPLs. Additionally, the solubility of PADI6 was dramatically increased in Mater(tm/tm oocytes following Triton extraction, suggesting that MATER is involved in CPL nucleation. This prediction is supported by transmission electron microscopic analysis of Mater(+/+ and Mater(tm/tm germinal vesicle stage oocytes which illustrated that volume fraction of CPLs was reduced by 90% in Mater(tm/tm oocytes compared to Mater(+/+ oocytes. CONCLUSIONS: Taken together, these results suggest that, similar to PADI6, MATER is also required for CPL formation. Given that PADI6 and MATER are essential for female fertility, these results not only strengthen the hypothesis that the lattices play a critical role in mediating events during the oocyte-to-embryo transition but also increase our understanding of the molecular nature of the CPLs.

  5. The first observations of oocyte diameters for the pipefish from the southern Black Sea

    OpenAIRE

    GÜRKAN, Şule; Taylan, Burcu

    2013-01-01

    Abstract Syngnathid specimens sample between July 2001˗May 2002 was examined about measurements of oocyte diameter in the coastal waters of Sinop peninsula, southern Back Sea. The specimens of Syngnathid fishes (Syngnathus acus, Syngnathus typhle, Syngnathus variegatus, Syngnathus tenuirostris, Nerophis ophidion) catched with using hand dredge from the vegetated coastal area. According to results, the mean of hydrated oocyte diameter was calculated as, respectively: Syngnathus acus: 1.96 mm, ...

  6. Comparison of ethylene glycol and propylene glycol for the vitrification of immature porcine oocytes.

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    Somfai, Tamás; Nakai, Michiko; Tanihara, Fuminori; Noguchi, Junko; Kaneko, Hiroyuki; Kashiwazaki, Naomi; Egerszegi, István; Nagai, Takashi; Kikuchi, Kazuhiro

    2013-01-01

    Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.

  7. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method.

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    Cobo, Ana; Kuwayama, Masashigue; Pérez, Sonia; Ruiz, Amparo; Pellicer, Antonio; Remohí, José

    2008-06-01

    To evaluate the outcome of oocyte vitrification using the Cryotop method, observed in an egg donation program by simultaneously evaluating embryos derived from vitrified and fresh oocytes coming from the same stimulated cycle. Cohort prospective randomized study. Instituto Valenciano de Infertilidad (IVI) Valencia, Spain. Thirty oocyte donors and 30 recipients with informed consents. Vitrification by the Cryotop method. Warming 1 hour after vitrification. Microinjection of surviving MII and fresh oocytes, evaluation of fertilization, embryo development, and clinical results. Survival, fertilization, and cleavage rate. Embryo quality, pregnancy rate (PR), and implantation rate. Survival rate observed was 96.7%. There was no difference in fertilization rates (76.3% and 82.2%), day 2 cleavage (94.2% and 97.8%), day 3 cleavage (80.8% and 80.5%), and blastocyst formation (48.7% and 47.5%) for vitrified and fresh oocytes, respectively. Embryo quality on day 3 and on day 5-6 were similar for vitrification and fresh oocyte group (80.8% vs. 80.5% and 81.1% vs. 70%, respectively). A total of 23 embryo transfers were carried out in the vitrification group. Pregnancy rates, implantation rates, miscarriage rates, and ongoing PR were 65.2%, 40.8%, 20%, and 47.8%, respectively. The Cryotop method preserves the potential of vitrified oocytes to fertilize and further develop, which is similar, when evaluated simultaneously, to fresh counterparts. Excellent clinical outcome indicates the possible use of this technology for egg donation programs, as well as a high potential for establishing oocyte banking.

  8. Obstetric outcome in donor oocyte pregnancies: a matched-pair analysis

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    Stoop Dominic

    2012-06-01

    Full Text Available Abstract Background To investigate the obstetrical and perinatal impact of oocyte donation, a cohort of women who conceived after OD was compared with a matched control group of women who became pregnant through in vitro fertilisation with autologous oocytes (AO. Methods A matched-pair analysis has been performed at the Centre for Reproductive Medicine of the UZ Brussel, Dutch speaking Free University of Brussel. A total of 410 pregnancies resulted in birth beyond 20 weeks of gestation occurring over a period of 10 years, including 205 oocyte donation pregnancies and 205 ICSI pregnancies with autologous oocytes (AO. Patients in the OD group were matched on a one-to-one basis with the AO group in terms of age, ethnicity, parity and plurality. Matched groups were compared using paired t-tests for continuous variables and McNemar test for categorical variables. A conditional logistic regression analyses was performed adjusting for paternal age, age of the oocyte donor, number of embryos transferred, and singleton/twin pregnancy. Results Oocyte donation was associated with an increased risk of pregnancy induced hypertension (PIH (matched OR: 1.502 CI: 1.024-2.204, and first trimester bleeding (matched OR: 1.493 CI: 1.036-2.15. No differences were observed between the two matched groups with regard to gestational age, mean birth weight and length, head circumference and Apgar scores. Conclusions Oocyte donation is associated with an increased risk for PIH and first trimester bleeding independent of the recipients’ age, parity and plurality, and independent of the age of the donor or the partner. However, oocyte donation has no impact on the overall perinatal outcome.

  9. Live, full-term mouse pups from oocytes grown and matured in vitro with serum substitutes.

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    Motohashi, Hideyuki H; Taniguchi, Ryoma; Sakamoto, Junpei; Sankai, Tadashi; Kada, Hidemi

    2017-06-01

    For in vitro growth and maturation of mouse oocytes (IVG-IVM), serum is added to media up to and including the stage of oocyte maturation; this subsequently supports oocytes through fertilization and early embryo development. However, problems may occur with sera, such as batch differences and issues of biosafety. The purpose of the present study was to determine the capacity for fertilization and pre- and post-implantation development of oocytes that underwent IVG-IVM with a serum substitute. Oocyte-granulosa cell complexes from preantral follicles were cultured in medium with either fetal bovine serum (FBS), Serum Substitute Supplement™ (SSS), or Knockout™ Serum Replacement (KSR) for 10days, and were then allowed to mature for 17 h. Subsequently, more than 90% of oocytes underwent germinal vesicle breakdown (GVBD) and more than 70% reached metaphase II, with no significant difference between the groups. A lower fertilization rate, presumably due to zona hardening, was found in the serum substitute groups. Nevertheless, more than 50% of the inseminated oocytes were fertilized and 35%-45% of them underwent first cleavage and developed to the blastocyst stage. Following embryo transfer, one and four live offspring were produced from the SSS and KSR groups, respectively. The present study demonstrated that murine IVG-IVM oocytes cultured in media with a serum substitute, achieved fertilization in vitro, pre- and post-implantation development, and the delivery of live pups, although the efficiency of the process is reduced compared to FBS supplementation. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  10. How oocytes try to get it right: spindle checkpoint control in meiosis.

    Science.gov (United States)

    Touati, Sandra A; Wassmann, Katja

    2016-06-01

    The generation of a viable, diploid organism depends on the formation of haploid gametes, oocytes, and spermatocytes, with the correct number of chromosomes. Halving the genome requires the execution of two consecutive specialized cell divisions named meiosis I and II. Unfortunately, and in contrast to male meiosis, chromosome segregation in oocytes is error prone, with human oocytes being extraordinarily "meiotically challenged". Aneuploid oocytes, that are with the wrong number of chromosomes, give rise to aneuploid embryos when fertilized. In humans, most aneuploidies are lethal and result in spontaneous abortions. However, some trisomies survive to birth or even adulthood, such as the well-known trisomy 21, which gives rise to Down syndrome (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012). A staggering 20-25 % of oocytes ready to be fertilized are aneuploid in humans. If this were not bad enough, there is an additional increase in meiotic missegregations as women get closer to menopause. A woman above 40 has a risk of more than 30 % of getting pregnant with a trisomic child. Worse still, in industrialized western societies, child birth is delayed, with women getting their first child later in life than ever. This trend has led to an increase of trisomic pregnancies by 70 % in the last 30 years (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012; Schmidt et al. in Hum Reprod Update 18:29-43, 2012). To understand why errors occur so frequently during the meiotic divisions in oocytes, we review here the molecular mechanisms at works to control chromosome segregation during meiosis. An important mitotic control mechanism, namely the spindle assembly checkpoint or SAC, has been adapted to the special requirements of the meiotic divisions, and this review will focus on our current knowledge of SAC control in mammalian oocytes. Knowledge on how chromosome segregation is controlled in mammalian oocytes may help to identify risk factors important for questions

  11. The Role of Glucose Metabolism on Porcine Oocyte Cytoplasmic Maturation and Its Possible Mechanisms

    Science.gov (United States)

    Kwon, Jeong-Woo; Jin, Yong-Xun; Park, Shun-Ha; Wang, Hai-Yang; Sun, Tian-Yi; Zhang, Jia-Bao; Kim, Nam-Hyung

    2016-01-01

    In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 μM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 μM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity. PMID:27997591

  12. Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy.

    Science.gov (United States)

    Funahashi, H; Ekwall, H; Rodriguez-Martinez, H

    2000-11-01

    Morphological changes in zona pellucidae (ZP) isolated from in vitro-matured (IVM) and ovulated porcine oocytes were compared before or after fertilization in vitro and in vivo, respectively, by using scanning electron microscopy (SEM). The ZP of some ovulated or IVM oocytes and in vivo- or in vitro-fertilized (IVF) zygotes were equally split into two halves while immersed in an enzyme-inhibitor solution, using a surgical blade. After washing, intact and ZP halves were fixed in 1% glutaraldehyde solution in 0.1 M cacodylate buffer, processed, and examined using SEM. The outer surface of ZP in ovulated oocytes had a mesh-like structure. The outer morphology in IVM oocytes was more smooth although the mesh-like structure was still visible at high magnification. In in vivo zygotes and IVM-IVF zygotes, this lysed, mesh-like structure was more obvious. The inner surface of ZP had some small depressions (orifices). The mean number of orifices per 100 micrometer(2) of ZP surface was larger in IVM oocytes as compared to ovulated ones. The number of orifices per 100 micrometer(2) decreased in IVM-IVF zygotes as compared to IVM oocytes; whereas, in vivo zygotes did not differ from ovulated oocytes. The mean diameter of intact ZP as well as their mean thickness was greater in ovulated oocytes than IVM oocytes. The mean thickness of the ZP was larger in ovulated oocytes than IVM ones. The ZP thickness was larger in zygotes than in in vivo oocytes, whereas that of IVM-IVF zygotes did not differ from that of IVM oocytes. These results indicate that the morphology of ZP and the ZP reaction at sperm penetration appears to be much different between IVM oocytes and ovulated ones.

  13. Lipid characterization of individual porcine oocytes by dual mode DESI-MS and data fusion.

    Science.gov (United States)

    Pirro, Valentina; Oliveri, Paolo; Ferreira, Christina Ramires; González-Serrano, Andrés Felipe; Machaty, Zoltan; Cooks, Robert Graham

    2014-10-27

    The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo production for porcine species.

  14. Adenosine Modulates the Oocyte Developmental Competence by Exposing Stages and Synthetic Blocking during In Vitro Maturation.

    Science.gov (United States)

    Cheon, Yong-Pil

    2016-06-01

    Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. By the inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage caused of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence..

  15. Lipid content and composition of oocytes from five coral species: potential implications for future cryopreservation efforts.

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    Chiahsin Lin

    Full Text Available Given the previously documented importance of lipid concentration and composition in the successful cryopreservation of gorgonian corals, these parameters were assessed in oocytes of five species of scleractinian coral; Platygyra daedalea, Echinopora gemmacea, Echinophyllia aspera, Oxypora lacera and Astreopora expansa. Wax esters, phosphatidylethanolamine, phosphatidylcholine, and fatty acids were all measured at detectable levels, and the latter were produced at significantly elevated quantities in E. gemmacea, E. aspera, and O. lacera. On the other hand, phosphatidylethanolamine, phosphatidylcholine, and wax ester were found at significantly higher concentrations in A. expansa oocytes. Triacylglycerol was not present in any species. Interestingly, the total lipid content of oocytes from all five scleractinians was significantly lower than that of oocytes of two gorgonian species, Junceella juncea and Junceella fragilis. As higher total lipid concentrations may be correlated with greater degrees of cellular membrane fluidity at lower temperatures, it stands to reason that gorgonian coral oocytes may be more likely to survive the cryopreservation process than oocytes of scleractinian corals.

  16. Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity.

    Science.gov (United States)

    Morais-de-Sá, Eurico; Mukherjee, Avik; Lowe, Nick; St Johnston, Daniel

    2014-08-01

    The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. Here we show that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl.

  17. Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals.

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    Jeffrey M Cloutier

    2015-10-01

    Full Text Available Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX. We find that DNA double-strand break (DSB foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities.

  18. Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals.

    Science.gov (United States)

    Cloutier, Jeffrey M; Mahadevaiah, Shantha K; ElInati, Elias; Nussenzweig, André; Tóth, Attila; Turner, James M A

    2015-10-01

    Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO) and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX). We find that DNA double-strand break (DSB) foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX) levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities.

  19. The cohesion stabilizer sororin favors DNA repair and chromosome segregation during mouse oocyte meiosis.

    Science.gov (United States)

    Huang, Chun-Jie; Yuan, Yi-Feng; Wu, Di; Khan, Faheem Ahmed; Jiao, Xiao-Fei; Huo, Li-Jun

    2017-03-01

    Maintenance and timely termination of cohesion on chromosomes ensures accurate chromosome segregation to guard against aneuploidy in mammalian oocytes and subsequent chromosomally abnormal pregnancies. Sororin, a cohesion stabilizer whose relevance in antagonizing the anti-cohesive property of Wings-apart like protein (Wapl), has been characterized in mitosis; however, the role of Sororin remains unclear during mammalian oocyte meiosis. Here, we show that Sororin is required for DNA damage repair and cohesion maintenance on chromosomes, and consequently, for mouse oocyte meiotic program. Sororin is constantly expressed throughout meiosis and accumulates on chromatins at germinal vesicle (GV) stage/G2 phase. It localizes onto centromeres from germinal vesicle breakdown (GVBD) to metaphase II stage. Inactivation of Sororin compromises the GVBD and first polar body extrusion (PBE). Furthermore, Sororin inactivation induces DNA damage indicated by positive γH2AX foci in GV oocytes and precocious chromatin segregation in MII oocytes. Finally, our data indicate that PlK1 and MPF dissociate Sororin from chromosome arms without affecting its centromeric localization. Our results define Sororin as a determinant during mouse oocyte meiotic maturation by favoring DNA damage repair and chromosome separation, and thereby, maintaining the genome stability and generating haploid gametes.

  20. Elevated non-esterified fatty acid concentrations during bovine oocyte maturation compromise early embryo physiology.

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    Veerle Van Hoeck

    Full Text Available Elevated concentrations of serum non-esterified fatty acids (NEFA, associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism.

  1. Adverse Effect of Superovulation Treatment on Maturation, Function and Ultrastructural Integrity of Murine Oocytes.

    Science.gov (United States)

    Lee, Myungook; Ahn, Jong Il; Lee, Ah Ran; Ko, Dong Woo; Yang, Woo Sub; Lee, Gene; Ahn, Ji Yeon; Lim, Jeong Mook

    2017-08-01

    Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve ultrastructural integrity and developmental competence of oocytes following oocyte activation and embryo culture. A randomized study using mouse model was designed and in vitro development (experiment 1), ultrastructural morphology (experiment 2) and functional integrity of the oocytes (experiment 3) retrieved after PMSG/hCG injection (superovulation group) or not (natural ovulation; control group) were evaluated. In experiment 1, more oocytes were retrieved following superovulation than following natural ovulation, but natural ovulation yielded higher (p superovulation. The capacity of mature oocytes to form pronucleus and to develop into blastocysts in vitro was similar. In experiment 2, a notable (p superovulation group. Multivesicular body formation was also increased, whereas early endosome formation was significantly decreased. No obvious changes in other microorganelles, however, were detected, which included the formation and distribution of mitochondria, cortical granules, microvilli, and smooth and rough endoplasmic reticulum. In experiment 3, significant decreases in mitochondrial activity, ATP production and dextran uptake were detected in the superovulation group. In conclusion, superovulation treatment may change both maturational status and functional and ultrastuctural integrity of oocytes. Superovulation effect on preimplantation development can be discussed.

  2. Role of ataxia-telangiectasia mutated (ATM) in porcine oocyte in vitro maturation.

    Science.gov (United States)

    Lin, Zi-Li; Kim, Nam-Hyung

    2015-06-01

    Ataxia-telangiectasia mutated (ATM) is critical for the DNA damage response, cell cycle checkpoints, and apoptosis. Significant effort has focused on elucidating the relationship between ATM and other nuclear signal transducers; however, little is known about the connection between ATM and oocyte meiotic maturation. We investigated the function of ATM in porcine oocytes. ATM was expressed at all stages of oocyte maturation and localized predominantly in the nucleus. Furthermore, the ATM-specific inhibitor KU-55933 blocked porcine oocyte maturation, reducing the percentages of oocytes that underwent germinal vesicle breakdown (GVBD) and first polar body extrusion. KU-55933 also decreased the expression of DNA damage-related genes (breast cancer 1, budding uninhibited by benzimidazoles 1, and P53) and reduced the mRNA and protein levels of AKT and other cell cycle-regulated genes that are predominantly expressed during G2/M phase, including bone morphogenetic protein 15, growth differentiation factor 9, cell division cycle protein 2, cyclinB1, and AKT. KU-55933 treatment decreased the developmental potential of blastocysts following parthenogenetic activation and increased the level of apoptosis. Together, these data suggested that ATM influenced the meiotic and cytoplasmic maturation of porcine oocytes, potentially by decreasing their sensitivity to DNA strand breaks, stimulating the AKT pathway, and/or altering the expression of other maternal genes.

  3. In vitro production of buffalo embryos from stepwise vitrified immature oocytes

    Directory of Open Access Journals (Sweden)

    Saber Mohammed Abd-Allah

    2009-09-01

    Full Text Available This study was conducted to produce buffalo embryos in vitro from stepwise vitrified immature oocytes. Cumulus oocyte complexes (COCs were obtained from the ovaries of slaughtered buffalo and were collected from the local abattoir. Selected COCs were exposed to a vitrification solution consisting of 40% ethylene glycol (EG plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP for 1 min and loaded in 0.25 ml plastic mini-straws containing 100 µl of 10% sucrose. The loaded cryostraws were cryopreserved by stepwise vitrification and were stored in liquid nitrogen for 4 to 6 months. Data analysis revealed a high percentage of post-thawing morphologically normal immature oocytes (80.7% with a low percentage of damaged oocytes. There were no significant differences in the maturation (82.1%, cleavage (47.6% and buffalo embryo development (15.4% produced by the stepwise vitrified immature oocytes in comparison to the three observations in fresh oocytes (88.3%, 50.4% and 19.4%, respectively, p<0.05.

  4. Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

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    Petter Franco Entringer

    2013-11-01

    Full Text Available Lipophorin (Lp is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp. Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.

  5. Studies on the physiological function of spermine in the process of progesterone induced toad oocyte maturation

    Institute of Scientific and Technical Information of China (English)

    LIRUNSHENG; JIAKETSO

    1992-01-01

    Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation.The ID50 for spermine inhibition via intra -oocyte microinjection on maturation induced by progesterone was 6.8mM(100nl).Spermine could inhibit MPF induced toad oocyte maturation with a much higher ID50.A 55 kD protein was dephosphorylated during the process of progesterone induced oocyte maturation .Spermine selectively promoted the level of phosphorylation of this protein in both progesterone-stimulated and hormone-untreated oocytes.The extent of its dephosphorylation was fairly Correlated with the percentage of GVBD in the hormone stimulated oocytes.The level of endogenous spermine was reduced by 28% between the perod of 0.40 GVBD50 and 0.60 GVBD50,at which 55 kD protein was dephosphorylated.Spermine inhibited progesterone-stimulated protein synthesis in almost the same dose dependent manner as its inhititory effect on the hormone-induced maturation,The endogenous spermine regulated 55 kD protein dephosphorylation which may trigger the increase of protein dephosphorylation which may trigger the increase of protein synthesis and in turn promote the activation of MPF,It is possible that 55 kD protein may be one of the components of messenger ribonucleoprotein(mRNP) particles.

  6. Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors.

    Science.gov (United States)

    Perkins, Adrienne T; Das, Thomas M; Panzera, Lauren C; Bickel, Sharon E

    2016-11-01

    In humans, errors in meiotic chromosome segregation that produce aneuploid gametes increase dramatically as women age, a phenomenon termed the "maternal age effect." During meiosis, cohesion between sister chromatids keeps recombinant homologs physically attached and premature loss of cohesion can lead to missegregation of homologs during meiosis I. A growing body of evidence suggests that meiotic cohesion deteriorates as oocytes age and contributes to the maternal age effect. One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Therefore, increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of cohesion and segregation errors. To test this hypothesis, we used an RNAi strategy to induce oxidative stress in Drosophila oocytes and measured the fidelity of chromosome segregation during meiosis. Knockdown of either the cytoplasmic or mitochondrial ROS scavenger superoxide dismutase (SOD) caused a significant increase in segregation errors, and heterozygosity for an smc1 deletion enhanced this phenotype. FISH analysis indicated that SOD knockdown moderately increased the percentage of oocytes with arm cohesion defects. Consistent with premature loss of arm cohesion and destabilization of chiasmata, the frequency at which recombinant homologs missegregate during meiosis I is significantly greater in SOD knockdown oocytes than in controls. Together these results provide an in vivo demonstration that oxidative stress during meiotic prophase induces chromosome segregation errors and support the model that accelerated loss of cohesion in aging human oocytes is caused, at least in part, by oxidative damage.

  7. The beneficial effect of repaglinide on in vitro maturation and development ability of immature mouse oocytes.

    Science.gov (United States)

    Kalehoei, Eshrat; Azadbakht, Mehri

    2017-08-01

    Repaglinide is a hypoglycemic drug, causing depolarization of the cell membrane, opening the voltage-gated calcium channels, and then increasing intracellular calcium in the pancreatic B cells by inhibition of the K-ATP-sensitive channels. Oocyte in vitro maturation (IVM) is influenced by different factors such as calcium signaling. In this study, we examined the effects of repaglinide on in vitro maturation and fertilization ability of mouse oocyte. Immature oocytes were isolated from female Naval Medical Research Institute mice which are 6-8 wk old mechanically and then cultured in 30 μl droplets of T6 medium with different concentrations of repaglinide. The control group did not receive repaglinide (R0). Treatment groups received different concentrations (5, 10, and 100 nM and 1 and 10 μM) of repaglinide (R1, R2, R3, R4, and R5, respectively). Oocyte in vitro maturation rate was assessed after 24 h. In vitro fertilization was performed using metaphase II oocytes obtained from R0 and R4 treatments. Embryo cleavage rate was calculated at 48 h post-IVF. Chi-square test was used for evaluating difference between control and treatment groups (p vitro oocyte maturation and embryo cleavage.

  8. In-vitro maturation and cryopreservation of oocytes at the time of oophorectomy

    Directory of Open Access Journals (Sweden)

    Melanie L. Walls

    2015-08-01

    Full Text Available A 27 year old female presented for fertility preservation prior to undergoing pelvic radiotherapy. She had previously undergone a radical laparoscopic hysterectomy for cervical carcinoma seven months earlier. A trans-vaginal oocyte aspiration was not advisable due to a vaginal recurrence of the disease. Due to a polycystic ovarian morphology (PCO, follicle stimulating hormone (FSH priming with no human chorionic gonadotrophin (hCG trigger was performed prior to oophorectomy followed by ex-vivo oocyte aspiration and in vitro maturation (IVM. All visualized follicles were punctured and follicular fluid aspirated. There were 22 immature oocytes identified and placed into maturation culture for 24 h. After this time, 15 oocytes were deemed to be mature and suitable for vitrification. Following an additional 24 h in maturation culture of the remaining 7 oocytes, three more were suitable for cryopreservation. The patient recovered well and progressed to radiotherapy three days later. This report demonstrates the use of IVM treatment to store oocytes for oncology patients in time-limited circumstances.

  9. ERK3 is required for metaphase-anaphase transition in mouse oocyte meiosis.

    Directory of Open Access Journals (Sweden)

    Sen Li

    Full Text Available ERK3 (extracellular signal-regulated kinase 3 is an atypical member of the mitogen-activated protein (MAP kinase family of serine/threonine kinases. Little is known about its function in mitosis, and even less about its roles in mammalian oocyte meiosis. In the present study, we examined the localization, expression and functions of ERK3 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that ERK3 localized to the spindles from the pre-MI stage to the MII stage. ERK3 co-localized with α-tubulin on the spindle fibers and asters in oocytes after taxol treatment. Deletion of ERK3 by microinjection of ERK3 morpholino (ERK3 MO resulted in oocyte arrest at the MI stage with severely impaired spindles and misaligned chromosomes. Most importantly, the spindle assembly checkpoint protein BubR1 could be detected on kinetochores even in oocytes cultured for 10 h. Low temperature treatment experiments indicated that ERK3 deletion disrupted kinetochore-microtubule (K-MT attachments. Chromosome spreading experiments showed that knock-down of ERK3 prevented the segregation of homologous chromosomes. Our data suggest that ERK3 is crucial for spindle stability and required for the metaphase-anaphase transition in mouse oocyte maturation.

  10. Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells.

    Science.gov (United States)

    Yamada, Mitsutoshi; Johannesson, Bjarki; Sagi, Ido; Burnett, Lisa Cole; Kort, Daniel H; Prosser, Robert W; Paull, Daniel; Nestor, Michael W; Freeby, Matthew; Greenberg, Ellen; Goland, Robin S; Leibel, Rudolph L; Solomon, Susan L; Benvenisty, Nissim; Sauer, Mark V; Egli, Dieter

    2014-06-26

    The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

  11. Effects of colchicine or demecolcine on cytoplasmic protrusions and assisted enucleation of golden hamster oocytes.

    Science.gov (United States)

    Wang, Lingyan; Jiang, Han; Su, Li; Tang, Bo; Li, Dexue; Li, Ziyi

    2009-12-01

    To establish experimental protocols for cloning golden hamsters, optimal concentrations of colchicine and demecolcine were determined for inducing cytoplasmic protrusion (containing chromosomes) and assisting enucleation of their oocytes. Denuded oocytes at different ages were treated with 2.5-10 microg/ml of colchicine for 1-4h or 0.02-0.6 microg/ml of demecolcine for 15-60 min. Cytoplasmic protrusions of oocytes were removed with a micromanipulation pipette. The results show that: 1) at 13.5-18h post-hCG injection, approximately 90% of oocytes treated for with 10 microg/ml of colchicine formed cytoplasmic protrusions, and in some oocytes enucleation occurred; 2) when treated with 0.4 microg/ml of demecolcine for 1h, cytoplasmic protrusions 13.5-18h post-hCG treatment were present in almost all oocytes; 3) after the protrusions induced by either treatment had been removed, the assisted enucleation rate was >80%, whereas it was approximately 32% with blind enucleation.

  12. Toxic effects of atrazine on porcine oocytes and possible mechanisms of action.

    Science.gov (United States)

    Yuan, Bao; Liang, Shuang; Jin, Yong-Xun; Zhang, Ming-Jun; Zhang, Jia-Bao; Kim, Nam-Hyung

    2017-01-01

    Because atrazine is a widely used herbicide, its adverse effects on the reproductive system have been extensively researched. In this study, we investigated the effects of atrazine exposure on porcine oocyte maturation and the possible mechanisms. Our results showed that the rates of oocyte maturation significantly decreased after treatment with 200 μM atrazine in vitro. Atrazine treatment resulted in abnormal spindle morphology but did not affect actin distribution. Atrazine exposure not only triggered a DNA damage response but also decreased MPF levels in porcine oocytes. Our results also revealed that atrazine worsened porcine oocyte quality by causing excessive accumulation of superoxide radicals, increasing cathepsin B activity, and decreasing the GSH level and mitochondrial membrane potential. Furthermore, atrazine decreased developmental competence of porcine oocytes up to the blastocyst stage and changed some properties: cell numbers, apoptosis, and related gene expression levels. Collectively, our results indicate that porcine oocyte maturation is defective after atrazine treatment at least through disruption of spindle morphology, MPF activity, and mitochondrial function and via induction of DNA damage, which probably reduces developmental competence.

  13. Roles of protein kinase C in oocyte meiotic maturation and fertilization

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Protein kinase C (PKC) is a superfamily of Ser/Thr protein kinases that is distributed widely in eukaryotes. It plays key regulatory roles at multiple steps of oocyte meiotic maturation and fertilization. During the process of meiotic maturation, the activation of PKC in cumulus cells stimulates meiotic maturation, whereas the activation of PKC in oocytes results in the inhibition of germinal vesicle breakdown. PKC activity increases following the meiotic maturation, and decreases at the transition of metaphase/anaphase in meiosis I, so as to facilitate the release of the first polar body and the entry of meiosis II. In fertilization of mammalian oocytes, PKC may act as one of the downstream targets of Ca2+ to stimulate the cortical granule exocytosis, release the oocytes from MII arrest and to induce pronucleus formation. PKC is also involved in the regulation of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Several PKC isoforms have been identified in mammalian oocytes, and there is evidence showing that classical PKCs may be the principal mediator of oocyte cortical reaction.

  14. Analysis of cat oocyte activation methods for the generation of feline disease models by nuclear transfer

    Directory of Open Access Journals (Sweden)

    Herrick Jason R

    2009-12-01

    Full Text Available Abstract Background Somatic cell nuclear transfer in cats offers a useful tool for the generation of valuable research models. However, low birth rates after nuclear transfer hamper exploitation of the full potential of the technology. Poor embryo development after activation of the reconstructed oocytes seems to be responsible, at least in part, for the low efficiency. The objective of this study was to characterize the response of cat oocytes to various stimuli in order to fine-tune existing and possibly develop new activation methods for the generation of cat disease models by somatic cell nuclear transfer. Methods First, changes in the intracellular free calcium concentration [Ca2+]i in the oocytes induced by a number of artificial stimuli were characterized. The stimuli included electroporation, ethanol, ionomycin, thimerosal, strontium-chloride and sodium (Na+-free medium. The potential of the most promising treatments (with or without subsequent incubation in the presence of cycloheximide and cytochalasin B to stimulate oocyte activation and support development of the resultant parthenogenetic embryos was then evaluated. Finally, the most effective methods were selected to activate oocytes reconstructed during nuclear transfer with fibroblasts from mucopolysaccharidosis I- and alpha-mannosidosis-affected cats. Results All treatments were able to elicit a [Ca2+]i elevation in the ooplasm with various characteristics. Pronuclear formation and development up to the blastocyst stage was most efficiently triggered by electroporation (60.5 +/- 2.9 and 11.5 +/- 1.7% and the combined thimerosal/DTT treatment (67.7 +/- 1.8 and 10.6 +/- 1.9%; incubation of the stimulated oocytes with cycloheximide and cytochalasin B had a positive effect on embryo development. When these two methods were used to activate oocytes reconstructed during nuclear transfer, up to 84.9% of the reconstructed oocytes cleaved. When the 2 to 4-cell embryos (a total of 220 were

  15. Effect of soybean phosphatidylcholine on lipid profile of bovine oocytes matured in vitro.

    Science.gov (United States)

    Pitangui-Molina, Caroline P; Vireque, Alessandra A; Tata, Alessandra; Belaz, Katia Roberta A; Santos, Vanessa G; Ferreira, Christina R; Eberlin, Marcos N; Silva-de-Sá, Marcos Felipe; Ferriani, Rui A; Rosa-E-Silva, Ana Carolina J S

    2017-04-01

    The phospholipid (PL) composition of embryo and oocyte membranes affects thermal phase behavior and several physicochemical properties such as fluidity and permeability. The characterization of PL profiles and the development of suitable in vitro maturation (IVM) protocols, that are able to modify membrane's composition, may result in significant improvements in oocyte developmental potential and cryotolerance. Using soybean phosphatidylcholine (PC) as a model supplement, we evaluated the effect of PL supplementation during IVM on bovine cumulus-oocyte-complex (COC). Substantial changes in the lipid profiles of oocyte membrane were observed and associated with pre-implantation data. The propensity of the PC supplement to become soluble in the maturation medium and/or diffuse into mineral oil was also assessed. Oocytes were matured in TCM without supplementation, i.e. control, (n=922) or supplemented with 50 or 100μM PC (n=994). The maturation media and mineral oil pre- and post- IVM, along with control and PC-treated oocytes were then analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the lipid profiles were compared via principal component analysis (PCA). Soybean PCs are bioavailable and stable in IVM medium; further, PCs did not diffuse to the mineral oil, which also remained unaltered by the metabolism of treated oocytes. PC supplementation at 100μM resulted in substantially greater relative abundances of polyunsatured PL, namely PC (32:1), PC (34:2), PC (36:6), PC (36:4), and PC (38:6), in oocyte membrane. These differences indicated that short-term exposure to the PC supplement could indeed modify the lipid composition of IVM-oocytes in a dose-dependent manner. Membrane incorporation of polyunsaturated molecular species of PC was favored, and does so without compromising the viability of the subsequent embryo in regards to cleavage, blastocyst development and hatching rate. The reported approach will allow for the

  16. Metabolomics: approaches to assessing oocyte and embryo quality.

    Science.gov (United States)

    Singh, R; Sinclair, K D

    2007-09-01

    Morphological evaluation remains the primary method of embryo assessment during IVF cycles, but its modest predictive power and inherent inter- and intra-observer variability limits its value. Low-molecular weight metabolites represent the end products of cell regulatory processes and therefore reveal the response of biological systems to a variety of genetic, nutrient or environmental influences. It follows that the non-invasive quantification of oocyte and embryo metabolism, from the analyses of follicular fluid or culture media, may be a useful predictor of pregnancy outcome following embryo transfer, a potential supported by recent clinical studies working with specific classes of metabolites such as glycolytic intermediates and amino acids. Such selective approaches, however, whilst adhering closely to known cellular processes, may fail to harness the full potential of contemporary metabolomic methodologies, which can measure a wider spectrum of metabolites. However, an important technical drawback with many existing methodologies is the limited number of metabolites that can be determined by a single analytical platform. Vibrational spectroscopy methodologies such as Fourier transform infrared and near infrared spectroscopy may overcome these limitations by generating unique spectral signatures of functional groups and bonds, but their application in embryo quality assessment remains to be fully validated. Ultimately, a combination of evaluation criteria that include morphometry with metabolomics may provide the best predictive assessment of embryo viability.

  17. New insights into human nondisjunction of chromosome 21 in oocytes.

    Directory of Open Access Journals (Sweden)

    Tiffany Renee Oliver

    2008-03-01

    Full Text Available Nondisjunction of chromosome 21 is the leading cause of Down syndrome. Two risk factors for maternal nondisjunction of chromosome 21 are increased maternal age and altered recombination. In order to provide further insight on mechanisms underlying nondisjunction, we examined the association between these two well established risk factors for chromosome 21 nondisjunction. In our approach, short tandem repeat markers along chromosome 21 were genotyped in DNA collected from individuals with free trisomy 21 and their parents. This information was used to determine the origin of the nondisjunction error and the maternal recombination profile. We analyzed 615 maternal meiosis I and 253 maternal meiosis II cases stratified by maternal age. The examination of meiosis II errors, the first of its type, suggests that the presence of a single exchange within the pericentromeric region of 21q interacts with maternal age-related risk factors. This observation could be explained in two general ways: 1 a pericentromeric exchange initiates or exacerbates the susceptibility to maternal age risk factors or 2 a pericentromeric exchange protects the bivalent against age-related risk factors allowing proper segregation of homologues at meiosis I, but not segregation of sisters at meiosis II. In contrast, analysis of maternal meiosis I errors indicates that a single telomeric exchange imposes the same risk for nondisjunction, irrespective of the age of the oocyte. Our results emphasize the fact that human nondisjunction is a multifactorial trait that must be dissected into its component parts to identify specific associated risk factors.

  18. Obesity and the risk of spontaneous abortion after oocyte donation.

    Science.gov (United States)

    Bellver, José; Rossal, Luis P; Bosch, Ernesto; Zúñiga, Andrés; Corona, José T; Meléndez, Fernando; Gómez, Emilio; Simón, Carlos; Remohí, José; Pellicer, Antonio

    2003-05-01

    To determine whether obesity increases the risk of spontaneous abortion. Retrospective study. Oocyte donation program at the Instituto Valenciano de Infertilidad in Spain. Seven hundred twelve cycles of recipients of ovum donation with known body mass index (BMI), good-quality embryo transfer, and absence of uterine pathology or clinical history of antiphospholipid antibodies or recurrent abortion. Recipients were divided in four BMI (kg/m(2)) groups: lean, with BMI /=30 (n = 50; 7%). Clinical parameters were compared among the groups. Spontaneous abortion rates according to BMI. No difference was found among the four BMI groups in any of the parameters of the cycle analyzed. The overall abortion rate was 15.8% (57 of 360). There were significant differences in abortion rates between the obese (38.1%), and the normal (13.3%) and overweight (15.5%) groups. When several cutoff BMI values were established (20, 25, and 30), only the obese women demonstrated a greater risk of abortion. Compared with the normal population, the obese group showed a significant fourfold increase in the risk of spontaneous abortion. Our findings confirm that obesity (BMI >/=30) is an independent risk factor for spontaneous abortion. Therefore, it would be advisable for obese patients to reduce weight before becoming pregnant.

  19. Microtransplantation of membranes from cultured cells to Xenopus oocytes: A method to study neurotransmitter receptors embedded in native lipids

    OpenAIRE

    Palma, Eleonora; Trettel, Flavia; Fucile, Sergio; Renzi, Massimiliano; Miledi, Ricardo; Eusebi, Fabrizio

    2003-01-01

    The Xenopus oocyte is used as a convenient cell expression system to study the structure and function of heterogenic transmitter receptors and ion channels. Recently, we introduced a method to microtransplant already assembled neurotransmitter receptors from the human brain to the plasma membrane of Xenopus oocytes. The same approach was used here to transplant neurotransmitter receptors expressed from cultured cells to the oocytes. Membrane vesicles prepared from a human embryonic kidney cel...

  20. The effect of cumulus cells on domestic cat (Felis catus) oocytes during in vitro maturation and fertilization.

    Science.gov (United States)

    Sowińska, N; Frankowska, K; Filipczyk, A; Adamaszek, A; Nalik, K; Fic, K; Pietsch-Fulbiszewska, A

    2017-04-01

    The aim of this study was to evaluate the effect of co-culture of denuded oocytes with cumulus cells (CC) or cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) and in vitro fertilization (IVF). Immature oocytes were collected from ovaries of domestic cats following a routine ovariectomy. Oocytes were matured in vitro for 24 hr within four groups: (i) denuded oocytes (DO), (ii) DO co-cultured with CC, (iii) DO co-cultured with COC and (iv) COC as a control group. In further experiments, COCs were matured in vitro for 24 hr, and then, oocytes were randomly divided into four groups as previously described and fertilized in vitro. Embryos were cultured for up to 7 days. At the end of each experiment, oocytes/embryos were stained with Hoechst 33342 solution and observed under an inverted fluorescence microscope. The results of oocyte maturation showed that their meiotic competence decreased significantly in all experimental groups, compared to the control group. The maturation rates were approximately 45%, 24%, 43% and 76% in experiment 1, and 21%, 14%, 33% and 50% in experiment 2 in groups (i), (ii), (iii) and (iv), respectively. Examination of in vitro fertilization revealed that embryos developed up to the morula stage in all experimental groups. DO and oocytes cultured with COC during fertilization showed a lower cleavage rate-36% and 25% as opposed to those co-cultured with loose CC and the control group-43% and 42%, respectively. Results of this study indicate that cumulus cells connected with an oocyte into a cumulus-oocyte complex are irreplaceable for the maturation of domestic cat oocyte, but that the addition of loose CC may be beneficial for IVF. © 2016 Blackwell Verlag GmbH.

  1. Inhibition of calcineurin by FK506 stimulates germinal vesicle breakdown of mouse oocytes in hypoxanthine-supplemented medium

    Science.gov (United States)

    Wang, Li; Zhen, Yan-Hong; Liu, Xiao-Ming; Cao, Jing; Wang, Yan-Ling

    2017-01-01

    Calcineurin (CN) is a serine/threonine phosphatase which plays important roles in meiosis maturation in invertebrate oocytes; however, the role of CN in mouse oocytes is relatively unexplored. In this study, we examined the expression, localization and functional roles of CN in mouse oocytes and granulosa cells. The RT-PCR results showed that the β isoform of calcineurin A subunit (Cn A) expressed significantly higher than α and γ isoforms, and the expression of Cn Aβ mRNA obviously decreased in oocytes in which germinal vesicle breakdown (GVBD) occurred, while only B1 of calcineurin B subunit (Cn B) was detected in oocytes and stably expressed during oocytes maturation. The following fluorescence experiment showed that Cn A was mainly located in the nucleus of germinal vesicle (GV) stage oocytes and gruanlosa cells, and subsequently dispersed into the entire cytoplasm after GVBD. The decline of Cn A in oocytes suggested that it may play an important role in GVBD. To further clarify the role of calcineurin during meiotic maturation, FK506 (a calcineurin inhibitor) was used in the culture medium contained hypoxanthine (HX) which could keep mouse oocytes staying at GV stage. As expected, FK506 could induce a significant elevation of GVBD rate and increase the MPF level of denuded oocytes (DOs). Furthermore, FK506 could also play an induction role of GVBD of oocytes in COCs and follicles, and the process could be counteracted by MAPK kinase inhibitor (U0126). Above all, the results implied that calcineurin might play a crucial role in development of mouse oocytes and MPF and MAPK pathways are involved in this process. PMID:28243539

  2. Role of G protein-coupled estrogen receptor 1, GPER, in inhibition of oocyte maturation by endogenous estrogens in zebrafish

    OpenAIRE

    2010-01-01

    Estrogen inhibition of oocyte maturation (OM) and the role of GPER (formerly known as GPR30) were investigated in zebrafish. Estradiol-17β (E2) and G-1, a GPER-selective agonist, bound to zebrafish oocyte membranes suggesting the presence of GPER which was confirmed by immunocytochemistry using a specific GPER antibody. Incubation of follicle-enclosed oocytes with an aromatase inhibitor, ATD, and enzymatic and manual removal of the ovarian follicle cell layers significantly increased spontane...

  3. The lipid peroxidation product 4-hydroxynonenal contributes to oxidative stress-mediated deterioration of the ageing oocyte.

    Science.gov (United States)

    Mihalas, Bettina P; De Iuliis, Geoffry N; Redgrove, Kate A; McLaughlin, Eileen A; Nixon, Brett

    2017-07-24

    An increase in intraovarian reactive oxygen species (ROS) has long been implicated in the decline in oocyte quality associated with maternal ageing. Oxidative stress (OS)-induced lipid peroxidation and the consequent generation of highly electrophilic aldehydes, such as 4-hydroxynonenal (4-HNE), represents a potential mechanism by which ROS can inflict damage in the ageing oocyte. In this study, we have established that aged oocytes are vulnerable to damage by 4-HNE resulting from increased cytosolic ROS production within the oocyte itself. Further, we demonstrated that the age-related induction of OS can be recapitulated by exposure of germinal vesicle (GV) oocytes to exogenous H2O2. Such treatments stimulated an increase in 4-HNE generation, which remained elevated during in vitro oocyte maturation to metaphase II. Additionally, exposure of GV oocytes to either H2O2 or 4-HNE resulted in decreased meiotic completion, increased spindle abnormalities, chromosome misalignments and aneuploidy. In seeking to account for these data, we revealed that proteins essential for oocyte health and meiotic development, namely α-, β-, and γ-tubulin are vulnerable to adduction via 4-HNE. Importantly, 4-HNE-tubulin adduction, as well as increased aneuploidy rates, were resolved by co-treatment with the antioxidant penicillamine, demonstrating a possible therapeutic mechanism to improve oocyte quality in older females.

  4. Birth of common marmoset (Callithrix jacchus) offspring derived from in vitro-matured oocytes in chemically defined medium.

    Science.gov (United States)

    Tomioka, I; Takahashi, T; Shimada, A; Yoshioka, K; Sasaki, E

    2012-10-15

    Optimization of oocyte culture conditions is a crucial aspect of reproductive biology and technology. In the present study, maturation of germinal vesicle-stage marmoset oocytes were evaluated in the following media: Waymouth medium, Waymouth medium containing porcine follicular fluid (pFF) (Waymouth-pFF medium), and porcine oocyte medium (POM). Oocytes cultured in Waymouth-pFF medium had higher maturation rates to the metaphase II stage than those cultured in Waymouth medium (36.1% vs. 24.8%, respectively, P marmoset oocytes. Hence, maturation of marmoset oocytes cultured in POM was subsequently evaluated. The rate of maturation to the metaphase I stage was significantly higher and degradation rates were significantly lower in oocytes cultured in POM than those cultured in Waymouth medium. In addition, three offspring were successfully obtained after transfer of embryos matured in chemically defined medium. Therefore, we concluded that POM was suitable for marmoset oocyte culture. Furthermore, this was apparently the first report of marmoset offspring derived from oocytes cultured in chemically defined medium.

  5. Translocation of classical PKC and cortical granule exocytosis of human oocyte in germinal vesicle and metaphase Ⅱ stage

    Institute of Scientific and Technical Information of China (English)

    Xue-qing WU; Xiao ZHANG; Xiao-hong LI; Hui-hong CHENG; Yan-rong KUAI; Song WANG; Ying-lu GUO

    2006-01-01

    Aim: Protein kinase C (PKC) is as a family of serine/threonine kinases that can be activated by Ca2+, phospholipid and diacylglycerol. PKC plays an important role in oocyte maturation and activation. This study was undertaken to investigate classical PKC (cPKC) in human oocyte maturation and activation. Methods: Germinal vesicle (GV) and metaphase Ⅱ (MII) stage oocytes were collected from healthy women. The expression and distribution of cPKC were investigated by immunoflourescence. MII oocytes were treated with PKC activator or inhibitor and imaged using a laser confocal scanning microscope (LCSM). Results: In GV oocytes, PKC α,β1 and γ were localized to the germinal vesicles, with a weak expression in ooplasm. In MII oocytes, PKCα, β1 and γ were distributed evenly in ooplasm. After treatment with PKC activator, phorbol 12-myristate 13-acetate (PMA), cPKC translocated to the periphery of oocyte, and cortical granules (CG) exocytosis was found. When the oocytes were treated with PKC inhibitor, staurosporine, no translocation of cPKC and CG exocytosis were found. Conclusion: PKCα, β1 and γ exist in human oocytes and activation of these subunits could induce CG exocytosis in MII stage.

  6. The effect of the culture vessel and insemination method on the in vitro fertilization and development of human oocytes

    OpenAIRE

    Boone, William R.; Johnson, Jane E.

    1997-01-01

    Our laboratory has corroborated previously published work demonstrating that tissue culture tubes and microdrops perform equally well for in vitro fertilization and culture of human oocytes and embryos.

  7. mtDNA copy number in oocytes of different sizes from individual pre- and post-pubertal pigs

    DEFF Research Database (Denmark)

    Pedersen, Hanne Skovsgaard; Løvendahl, Peter; Larsen, Knud Erik

    2014-01-01

    Reproduction 131, 233–245). However, the correlation between size and mtDNA copy number in single oocytes has not been determined. This study describes the relation between oocytes of defined diameters from individual pre- and postpubertal pigs and mtDNA copy number. Cumulus-oocyte complexes were aspirated......Oocyte competence has been related to mtDNA copy number, but a large variation in mtDNA copy number between oocytes has been observed, caused by, e.g. oocyte donor and oocyte size (Sato et al. 2014 PLOS ONE 9, e94488; Cotterill et al. 2013 Mol. Hum. Reprod. 19, 444–450; El Shourbagy et al. 2006...... from ovaries of 10 pre- and 10 post-pubertal pigs. Cumulus cells were removed and the oocytes were measured (inside-ZP-diameter). Oocytes were transferred to DNAase-free tubes, snap-frozen, and stored at –80°C. The genes ND1 and COX1 were used to determine the mtDNA copy number. Plasmid preparations...

  8. Cryopreservation of ovarian tissue and in vitro matured oocytes in a female with mosaic Turner syndrome: Case Report.

    Science.gov (United States)

    Huang, J Y J; Tulandi, T; Holzer, H; Lau, N M; Macdonald, S; Tan, S L; Chian, R C

    2008-02-01

    We report a novel approach of fertility preservation in a young woman with mosaic Turner syndrome. A 16-year-old female with 20% 45XO and 80% 46XX karyotype underwent laparoscopic ovarian wedge resection. Before performing ovarian tissue cryopreservation, all visible follicles on the ovarian surface were aspirated. We recovered 11 immature germinal vesicle stage oocytes, which were subjected to in vitro maturation (IVM). Eight oocytes that matured (73% maturation rate) were cryopreserved by vitrification. The combination of ovarian tissue cryobanking and immature oocyte collection from the tissue followed by IVM and vitrification of matured oocytes represent a promising approach of fertility preservation for young women with mosaic Turner syndrome.

  9. Effect of Equilibration Temperature on In vitro Viability and Subsequent Embryo Development of Vitrified-Warmed Immature Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    Hajarian Hadi

    2010-01-01

    Full Text Available Problem statement: Vitrification is replacing conventional slow freezing to cryopreserve gametes and embryos especially for in vitro production of embryo in domestic animal species. However, the results are still not satisfactory. The aim of this experiment was to study the effect of different equilibration temperatures on in vitro viability of immature bovine oocytes after vitrification. Approach: Oocytes were obtained from slaughterhouse ovaries. Only grade one oocytes were used. Oocytes were equilibrated in three different temperatures: 32, 37, or 41°C. Immature oocytes were equilibrated in VS1 (7.5 Ethylene Glycol (EG + 7.5% DMSO for 10-12 min and then exposed to VS2 (15% EG + 15%DMSO + 0.5M sucrose for one min. Thereafter oocytes were loaded on hand-made Cryotop and directly plunged into liquid nitrogen. After warming, oocytes were examined for viability, maturation, cleavage and blastocyst production. Results: Oocytes that were equilibrated at 37°C had significantly higher (pConclusion: In conclusion, these results indicated that immature bovine oocytes can be equilibrated successfully at 37°C while higher or lower temperature can significantly decrease their subsequent viability and development.

  10. Endometriosis-induced alterations in mouse metaphase II oocyte microtubules and chromosomal alignment: a possible cause of infertility.

    Science.gov (United States)

    Mansour, Gihan; Sharma, Rakesh K; Agarwal, Ashok; Falcone, Tommaso

    2010-10-01

    To examine the effect of peritoneal fluid (PF) of patients with endometriosis on the cytoskeleton of metaphase II oocytes and correlate the results with the stage of endometriosis and the duration of infertility. Prospective-controlled study. Center for reproductive medicine at a tertiary-care hospital. Women with endometriosis (n=23) and tubal ligation/reversal (n=15). Peritoneal fluid obtained from 38 women (23 with endometriosis and 15 tubal ligation/reversal) after laparoscopy. Four hundred metaphase II oocytes were used: 165 frozen metaphase II oocytes were incubated in the PF of patients with endometriosis, 135 oocytes incubated in the PF of nonendometriosis patients (control subjects) and 100 oocytes incubated in human tubal fluid (HTF) media. Spindle abnormalities (microtubule and chromosomal) were evaluated by confocal imaging. In the endometriosis group, the cytoskeleton had a higher frequency of abnormal meiotic spindle and chromosomal misalignment (score ≥3), indicating severe damage compared with the control groups. The proportions of abnormalities in microtubule and chromosome alterations in endometriosis (67.9% and 63.6%, respectively) were significantly higher than for oocytes incubated with PF of the nonendometriosis group (24.4% and 14.8%) as well as the HTF group (13% and 13%). Oocyte cytoskeleton damage positively correlated with the duration of infertility and the stage of endometriosis. Alteration of oocyte cytoskeleton might be one of the causes of poor oocyte quality in patients with endometriosis. Copyright © 2010. Published by Elsevier Inc.

  11. Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation.

    Science.gov (United States)

    Yoon, Jeong; Juhn, Kyoung-Mi; Yoon, San-Hyun; Ko, Yong; Lim, Jin-Ho

    2017-03-01

    The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of Ca(2)(+) chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a Ca(2)(+) chelator to investigate the effect of Ca(2)(+) oscillations on their maturation. As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the Ca(2)(+) chelator-treated group. The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by Ca(2)(+) chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular Ca(2)(+) oscillations driven by fertilization.

  12. Superoxide dismutase and taurine supplementation improves in vitro blastocyst yield from poor-quality feline oocytes.

    Science.gov (United States)

    Ochota, Małgorzata; Pasieka, Anna; Niżański, Wojciech

    2016-03-15

    Blastocyst production in vitro seems to be crucial part of assisted reproduction techniques in feline species. However, the results of cats' oocyte maturation and embryo development are still lower than those in other species. The aim of this study was to evaluate whether the supplementation with superoxide dismutase (SOD) and taurine during maturation or culture would improve the blastocyst yield obtained from lower grades of oocytes, that are usually discarded, as not suitable for further in vitro purposes. To investigate the effect of antioxidants' addition, the good- and poor-quality oocytes, were cultured with the addition of 10-mmol taurine and 600 UI/mL SOD. The nuclear maturity, embryo development, and blastocyst quality were subsequently assessed. In control group, without antioxidant supplementation, significantly less poor-quality oocytes matured (42% vs. 62%) and more degenerated (35% vs. 20%), comparing to the experimental group supplemented with SOD and taurine. The amount of obtained blastocyst was much higher, when poor quality oocytes were supplemented with SOD and taurine (supplementation to IVM-4%; supplementation to IVC-5.5%; supplementation to IVM and IVC-5.9% of blastocyst), comparing to not supplemented control group (1.3%). The best blastocysts were obtained when poor oocytes had antioxidants added only during embryo culture (185 ± 13.4 blastomeres vs. 100 ± 1.5 in control). In the present study, we reported that the lower grades of oocytes can better mature and form significantly more blastocysts with better quality, when cultured with addition of SOD and taurine. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine.

    Science.gov (United States)

    Crocomo, L F; Ariu, F; Bogliolo, L; Bebbere, D; Ledda, S; Bicudo, S D

    2016-04-01

    The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos. © 2016 Blackwell Verlag GmbH.

  14. Modulation of glycolysis and the pentose phosphate pathway influences porcine oocyte in vitro maturation.

    Science.gov (United States)

    Alvarez, G M; Ferretti, E L; Gutnisky, C; Dalvit, G C; Cetica, P D

    2013-08-01

    Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.

  15. Scrambled and fried: Cigarette smoke exposure causes antral follicle destruction and oocyte dysfunction through oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Sobinoff, A.P. [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 (Australia); Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia); Beckett, E.L.; Jarnicki, A.G. [Centre for Asthma and Respiratory Disease, The University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308 (Australia); Sutherland, J.M. [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 (Australia); Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia); McCluskey, A. [Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia); Hansbro, P.M. [Centre for Asthma and Respiratory Disease, The University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308 (Australia); McLaughlin, E.A., E-mail: eileen.mclaughlin@newcastle.edu.au [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 (Australia); Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia)

    2013-09-01

    Cigarette smoke is a reproductive hazard associated with pre-mature reproductive senescence and reduced clinical pregnancy rates in female smokers. Despite an increased awareness of the adverse effects of cigarette smoke exposure on systemic health, many women remain unaware of the adverse effects of cigarette smoke on female fertility. This issue is compounded by our limited understanding of the molecular mechanisms behind cigarette smoke induced infertility. In this study we used a direct nasal exposure mouse model of cigarette smoke-induced chronic obstructive pulmonary disease to characterise mechanisms of cigarette-smoke induced ovotoxicity. Cigarette smoke exposure caused increased levels of primordial follicle depletion, antral follicle oocyte apoptosis and oxidative stress in exposed ovaries, resulting in fewer follicles available for ovulation. Evidence of oxidative stress also persisted in ovulated oocytes which escaped destruction, with increased levels of mitochondrial ROS and lipid peroxidation resulting in reduced fertilisation potential. Microarray analysis of ovarian tissue correlated these insults with a complex mechanism of ovotoxicity involving genes associated with detoxification, inflammation, follicular activation, immune cell mediated apoptosis and membrane organisation. In particular, the phase I detoxifying enzyme cyp2e1 was found to be significantly up-regulated in developing oocytes; an enzyme known to cause molecular bioactivation resulting in oxidative stress. Our results provide a preliminary model of cigarette smoke induced sub-fertility through cyp2e1 bioactivation and oxidative stress, resulting in developing follicle depletion and oocyte dysfunction. - Highlights: • Cigarette smoke exposure targets developing follicle oocytes. • The antral follicle oocyte is a primary site of ovarian cigarette smoke metabolism. • Cyp2e1 is a major enzyme involved in ameliorating smoke-induced ovotoxicity. • Cigarette smoke causes oocyte

  16. Enhancing survival of mouse oocytes following chemotherapy or aging by targeting Bax and Rad51.

    Directory of Open Access Journals (Sweden)

    Loro L Kujjo

    Full Text Available BACKGROUND: Therapeutic approaches to preserve fertility in females undergoing cancer treatments are currently ineffective. This is partly due to limited knowledge of the molecular mechanisms that injured germ cells elicit to repair damage and survive or to abort repair and activate biochemical pathways leading to death. So far, we know that following spontaneously occurring or drug-induced DNA damage, the efficiency of DNA repair is a critical determinant of the cell's fate. The protein encoded by the Rad51 gene is one of several components recruited for homologous recombination-dependent DNA double-strand break repair in both somatic cells and germ cells. Recently, we showed that microinjection of recombinant Rad51 into AKR/J mouse oocytes decreased the extent of spontaneous DNA double-strand breaks, suppressed apoptosis, and restored the developmental competence in AKR/J embryos. Herein we characterized the nature of chemotherapy-induced lesions in oocytes, and the associated individual components of the DNA damage sensor and repair apparatus. For comparison, we also assessed parallel spontaneous changes in aging oocytes. METHODS: Data collected were derived from: analysis of apoptosis; immunodepletion; oocyte microinjections; immunocytochemistry; immunofluorescence; and CHIP-like assays. RESULTS: Our data show that: (i DNA damage in oocytes can be induced by both chemotherapy and spontaneously by the aging process; (ii oocytes possess the machinery and capability for repairing such DNA damage; (iii Rad51 is a critical player in the repair of both chemotherapy-induced and spontaneously-sustained DNA damage; and (iv in response to damage, oocytes exhibit an inverse functional relationship between presence of Bax and activity of Rad51. CONCLUSION/SIGNIFICANCE: Our results establish Rad51 and/or Bax as potential candidates that can be targeted for development of individualized chemotherapeutic interventions that are effective, but minimal in

  17. Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes.

    Science.gov (United States)

    Suttirojpattana, Tayita; Somfai, Tamas; Matoba, Satoko; Parnpai, Rangsun; Nagai, Takashi; Geshi, Masaya

    2017-02-01

    Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non-stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P < 0.05). Supplementation of the holding medium with dithiothreitol (DTT) at any concentrations did not improve embryo development from stored oocytes. Although supplementation with cyclosporine A (CsA) significantly reduced apoptosis and membrane damage rates during storage, it did not improve the developmental competence of oocytes. 1,2-bis(2-aminophenoxy) ethane N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester and ruthenium red had no effect on oocyte apoptotic rates. Blastocyst formation rates in all stored groups remained significantly lower than that of the control. In conclusion, pyruvate and serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence. © 2016 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.

  18. Mitosis-meiosis and sperm-oocyte fate decisions are separable regulatory events.

    Science.gov (United States)

    Morgan, Clinton T; Noble, Daniel; Kimble, Judith

    2013-02-26

    Germ cell fate decisions are poorly understood, despite their central role in reproduction. One fundamental question has been whether germ cells are regulated to enter the meiotic cell cycle (i.e., mitosis-meiosis decision) and to be sperm or oocyte (i.e., sperm-oocyte decision) through one or two cell fate choices. If a single decision is used, a male-specific or female-specific meiotic entry would lead necessarily toward spermatogenesis or oogenesis, respectively. If two distinct decisions are used, meiotic entry should be separable from specification as sperm or oocyte. Here, we investigate the relationship of these two decisions with tools uniquely available in the nematode Caenorhabditis elegans. Specifically, we used a temperature-sensitive Notch allele to drive germ-line stem cells into the meiotic cell cycle, followed by chemical inhibition of the Ras/ERK pathway to reprogram the sperm-oocyte decision. We found that germ cells already in meiotic prophase can nonetheless be sexually transformed from a spermatogenic to an oogenic fate. This finding cleanly uncouples the mitosis-meiosis decision from the sperm-oocyte decision. In addition, we show that chemical reprogramming occurs in a germ-line region where germ cells normally transition from the mitotic to the meiotic cell cycle and that it dramatically changes the abundance of key sperm-oocyte fate regulators in meiotic germ cells. We conclude that the C. elegans mitosis-meiosis and sperm-oocyte decisions are separable regulatory events and suggest that this fundamental conclusion will hold true for germ cells throughout the animal kingdom.

  19. Efficacy of Simple Assessment System of Oocyte Maturity in IVF-ET Cycles

    Institute of Scientific and Technical Information of China (English)

    Kee Sang Park; Gun Ho Song; Hang Jin Kim; Hai Bum Song; Taek Hoo Lee; Sang Sik Chun

    2006-01-01

    Objective To establish and evaluate the efficacy of the simple assessment system of oocyte maturity.Methods A total of 251 couples were enrolled in this study and female patients were divided into two groups. In group Ⅰ, oocytes were inseminated at 6 h after ovum pickup. In group Ⅱ, oocyte maturity was rapidly categorized by simple assessment system.Mature oocytes were inseminated at 3-4 h after ovum pick-up when oocyte-corona complexes (OCC) exhibited clear thick ring-like halo (RLH) and expanded cumulus cells (CC) or 5-6 h when OCC exhibited RLH and a few clumped and/or dark CC,respectively. Intermediate oocytes were inseminated at 7-8 h when RLH was not found around the OCC and CC were compacted and clumped and/or dark.Results Normal fertilization rate was higher in group Ⅱ (76.5%) than that in group Ⅰ(58.0%) (P<0. 001). However, abnormal fertilization rate was higher in group Ⅰ(11.3%) than that in group Ⅱ (3.6%) (P<0. 001). The cleavage (82.6% vs 90.0%),chemical pregnancy (4. 8% vs 3.9%), twin pregnancy (6. 7% vs 3. 9%) and implantation rate (8.4% vs 10. 6%) had no statistically differences between group Ⅰ and Ⅱ. Rate of clinical and singleton pregnancy was higher in group Ⅱ (35.3% and 31.4%) than those in group Ⅰ (24.8% and 18.2%) (P<0. 05).Conclusion This simple assessment system is useful and effective for evaluation and categorization of the oocyte maturity with better reproductive outcomes.

  20. PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

    Science.gov (United States)

    Terzaghi, L; Tessaro, I; Raucci, F; Merico, V; Mazzini, G; Garagna, S; Zuccotti, M; Franciosi, F; Lodde, V

    2016-08-02

    Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

  1. Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation.

    Science.gov (United States)

    Ajmat, M T; Bonilla, F; Zelarayán, L; Bühler, M I

    2011-05-01

    Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP₃Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP₃Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP₃Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP₃Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP₃Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear.

  2. Lipid characterization of individual porcine oocytes by dual mode DESI-MS and data fusion

    Energy Technology Data Exchange (ETDEWEB)

    Pirro, Valentina, E-mail: valentina.pirro@unito.it [Department of Chemistry, University of Turin, Via Pietro Giuria 7, Turin 10125 (Italy); Oliveri, Paolo [Department of Pharmacy, University of Genoa, Via Brigata Salerno 13, Genoa 16147 (Italy); Ferreira, Christina Ramires [Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907 (United States); González-Serrano, Andrés Felipe [Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Hoeltystrasse 10, 31535 Neustadt, Mariensee (Germany); Machaty, Zoltan [Department of Animal Sciences, Purdue University, 915 W. State St., West Lafayette, IN 47907 (United States); Cooks, Robert Graham [Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907 (United States)

    2014-10-27

    Highlights: • Repeated analysis by DESI(±)-MS of intact single oocytes for lipid characterization. • Deployment of a data fusion strategy to merge positive and negative ion mode data. • Enhanced interpretation of metabolic changes by more efficient analysis of spectral data. • Discovery of increased fatty acid metabolism and membrane complexity during maturation. • Assistance in the improvement of in vitro embryo production for porcine species. - Abstract: The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo

  3. Recipient screening in IVF: First data from women undergoing anonymous oocyte donation in Dublin

    LENUS (Irish Health Repository)

    Walsh, Anthony PH

    2011-04-20

    Abstract Background Guidelines for safe gamete donation have emphasised donor screening, although none exist specifically for testing oocyte recipients. Pre-treatment assessment of anonymous donor oocyte IVF treatment in Ireland must comply with the European Union Tissues and Cells Directive (Directive 2004\\/23\\/EC). To determine the effectiveness of this Directive when applied to anonymous oocyte recipients in IVF, we reviewed data derived from selected screening tests performed in this clinical setting. Methods Data from tests conducted at baseline for all women enrolling as recipients (n = 225) in the anonymous oocyte donor IVF programme at an urban IVF referral centre during a 24-month period were analysed. Patient age at programme entry and clinical pregnancy rate were also tabulated. All recipients had at least one prior negative test for HIV, Hepatitis B\\/C, chlamydia, gonorrhoea and syphilis performed by her GP or other primary care provider before reproductive endocrinology consultation. Results Mean (±SD) age for donor egg IVF recipients was 40.7 ± 4.2 yrs. No baseline positive chlamydia, gonorrhoea or syphilis screening results were identified among recipients for anonymous oocyte donation IVF during the assessment interval. Mean pregnancy rate (per embryo transfer) in this group was 50.5%. Conclusion When tests for HIV, Hepatitis B\\/C, chlamydia, gonorrhoea and syphilis already have been confirmed to be negative before starting the anonymous donor oocyte IVF sequence, additional (repeat) testing on the recipient contributes no new clinical information that would influence treatment in this setting. Patient safety does not appear to be enhanced by application of Directive 2004\\/23\\/EC to recipients of anonymous donor oocyte IVF treatment. Given the absence of evidence to quantify risk, this practice is difficult to justify when applied to this low-risk population.

  4. Conforming with current regulation in Turkey regarding the freezing of oocytes: A case report of the first pregnancy in Turkey achieved through oocyte vitrification

    Directory of Open Access Journals (Sweden)

    Semra Kahraman MD

    2017-01-01

    Full Text Available Objectives: To present the first pregnancy achieved in Turkey with frozen–warmed oocytes in a case with previous nine unsuccessful assisted reproductive technology (ART attempts. Methods: The clinical follow-up of a 33-year-old female applying to our ART centre after a long and complicated history of infertility is described. Results: In April 2013, the woman attempted our centre for her 10th ART trial. She informed us on oocyte pick-up (OPU day that her husband had been hospitalized following a car crush in Albania and was unable to travel to our clinic to give a sperm sample. We were therefore placed in the position of having to make an emergency decision. OPU was done and seven oocytes were retrieved. Six metaphase II (MII oocytes out of seven Cumulus Oocyte Complexes (COCs were vitrified using the Kitazato Vitrification Cryotop Kit. Six months later, in November 2013, the patient applied for transfer. Two blastocysts were transferred and the ART trial resulted with a singleton pregnancy and the birth of a healthy new-born at term via cesarean section. Conclusion: Regulation Codes on Assisted Reproductive Procedures and Assisted Reproductive Technology Centres, published in the Official Gazette of the Republic of Turkey, on 6 March 2010 forbade the freezing of gonad cells and tissues except when essential for medical reasons and stated that this would be specified later. However, the Regulation Codes published in the Official Gazette of the Republic of Turkey, on 30 September 2014 provided no further clarification. Unfortunately, the wording of the regulations did not specifically address this unexpected emergency situation. However, we saw our decision to cryopreserve the oocytes as a valid interpretation of the regulations, bearing in mind also the requirement that sperm and oocyte in the IVF process must be those of a married couple. Turkish medicolegal regulations should be revised to increase the chances of more women taking advantage

  5. Conforming with current regulation in Turkey regarding the freezing of oocytes: A case report of the first pregnancy in Turkey achieved through oocyte vitrification

    Science.gov (United States)

    Kahraman, Semra; Ulker, Kahraman; Pirkevi Çetinkaya, Caroline; Kumtepe Colakoglu, Yesim; Yelke, Hakan K.; Yilanlioglu, Cihangir

    2017-01-01

    Objectives: To present the first pregnancy achieved in Turkey with frozen–warmed oocytes in a case with previous nine unsuccessful assisted reproductive technology (ART) attempts. Methods: The clinical follow-up of a 33-year-old female applying to our ART centre after a long and complicated history of infertility is described. Results: In April 2013, the woman attempted our centre for her 10th ART trial. She informed us on oocyte pick-up (OPU) day that her husband had been hospitalized following a car crush in Albania and was unable to travel to our clinic to give a sperm sample. We were therefore placed in the position of having to make an emergency decision. OPU was done and seven oocytes were retrieved. Six metaphase II (MII) oocytes out of seven Cumulus Oocyte Complexes (COCs) were vitrified using the Kitazato Vitrification Cryotop Kit. Six months later, in November 2013, the patient applied for transfer. Two blastocysts were transferred and the ART trial resulted with a singleton pregnancy and the birth of a healthy new-born at term via cesarean section. Conclusion: Regulation Codes on Assisted Reproductive Procedures and Assisted Reproductive Technology Centres, published in the Official Gazette of the Republic of Turkey, on 6 March 2010 forbade the freezing of gonad cells and tissues except when essential for medical reasons and stated that this would be specified later. However, the Regulation Codes published in the Official Gazette of the Republic of Turkey, on 30 September 2014 provided no further clarification. Unfortunately, the wording of the regulations did not specifically address this unexpected emergency situation. However, we saw our decision to cryopreserve the oocytes as a valid interpretation of the regulations, bearing in mind also the requirement that sperm and oocyte in the IVF process must be those of a married couple. Turkish medicolegal regulations should be revised to increase the chances of more women taking advantage of the procedure

  6. Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation

    Directory of Open Access Journals (Sweden)

    Barboni Barbara

    2003-05-01

    Full Text Available Abstract Background The possibility to predict the ability of a germ cell to properly sustain embryo development in vitro or in vivo as early as possible is undoubtedly the main problem of reproductive technologies. To date, only the achievement of nuclear maturation and cumulus expansion is feasible, as all the studies on cytoplasmic maturation are too invasive and have been complicated by the death of the cells analyzed. The authors studied the possibility to test the cytoplasmic quality of pig oocytes by evaluating their ability to produce steroidogenesis enabling factor(s. To this aim, oocytes matured under different culture conditions that allowed to obtain gradable level of cytoplasmic maturation, were used to produce conditioned media (OCM. The secretion of the factor(s in conditioned media was then recorded by evaluating the ability of the spent media to direct granulosa cells (GC steroidogenesis. Methods In order to obtain germ cells characterized by a different degree of developmental competence, selected pig oocytes from prepubertal gilts ovaries were cultured under different IVM protocols; part of the matured oocytes were used to produce OCM, while those remaining were submitted to in vitro fertilization trials to confirm their ability to sustain male pronuclear decondensation. The OCM collected were finally used on cumulus cells grown as monolayers for 5 days. The demonstration that oocytes secreted factor(s can influence GC steroidogenesis in the pig was confirmed in our lab by studying E2 and P4 production by cumulus cells monolayers using a radioimmunoassay technique. Results Monolayers obtained by growing GC surrounding the oocytes for five days represent a tool, which is practical, stable and available in most laboratories; by using this bioassay, we detected the antiluteal effect of immature oocytes, and for the first time, demonstrated that properly matured germ cells are able to direct cumulus cells steroidogenesis by

  7. Childless women's beliefs and knowledge about oocyte freezing for social and medical reasons.

    Science.gov (United States)

    Daniluk, J C; Koert, E

    2016-10-01

    What factors inform a woman's decision-making about oocyte freezing to preserve fertility for social and medical reasons? Women lacked knowledge about the costs and viability of oocyte freezing as a fertility preservation option for social and medical reasons, and identified health consequences, costs, and viability as being particularly influential in their decision-making. Having only recently become a viable fertility preservation option, relatively little is known about childless women's beliefs or knowledge about oocyte freezing for social or medical reasons. A cross sectional study of 500 childless women was conducted in August, 2015. A total of 500 childless, presumed fertile, women from 18 to 38 years of age completed an online, self-report questionnaire assessing beliefs and knowledge about oocyte freezing to preserve fertility for social or medical reasons. Financial costs (85.6%), health risks to themselves (86.4%) or their offspring (87.8%), and success rates (82%) were the primary factors that women felt would influence their decision to freeze their oocytes. Partner's feelings (88.6%), prognosis for a full recovery (85.4%), and concerns about the health effects of the hormones or oocyte retrieval procedure (85.4%) were identified as being particularly important when considering oocyte freezing for medical reasons. Consistent with their perceptions of having little or no knowledge about oocyte freezing, there was an overall correct response rate of 33% to the 12 knowledge questions. The online format and use of a survey company to recruit participants may have increased the risk of self-selection bias and limit the generalizability of these findings. The findings may also be limited by the fact that the participants were not facing cancer treatments, and the younger participants were not nearing the end of their reproductive lifespan, and therefore would not have had reason to learn about, or consider, fertility preservation for medical or social

  8. The RNA-binding protein, ZFP36L2, influences ovulation and oocyte maturation.

    Directory of Open Access Journals (Sweden)

    Christopher B Ball

    Full Text Available ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. In the C57BL/6NTac mouse, a mutation in Zfp36l2 that results in the decreased expression of a form of ZFP36L2 in which the 29 N-terminal amino acid residues have been deleted, ΔN-ZFP36L2, leads to fertilized eggs that arrest at the two-cell stage. Interestingly, homozygous ΔN-Zfp36l2 females in the C57BL/6NTac strain release 40% fewer eggs than the WT littermates (Ramos et al., 2004, suggesting an additional defect in ovulation and/or oocyte maturation. Curiously, the same ΔN-Zfp36l2 mutation into the SV129 strain resulted in anovulation, prompting us to investigate a potential problem in ovulation and oocyte maturation. Remarkably, only 20% of ΔN-Zfp36l2 oocytes in the 129S6/SvEvTac strain matured ex vivo, suggesting a defect on the oocyte meiotic maturation process. Treatment of ΔN-Zfp36l2 oocytes with a PKA inhibitor partially rescued the meiotic arrested oocytes. Furthermore, cAMP levels were increased in ΔN-Zfp36l2 oocytes, linking the cAMP/PKA pathway and ΔN-Zfp36l2 with meiotic arrest. Since ovulation and oocyte maturation are both triggered by LHR signaling, the downstream pathway was investigated. Adenylyl cyclase activity was increased in ΔN-Zfp36l2 ovaries only upon LH stimulation. Moreover, we discovered that ZFP36L2 interacts with the 3'UTR of LHR mRNA and that decreased expression levels of Zfp36l2 correlates with higher levels of LHR mRNA in synchronized ovaries. Furthermore, overexpression of ZFP36L2 decreases the endogenous expression of LHR mRNA in a cell line. Therefore, we propose that lack of the physiological down regulation of LHR mRNA levels by ZFP36L2 in the ovaries is associated with anovulation and oocyte meiotic arrest.

  9. Role of ubiquitin C-terminal hydrolase-L1 in antipolyspermy defense of mammalian oocytes.

    Science.gov (United States)

    Susor, Andrej; Liskova, Lucie; Toralova, Tereza; Pavlok, Antonin; Pivonkova, Katerina; Karabinova, Pavla; Lopatarova, Miloslava; Sutovsky, Peter; Kubelka, Michal

    2010-06-01

    The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

  10. Antibody microarray analyses of signal transduction protein expression and phosphorylation during porcine oocyte maturation.

    Science.gov (United States)

    Pelech, Steven; Jelinkova, Lucie; Susor, Andrej; Zhang, Hong; Shi, Xiaoqing; Pavlok, Antonin; Kubelka, Michal; Kovarova, Hana

    2008-07-01

    Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.

  11. Are there optimal numbers of oocytes, spermatozoa and embryos in assisted reproduction?

    Science.gov (United States)

    Milachich, Tanya; Shterev, Atanas

    2016-08-01

    The aim of this overview is to discuss the current information about the search for the optimum yield of gametes in assisted reproduction, as one of the major pillars of IVF success. The first topic is focused on the number of male gametes and the possible impact of some genetic traits on these parameters. The number of spermatozoa did not seem to be crucial when there is no severe male factor of infertility. Genetic testing prior to using those sperm cells is very important. Different methods were applied in order to elect the "best" spermatozoa according to specific indications. The next problem discussed is the importance of the number of oocytes collected. Several studies have agreed that "15 oocytes is the perfect number," as the number of mature oocytes is more important. However, if elective single embryo transfer is performed, the optimal number of oocytes will enable a proper embryo selection. The third problem discussed concerns fertility preservation. Many educational programs promote and encourage procreation at maternal ages between 20-35 years, since assisted reproduction is unable to fully overcome the effects of female aging and fertility loss after that age. It is also strongly recommended to ensure a reasonable number of cryopreserved mature oocytes, preferably in younger ages (<35), for which an average of two stimulation cycles are likely required. For embryo cryopreservation, the "freeze all" strategy suggests the vitrification of good embryos, therefore quality is prior to number and patient recruitment for this strategy should be performed cautiously.

  12. Nitric oxide acts through different signaling pathways in maturation of cumulus cell-enclosed mouse oocytes

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    M Abbasi

    2009-03-01

    Full Text Available ABSTRACT Background: Nitric oxide (NO have a dual action in mouse oocyte meiotic maturation which depends on its concentration, but the mechanisms by which it influences oocyte maturation has not been exactly clarified. In this study different signaling mechanisms which exist for in vitro maturation of meiosis was examined in cumulus cell-enclosed oocytes (CEOs after injection of pregnant mare's serum gonadotropin (PMSG to immature female mice. Methods: The CEOs were cultured in spontaneous maturation and hypoxanthine (HX arrested model. Results: Sodium nitroprusside (SNP, an NO donor, 10mM delayed germinal vesicle breakdown (GVBD significantly during the first 5 hrs of incubation and inhibited the formation of first polar body (PB1 at the end of 24 hrs of incubation. SNP (10-5M stimulated the meiotic maturation of oocytes significantly by overcoming the inhibition of HX. Sildenafil (a cGMP stimulator, 100 nM, had a significant inhibitory effects on both spontaneous meiotic maturation and HX-arrested meiotic maturation. Forskolin (an adenylate cyclase stimulator, 6µM and SNP (10mM had the same effects on GVBD. Forskolin reversed the SNP (10-5M stimulated meiotic maturation. Conclusion: These results suggest that differences in pathways are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation in mouse oocytes

  13. Regulation of the formin Cappuccino is critical for polarity of Drosophila oocytes.

    Science.gov (United States)

    Bor, Batbileg; Bois, Justin S; Quinlan, Margot E

    2015-01-01

    The Drosophila formin Cappuccino (Capu) creates an actin mesh-like structure that traverses the oocyte during midoogenesis. This mesh is thought to prevent premature onset of fast cytoplasmic streaming which normally happens during late-oogenesis. Proper cytoskeletal organization and cytoplasmic streaming are crucial for localization of polarity determinants such as osk, grk, bcd, and nanos mRNAs. Capu mutants disrupt these events, leading to female sterility. Capu is regulated by another nucleator, Spire, as well as by autoinhibition in vitro. Studies in vivo confirm that Spire modulates Capu's function in oocytes; however, how autoinhibition contributes is still unclear. To study the role of autoinhibition in flies, we expressed a Capu construct that is missing the Capu Inhibitory Domain, CapuΔN. Consistent with a gain of activity due to loss of autoinhibition, the actin mesh was denser in CapuΔN oocytes. Further, cytoplasmic streaming was delayed and fertility levels decreased. Localization of osk mRNA in early stages, and bcd and nanos in late stages, were disrupted in CapuΔN-expressing oocytes. Finally, evidence that these phenotypes were due to a loss of autoinhibition comes from coexpression of the N-terminal half of Capu with CapuΔN, which suppressed the defects in actin, cytoplasmic streaming and fertility. From these results, we conclude that Capu can be autoinhibited during Drosophila oocyte development.

  14. The hippo pathway promotes Notch signaling in regulation of cell differentiation, proliferation, and oocyte polarity.

    Directory of Open Access Journals (Sweden)

    Jianzhong Yu

    Full Text Available Specification of the anterior-posterior axis in Drosophila oocytes requires proper communication between the germ-line cells and the somatically derived follicular epithelial cells. Multiple signaling pathways, including Notch, contribute to oocyte polarity formation by controlling the temporal and spatial pattern of follicle cell differentiation and proliferation. Here we show that the newly identified Hippo tumor-suppressor pathway plays a crucial role in the posterior follicle cells in the regulation of oocyte polarity. Disruption of the Hippo pathway, including major components Hippo, Salvador, and Warts, results in aberrant follicle-cell differentiation and proliferation and dramatic disruption of the oocyte anterior-posterior axis. These phenotypes are related to defective Notch signaling in follicle cells, because misexpression of a constitutively active form of Notch alleviates the oocyte polarity defects. We also find that follicle cells defective in Hippo signaling accumulate the Notch receptor and display defects in endocytosis markers. Our findings suggest that the interaction between Hippo and classic developmental pathways such as Notch is critical to spatial and temporal regulation of differentiation and proliferation and is essential for development of the body axes in Drosophila.

  15. Degradation of mitochondrial DNA in cryoprotectant-treated hard coral (Echinopora spp.) oocytes.

    Science.gov (United States)

    Tsai, Sujune; Chen, Jiann-Chu; Spikings, Emma; Li, Jan-Jung; Lin, Chiahsin

    2015-06-01

    A critical step for successful cryopreservation is to determine the optimal cryoprotectant treatment that can provide protective effects against cryoinjury during freezing and with minimal toxicity. Most cryoprotectants have chemical and osmotic effects when used at high concentrations. Cryoprotectants can damage coral mitochondrial distributions and membrane potentials, which results in reduced ATP production. As mitochondrial DNA (mtDNA) encodes for components of the electron transport chain (ETC) and plays a critical role in ATP synthesis capacity, we determined the effects of cryoprotectants on mtDNA in hard coral (Echinopora spp.) oocytes using quantitative real-time PCR. Our results showed that an insult from a cryoprotectant may be compensated for by the genetic defense mechanisms of these cells. Methanol was found to have the least effect on coral oocytes with regard to their energy status. A single oocyte without cryoprotectant treatment produced an average of 4,220,645 ± 169,990 mtDNA copies, which was greater than that in mammals. However, relatively lower mtDNA copy numbers (<2,000,000) were observed when oocytes were treated with dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), or glycerol at a concentration of 3 M for 20 min. These results provide direct evidence that hard coral (Echinopora spp.) oocytes are extremely susceptible to cryoprotectants and support the concerns with regard to the adverse effects of cryoprotectants.

  16. Improved low-CPA vitrification of mouse oocytes using quartz microcapillary.

    Science.gov (United States)

    Choi, Jung Kyu; Huang, Haishui; He, Xiaoming

    2015-06-01

    Cryopreservation by low-cryoprotectant (CPA) vitrification has the potential to combine all the advantages of the conventional high-CPA vitrification and slow-freezing approaches while avoiding their drawbacks. However, current low-CPA vitrification protocol for cryopreservation of oocytes requires a lengthy and multi-step procedure for unloading CPAs. In this study, we report a much-simplified procedure of using quartz microcapillary (QMC) for low-CPA vitrification of mouse oocytes with only one step for unloading CPAs. The immediate viability of oocytes after the improved low-CPA vitrification was determined to be more than 90%. Moreover, no significant difference was observed in terms of embryonic development from the two-cell to blastocyst stages between the fresh and vitrified oocytes after in vitro fertilization (IVF). This improved low-CPA vitrification technology has the potential for efficient cryopreservation of oocytes to preserve the fertility of mammals including humans for assisted reproductive medicine, maintenance of animal resource and endangered species, and livestock management.

  17. Insulin alone can lead to a withdrawal of meiotic arrest in the carp oocyte

    Indian Academy of Sciences (India)

    S Dasgupta; D Basu; L Ravi Kumar; S Bhattacharya

    2001-09-01

    Meiotic arrest of oocyte in an Indian carp, Labeo rohita Ham. has been found for the first time to be withdrawn by insulin only. Addition of insulin to oocytes in vitro caused germinal vesicle breakdown (GVBD), one of the first visual markers to determine initiation of the final maturational process. Under the influence of insulin the germinal vesicle (GV) of the oocyte migrated towards the animal pole, reached the micropyle and then dissolved (GVBD). By using different concentrations of insulin i.e., 0.063, 0.63, 6.3 and 12.6 M, optimum amount required was found to be 6.3 M. Induction of GVBD by insulin could be blocked by cycloheximide (Chx), a translation inhibitor, while actinomycin D (AcD) had no effect suggesting non-involvement of transcriptional activity in this process. Addition of the maturation-inducing steroid 17,20-dihydroxy-4-pregnen-3-one (DHP) stimulated ( < 0.01) GVBD of carp oocytes and its combination with insulin showed an additive effect. Gonadotropin (GtH) caused GVBD but its effect was greatly augmented by insulin. Our results demonstrate that not only can insulin alone induce GVBD in carp oocytes, but it also augments the stimulatory effect of DHP or IGF-I or GtH on GVBD. This information will be important in hormonal manipulation during induced breeding of carp.

  18. Acrylamide toxic effects on mouse oocyte quality and fertility in vivo.

    Science.gov (United States)

    Duan, Xing; Wang, Qiao-Chu; Chen, Kun-Lin; Zhu, Cheng-Cheng; Liu, Jun; Sun, Shao-Chen

    2015-06-25

    Acrylamide is an industrial chemical that has attracted considerable attention due to its presumed carcinogenic, neurotoxic, and cytotoxic effects. In this study we investigated possible acrylamide reproductive toxic effects in female mice. Mice were fed an acrylamide-containing diet for 6 weeks. Our results showed the following effects of an acrylamide-containing diet. (1) Ovary weights were reduced in acrylamide-treated mice and oocyte developmental competence was also reduced, as shown by reduced GVBD and polar body extrusion rates. (2) Acrylamide feeding resulted in aberrant oocyte cytoskeletons, as shown by an increased abnormal spindle rate and confirmed by disrupted γ-tubulin and p-MAPK localization. (3) Acrylamide feeding resulted in oxidative stress and oocyte early stage apoptosis, as shown by increased ROS levels and p-MAPK expression. (4) Fluorescence intensity analysis showed that DNA methylation levels were reduced in acrylamide-treated oocytes and histone methylation levels were also altered, as H3K9me2, H3K9me3, H3K4me2, and H3K27me3 levels were reduced after acrylamide treatment. (5) After acrylamide feeding, the litter sizes of acrylamide-treated mice were significantly smaller compared to thus of control mice. Thus, our results indicated that acrylamide might affect oocyte quality through its effects on cytoskeletal integrity, ROS generation, apoptosis induction, and epigenetic modifications.

  19. Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

    Science.gov (United States)

    Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

    2014-02-01

    During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

  20. Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

    Science.gov (United States)

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

    2016-01-01

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

  1. Smc1β is required for activation of SAC during mouse oocyte meiosis.

    Science.gov (United States)

    Miao, Yilong; Zhou, Changyin; Cui, Zhaokang; Dai, Xiaoxin; Zhang, Mianqun; Lu, Yajuan; Xiong, Bo

    2017-03-19

    Smc1β is a meiosis-specific cohesin subunit that is essential for sister chromatid cohesion and DNA recombination. Previous studies have shown that Smc1β-deficient mice in both sexes are sterile. Ablation of Smc1β during male meiosis leads to the blockage of spermatogenesis in pachytene stage, and ablation of Smc1β during female meiosis generates a highly error-prone oocyte although it could develop to metaphase II stage. However, the underlying mechanisms regarding how Smc1β maintains the correct meiotic progression in mouse oocytes have not been clearly defined. Here, we find that GFP-fused Smc1β is expressed and localized to the chromosomes from GV to MII stages during mouse oocyte meiotic maturation. Knockdown of Smc1β by microinjection of gene-specific morpholino causes the impaired spindle apparatus and chromosome alignment which are highly correlated with the defective kinetochore-microtubule attachments, consequently resulting in a prominently higher incidence of aneuploid eggs. In addition, the premature extrusion of polar bodies and escape of metaphase I arrest induced by low dose of nocodazole treatment in Smc1β-depleted oocytes indicates that Smc1β is essential for activation of spindle assembly checkpoint (SAC) activity. Collectively, we identify a novel function of Smc1β as a SAC participant beyond its role in chromosome cohesion during mouse oocyte meiosis.

  2. Heat stress and antioxidant enzyme activity in bubaline ( Bubalus bubalis) oocytes during in vitro maturation

    Science.gov (United States)

    Waiz, Syma Ashraf; Raies-ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T. Sridhar; Chauhan, M. S.; Upadhyay, R. C.

    2016-09-01

    In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly ( P peroxidase, and glutathione reductase were significantly ( P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

  3. Temporal dynamics of oocyte growth and vitellogenin gene expression in zebrafish (Danio rerio).

    Science.gov (United States)

    Connolly, Michelle H; Dutkosky, Rachel M; Heah, Tze P; Sayler, Gary S; Henry, Theodore B

    2014-04-01

    Little is known about how hepatic vitellogenin gene (vtg) expression relates to oogenesis in fish, especially among fractional spawners. The objective of this study was to relate hepatic vtg 1A/B expression to stage-specific oocyte development in zebrafish (Danio rerio), an asynchronous spawning fish. Liver samples were collected at seven time points postspawning (1-32 days) and fish were preserved for subsequent histological analyses. Relative vtg 1A/B expression among liver samples was quantified by reverse transcription-quantitative PCR and oogenesis was evaluated following standard hematoxylin and eosin staining of serial ovarian sections. Histological analyses indicate that a subset of previtellogenic oocytes (stages 1-2) transitioned into postvitellogenic oocytes (stages 3-4) within 4 days (96 h) postspawning. By 8 days postspawning (192 h), the majority of the ovary was occupied by mature (stage 4) oocytes, a trend that continued through 32 days postspawning. Hepatic vtg 1A/B gene expression was upregulated 3.89-fold 1-h postspawning relative to the average gene expression across all time points, but was not correlated to stage-specific oogenesis. Follicular atresia among fish sampled 32 days postspawning highlights the importance of regular spawning in zebrafish and suggests that the event of spawning itself may be integral to the regulation of oocyte development.

  4. Hydrostatic Pressure Affects In Vitro Maturation of Oocytes and Follicles and Increases Granulosa Cell Death

    Directory of Open Access Journals (Sweden)

    Isac Karimi

    2013-01-01

    Full Text Available Objective: This study examines the effects of hydrostatic pressure on in vitro maturation (IVM of oocytes derived from in vitro grown follicles.Materials and Methods: In this experimental study, preantral follicles were isolated from 12-day-old female NMRI mice. Each follicle was cultured individually in Alpha Minimal Essential Medium (α-MEM under mineral oil for 12 days. Then, follicles were induced for IVM and divided into two groups, control and experiment. In the experiment group follicles were subjected to 20 mmHg pressure for 30 minutes and cultured for 24-48 hours. We assessed for viability and IVM of the oocytes. The percentage of apoptosis in cumulus cells was determined by the TUNEL assay. A comparison between groups was made using the student’s t test.Results: The percentage of metaphase II oocytes (MII increased in hydrostatic pressure-treated follicles compared to controls (p<0.05. Cumulus cell viability reduced in hydrostatic pressure-treated follicles compared to controls (p<0.05. Exposure of follicles to pressure increased apoptosis in cumulus cells compared to controls (p<0.05.Conclusion: Hydrostatic pressure, by inducing apoptosis in cumulus cells, participates in the cumulus oocyte coupled relationship with oocyte maturation.

  5. Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.

    Science.gov (United States)

    Bassez, T; Paris, J; Omilli, F; Dorel, C; Osborne, H B

    1990-11-01

    The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

  6. Development of Cryopreservation Techniques for Gorgonian (Junceella juncea Oocytes through Vitrification.

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    Sujune Tsai

    Full Text Available Gorgonian corals are slowly declining due to human interaction and environmental impacts. Cryopreservation of gorgonian corals is an ex-situ method of conservation, ensuring future reproduction. The present study assessed the vitrification properties of cryoprotectant (CPT mixtures using the cryotop, cryoloop and open pulled straw (OPS cryopereservation methods prior to experimentation on gorgonian (Junceella juncea oocytes. Investigations of the equilibration and vitrification solutions' (ES and VS effect on oocytes throughout different incubation periods were conducted. The cryotop method was found to be the most successful in ensuring vitrification. The most favourable VS was composed of propylene glycol (PG, ethylene glycol (EG and methanol with concentrations of 3.5 M, 1.5 M and 2 M respectively. Experiments were performed using the cryotop method to cryopreserve Junceella juncea oocytes using VS2, the solution had the least impact on oocytes at 5°C rather than at 26°C. The success of the vitrification procedures was determined by adenosine triphosphate (ATP levels in cooled-thaw oocytes and the highest viability obtained from the present study was 76.6 ± 6.2%. This study provides information regarding gorgonian corals' tolerance and viability throughout vitrification to further advance the vitrification protocol on whip corals.

  7. [Intensity loss of two-photon excitation fluorescence microscopy images of mouse oocyte chromosomes].

    Science.gov (United States)

    Zhao, Feng-Ying; Wu, Hong-Xin; Chen, Die-Yan; Ma, Wan-Yun

    2014-07-01

    As an optical microscope with high resolution, two-photon excitation (TPE) fluorescence microscope is widely used in noninvasive 3D optical imaging of biological samples. Compared with confocal laser scanning microscope, TPE fluorescence microscope provides a deeper detecting depth. In spite of that, the image quality of sample always declines as the detecting depth increases when a noninvasive 3D optical imaging of thicker samples is performed. Mouse oocytes with a large diameter, which play an important role in clinical and biological fields, have obvious absorption and scattering effects. In the present paper, we performed compensation for two-photon fluorescence images of mouse oocyte chromosomes. Using volume as a parameter, the attenuation degree of these chromosomes was also studied. The result of our data suggested that there exists a severe axial intensity loss in two-photon microscopic images of mouse oocytes due to the absorption and scattering effects. It is necessary to make compensation for these images of mouse oocyte chromosomes obtained from two-photon microscopic system. It will be specially needed in studying the quantitative three-dimensional information of mouse oocytes.

  8. Determination in oocytes of the reproductive modes for the brine shrimp Artemia parthenogenetica.

    Science.gov (United States)

    Dai, Zhong-Min; Li, Ran; Dai, Li; Yang, Jin-Shu; Chen, Su; Zeng, Qing-Guo; Yang, Fan; Yang, Wei-Jun

    2011-02-01

    The brine shrimp, Artemia, reproduces either oviparously, producing encysted embryos (diapause cysts), or ovoviviparously, producing free-swimming nauplii. Environmental factors, such as photoperiod, have been applied to control the reproduction mode of Artemia, but when the determination of a reproductive mode occurs remains unknown. We analysed the differential gene expression between oocytes from oviparous and ovoviviparous Artemia reared under different photoperiods. A total of 692 qualified cDNA clones were obtained by subtractive hybridization, 327 of which matched GenBank® Nucleotide Sequence Database entries. Gene expressions of 44 cDNAs (representing 56 clones) were analysed in oocytes using real-time PCR. Among these genes, 11 (21 clones) were significantly (PArtemia oocytes that subsequently enter diapause. Remarkably, known diapause-related proteins such as ArHsp22 (Artemia heat-shock protein 22) and chitin-binding proteins are found to be already differentially expressed. Furthermore, RNAi (RNA interference) knockdown of a differentially expressed gene, polo-like kinase 1, in oocyte of ovoviviparous Artemia led to the production of white embryos rather than free-swimming nauplii. In summary, our results provide evidence at the molecular level that the reproductive mode of Artemia is already determined at the oocyte stage of their life cycle.

  9. Live Birth from ICSI-TESA into In Vitro Matured Oocytes: A Case Report.

    Science.gov (United States)

    Hatırnaz, Şafak; Hatırnaz, Ebru; Bakacak, Murat; Bostancı, Mehmet Sühha; Atasever, Melahat

    2015-01-01

    Repeated in vitro Fertilization (IVF) failure together with Ovarian Hyperstimulation Syndrome (OHSS) is one of the distressing situations leading couples to search for alternative treatment options. For such patients with Polycystic Ovarian Syndrome (PCOS) who have experienced Ovarian Hyperstimulation Syndrome, mild ovarian stimulation with in vitro oocyte maturation could be a promising alternative. Testicular Sperm Aspiration (TESA) of spermatozoa from a known obstructive azoospermic patient is a limiting factor for IVM (in vitro maturation) but the couple reported here accepted mild IVF -IVM with TESA. In the treatment of a 32-year old woman, 9 immature oocytes were retrieved, 5 in vitro matured oocytes (maturation rate 55%) and after fertilization by Intracytoplasmic Sperm Injection (ICSI), 3 oocytes (fertilization rate 60%) which had two pronuclei and two good quality embryos on day 2, were transferred. A live pregnancy was observed by ultrasound scan and healthy infant was delivered. Although the number of births from ICSI of immature oocytes injected with surgically derived sperm is quite low, the selection of this mode of therapy as an alternative to conventional IVF will overcome the limitations and provides a new option in IVF practice.

  10. The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

    Institute of Scientific and Technical Information of China (English)

    TAN Xin; PENG An; WANG Yongchao; TANG Zuoqing

    2005-01-01

    In order to study the effects of ubiquitin-proteasome pathway (UPP) on mouse oocyte meiosis and cleavage, oocytes undergoing maturation and parthenogenetic activation and 1-cell embryos were treated with lactacystin, a specific inhibitor of proteasome. The results indicared that the rate of GVBD was not influenced by the treatment, but polar body extrusion, parthenogenesis and first cleavage were inhibited. Immunofluorescent staining using anti β-tubulin antibody indicated that the continuous treatment of lactacystin from GV stage disorganized microtubules and spindle assembly. When metaphase stage oocytes were treated with the drug,the already formed spindle structure was not affected, but the oocytes were arrested at metaphases. The 1-cell embryos were arrested at interphase or metaphase of first mitosis when they were incubated in the drug. Proteasome regulatory subunit PA700 was located in the spindle region, as indicated by immunofluorescence. These results suggest that UPP has effects on the process of oocyte meiosis and early cleavage in many aspects, including normal organization of spindle at prophase and segregation of chromosomes at anaphase for normal meiosis.

  11. Rotation of Meiotic Spindle Is Controlled by Microfilaments in Mouse Oocytes

    Institute of Scientific and Technical Information of China (English)

    Da-YuanChen; Jin-SongLi; LiLian; LeiLei; Zhi-MingHan; Qing-YuanSun

    2005-01-01

    The completion of meiosis requires the spatial and temporal coordination of cytokinesis and karyokirlesis. During meiotic maturation, many events, such as formation, location, and rotation of the meiotic spindle as well as chromosomal movement,Polar body extrusion,and pronuclear migration,are dependent on regulation of the cytoskeleton system.To study functions of microfilaments in meiosis,we induced metaphase Ⅱ(MII)mouse oocytes to resume meiosis by in vitro fertilization or parthenogenetic activation,and we treated such oocytes with cytochalasin B(CB).The changes of the meiotic spindle,as visualized in preparations stained for β-tubulin and chromation,were observed by fluorescent confocal microscopy.The meiotic spindle of Mll oocytes was observed to be parallel to the plasmalemma.After meiosis had resumed,the spindle rotated to the vertical position so that the second polar body could be extruded into the perivitelline space.When meiosis resumed and oocytes were treated with 10μg/ml of CB,the spindle rotation was inhibited.Consequently,the oocyte formed an extra pronucleus instead of extruding a second polar body.These results indicate that spindle rotation is essential for polar body extrusion;it is the microfilaments that play a crucial role in regulating rotation of the meiotic spindle.

  12. The dynamics of postovulatory follicle degeneration and oocyte growth in Baltic sprat

    Science.gov (United States)

    Haslob, H.; Kraus, G.; Saborido-Rey, F.

    2012-01-01

    Ovaries of Baltic sprat ( Sprattus sprattus balticus S.) were analysed histologically to identify stages of postovulatory follicles (POF) and to assess the oocyte development pattern. Samples were taken every 3 h during a 24 h trawl survey conducted in the Bornholm Basin in April 2007. Gonad histology revealed spawning of sprat throughout the day which hampered the exact ageing of POFs by the postovulatory follicle method and therefore did not allow direct estimation of spawning frequency. However, it was possible to define four stages of POFs, according to their histological features. The occurrence of these POF stages (I to IV) corresponded clearly to the development of the leading oocyte cohort. Further, the oocyte recruitment pattern revealed that the spawning batch can be identified prior to hydration. The POF stages I and II were present almost exclusively in vitellogenic ovaries, POF III were found in ovaries in the germinal vesicle stage, and the most deteriorated POF stage IV was found in actively spawning fish with hydrated oocytes. Since POF were absent only in very few ovaries (5%), and in each ovary in general only one POF stage was present, the duration of POF degeneration approximately equals the average batch interval, i.e. the time lag between subsequent spawning events. The results of the present study will serve as basis for future studies on Baltic sprat oocyte recruitment and daily spawning fraction.

  13. Histological characterization of oocyte developmental stages of suruvi Steindachneridion scriptum kept in captivity

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    Mariana Roza Abreu

    2015-10-01

    Full Text Available Stages of oocyte development were described for suruvi females (Steindachneridion scriptum kept in captivity. Oocytes of 21 mature or maturing females and ovaries of 12 immature females were fixed in Karnovsky solution for 4 and 12 hours, respectively. After, they were processed for routine histology, embedded in paraffin and stained with hematoxylin-eosin. Slides were examined under a light microscope and the oocytes were measured using the LAS EZ software, using the average between the largest and the smallest diameter of each oocyte for representation. There were six types of oocytes: Chromatin nucleolus: found in immature females, with average diameter of 9.5 ± 4.46 μm; Perinucleolar: present in all developmental stages with average diameter of 67.6 ± 15.61 μm; Cortical alveolar: measuring 175.6 ± 56.58 μm; Vitellogenic: average diameter of 797.65 ± 136.10 μm; Mature: with average diameter of 1184.98 ± 171.13 μm and Atretic, which were found in mature or maturing females of suruvi.

  14. Microtubule-microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes.

    Science.gov (United States)

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill; Gelfand, Vladimir I

    2016-08-23

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

  15. Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte

    Science.gov (United States)

    Dalton, Caroline M.; Carroll, John

    2013-01-01

    Summary A fundamental rule of cell division is that daughter cells inherit half the DNA complement and an appropriate proportion of cellular organelles. The highly asymmetric cell divisions of female meiosis present a different challenge because one of the daughters, the polar body, is destined to degenerate, putting at risk essential maternally inherited organelles such as mitochondria. We have therefore investigated mitochondrial inheritance during the meiotic divisions of the mouse oocyte. We find that mitochondria are aggregated around the spindle by a dynein-mediated mechanism during meiosis I, and migrate together with the spindle towards the oocyte cortex. However, at cell division they are not equally segregated and move instead towards the oocyte-directed spindle pole and are excluded from the polar body. We show that this asymmetrical inheritance in favour of the oocyte is not caused by bias in the spindle itself but is dependent on an intact actin cytoskeleton, spindle–cortex proximity, and cell cycle progression. Thus, oocyte-biased inheritance of mitochondria is a variation on rules that normally govern organelle segregation at cell division, and ensures that essential maternally inherited mitochondria are retained to provide ATP for early mammalian development. PMID:23659999

  16. Reduced developmental competence of immature, in-vitro matured and postovulatory aged mouse oocytes following IVF and ICSI

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    Trounson Alan

    2008-12-01

    Full Text Available Abstract Background The present study highlights basic physiological differences associated with oocyte maturation and ageing. The study explores the fertilizing capacity and resistance to injury of mouse oocytes at different stages of maturation and ageing following IVF and ICSI. Also, the study examines the developmental competence of embryos obtained from these oocytes. The outcome of the study supports views that the mouse can be a model for human IVF suggesting that utilizing in-vitro matured and failed fertilized oocytes to produce embryos mainly when limited number of oocytes is retrieved in a specific cycle, should be carefully considered. Methods Hybrid strain mouse oocytes were inseminated by in-vitro fertilization (IVF or intracytoplasmic sperm injection (ICSI. Oocytes groups that were used were germinal vesicle (GV in-vitro matured metaphase II (IVM-MII, freshly ovulated MII (OV-MII, 13 hrs in-vitro aged MII (13 hrs-MII and 24 hrs in-vitro aged MII (24 hrs-MII. Fertilization and embryo development to the blastocyst stage were monitored up to 5 days in culture for IVF and ICSI zygotes. Sperm head decondensation and pronuclear formation were examined up to 9 hrs in oocytes following ICSI. Apoptotic events in blocked embryos were examined using the TUNNEL assay. Differences between females for the number and quality of GV and OV-MII oocytes were examined by ANOVA analyses. Differences in survival after ICSI, fertilization by IVF and ICSI and embryo development were analysed by Chi-square test with Yates correction. Results No differences in number and quality of oocytes were identified between females. The findings suggest that inability of GV oocytes to participate in fertilization and embryo development initiates primarily from their inability to support initial post fertilization events such as sperm decondensation and pronuclei formation. These events occur in all MII oocytes in similar rates (87–98% for IVF and ICSI. Following

  17. Roscovitine in combination with calcium ionophore induces oocyte activation through reduction of M-phase promoting factor activity in mice.

    Science.gov (United States)

    Iba, Tomomi; Yano, Yuya; Umeno, Mayumi; Hinokio, Kenji; Kuwahara, Akira; Irahara, Minoru; Yamano, Shuji; Yasui, Toshiyuki

    2012-11-01

    The aim of the present study was to determine oocyte activation and change in M-phase promoting factor (MPF) activity induced by treatment with calcium ionophore and roscovitine in comparison with those induced by treatment with roscovitine alone and treatment with calcium ionophore and puromycin in mice. Freshly ovulated oocytes obtained from 6-8-week-old mice were divided into five groups (no activation treatment; 5 μM calcium ionophore A23187; 50 μM roscovitine; 5 μM calcium ionophore and 10 μg/ml puromycin; and 5 μM calcium ionophore and 50 μM roscovitine) and were incubated for 6 h. Oocyte activation, assessed by morphological changes, and changes in MPF activity in the five groups at 0, 2, 4 and 6 h of incubation were examined. Activated oocytes were defined as oocytes with at least one pronucleus. Oocytes treated with roscovitine alone were not activated during the 6-h incubation period. All of the oocytes in the calcium ionophore with puromycin group and in the calcium ionophore with roscovitine group were activated. The percentage activity of MPF in oocytes treated with roscovitine alone was decreased after 2 h and increased after 4 h of incubation. The percentage activity of MPF in oocytes treated with calcium ionophore and roscovitine was significantly decreased with suppression of MPF activity being maintained for 6 h, and this change was similar to that in oocytes treated with calcium ionophore and puromycin. Roscovitine with calcium ionophore is effective for induction of oocyte activation through suppression of MPF activity in mice.

  18. Compromised fertility disrupts Peg1 but not Snrpn and Peg3 imprinted methylation acquisition in mouse oocytes

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    Michelle M Denomme

    2012-07-01

    Full Text Available Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERβ and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20-80 μm control oocytes at three imprinted genes, Snrpn, Peg3 and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition, individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3 or Peg1 in oocytes from Ers2-deficient females, and no perturbation in Snrpn or Peg3 de novo methylation in oocytes from Gja4-null females. However, Gja4-deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further st