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Sample records for oocyte activation methods

  1. Analysis of cat oocyte activation methods for the generation of feline disease models by nuclear transfer

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    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

  2. 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.

  3. 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.

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

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    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.

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

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    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.

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

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    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.

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

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    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.

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

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    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.

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

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    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.

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

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    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.

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

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    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.

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

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    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

  13. Regulation of ALF promoter activity in Xenopus oocytes.

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    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.

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

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    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...

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

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    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.

  16. Development capacity of pre- and postpubertal pig oocytes evaluated by somatic cell nuclear transfer and parthenogenetic activation

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    Skovsgaard, Hanne; Li, Rong; Liu, Ying;

    2013-01-01

    Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear...... transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible...... perivitelline space) or bad before used for PA (good and bad) or SCNT (good). The PA and SCNT were performed as before with minor modifications (Cryobiol. 64, 60; Cell. Reprogr. 13, 521) before culture for 6 days in a standard or timelapse incubator. Rates of cleavage (CL%, Day 2), blastocyst (BL%, Day 6...

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

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    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.

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

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    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.

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

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    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.

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

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    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.

  1. In vitro and in vivo developmental competence of dromedary (Camelus dromedarius) oocytes following in vitro fertilization or parthenogenetic activation.

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    Khatir, H; Anouassi, A; Tibary, A

    2009-07-01

    Parthenogenetic activation of the oocyte represents an important step in the somatic cloning. The aim of the present study was to evaluate the effectiveness (in term of in vitro development) of different methods of parthenogenetic activation of dromedary oocytes. Selected cumulus-oocytes-complexes (n=1264) collected by follicular aspiration from ovaries obtained postmortem were matured in vitro (IVM) for 30 h then divided randomly into seven groups and submitted to artificial activation. Two groups were preactivated with 25 microM of calcium ionophore (CaI) for 20 min then incubated for 4h with either 2mM 6-dimethylaminopurine (6-DMAP) (group 1, n=202) or with 10 microg/mL cycloheximide (CHX) (group 2, n=194). Group 3 (n=172) and group 4 (n=184), oocytes were pretreated with 5 microM ionomycin (Iono) for 5 min then incubated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 5 (n=161) and group 6 (n=155) oocytes were preactivated with electrical stimulation (ES) then activated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 7 (n=196) oocytes were submitted to in vitro fertilization (IVF) and served as a control. All groups containing oocytes were cultured in vitro following activation or IVF, at 38.5 degrees C under 5% CO(2) in air with >95% humidity. The in vitro development rates of dromedary oocytes exposed to 6-DMAP after CaI (61%), ES (74%) and the IVF group (71%) were similar and significantly greater (Pdromedary embryos was examined by transfer to synchronized recipients. An embryonic vesicle was seen by ultrasonography at 15 days post transfer in four females (CaI/6-DMAP: 1/5; 20%, IVF: 3/10; 30%). The only pseudopregnancy obtained with an activated embryo resorbed at 25 days. One of the females receiving the IVF produced embryos aborted at 2 months and the other two females carried to term and gave birth to healthy calves (one female and one male). This study shows that artificial activation of

  2. 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.

  3. 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.

  4. 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.

  5. Successful pregnancy and childbirth after intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient.

    Science.gov (United States)

    Tejera, Alberto; Mollá, Marta; Muriel, Lourdes; Remohí, Jose; Pellicer, Antonio; De Pablo, Jose Luis

    2008-10-01

    To check the effectiveness of intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA) in a globozoospermic patient. Case report. Instituto Valenciano de Infertilidad, Valencia, Spain. A patient with globozoospermia. ICSI was administered in 14 oocytes. ICSI combined with AOA, in which a small amount of calcium was injected followed by calcium ionophore exposure, was done in 9 oocytes. Fertilization rate and embryo quality was assessed in both groups. Chemical activation increased fertilization rate (55.6% vs. 35.7%) and the number of embryos with less multinucleation on day 2 (0 vs. 60%). Two embryos generated from AOA were transferred into the uterus (on day 3), resulting in a pregnancy and a healthy newborn. The AOA with calcium ionophore treatment improved fertilization rate and quality of the embryos, and was found to be an effective method for AOA in this patient with a low fertilization rate after previous ICSI treatment.

  6. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method.

    Science.gov (United States)

    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.

  7. Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

    Science.gov (United States)

    Ebner, Thomas; Montag, Markus; Montag, M; Van der Ven, K; Van der Ven, H; Ebner, T; Shebl, O; Oppelt, P; Hirchenhain, J; Krüssel, J; Maxrath, B; Gnoth, C; Friol, K; Tigges, J; Wünsch, E; Luckhaus, J; Beerkotte, A; Weiss, D; Grunwald, K; Struller, D; Etien, C

    2015-04-01

    Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Artificial oocyte activation in intracytoplasmic sperm injection cycles using testicular sperm in human in vitro fertilization.

    Science.gov (United States)

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

    2015-06-01

    Artificial oocyte activation (AOA) is an effective method to avoid total fertilization failure in human in vitro fertilization-embryo transfer (IVF-ET) cycles. AOA performed using a calcium ionophore can induce calcium oscillation in oocytes and initiate the fertilization process. We evaluated the usefulness of AOA with a calcium ionophore in cases of total fertilization failure in previous cycles and in cases of severe male factor infertility patients with non-motile spermatozoa after pentoxifylline (PF) treatment. The present study describes 29 intracytoplasmic sperm injection (ICSI)-AOA cycles involving male factor infertility at Cheil General Hospital from January 2006 to June 2013. Patients were divided into two groups (control, n=480; AOA, n=29) depending on whether or not AOA using a calcium ionophore (A23187) was performed after testicular sperm extraction-ICSI (TESE-ICSI). The AOA group was further split into subgroups according to sperm motility after PF treatment: i.e., motile sperm-injected (n=12) and non-motile sperm-injected (n=17) groups (total n=29 cycles). The good embryo rate (52.3% vs. 66.9%), pregnancy rate (20.7% vs. 52.1%), and delivery rate (10.3% vs. 40.8%) were lower in the PF/AOA group than in the control group. When evaluating the effects of restoration of sperm motility after PF treatment on clinical outcomes there was no difference in fertilization rate (66.6% vs. 64.7% in non-motile and motile sperm, respectively), pregnancy rate (17.6% vs. 33.3%), or delivery rate (5.9% vs. 16.7%) between the two groups. We suggest that oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles regardless of restoration of sperm motility after PF treatment. AOA may be useful in selected patients who have a low fertilization rate or total fertilization failure.

  10. 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.

  11. A method for mouse oocyte enucleation assisted with zona pellucida dilating

    Institute of Scientific and Technical Information of China (English)

    Bai Zhao-dai; Liu Kai; Bing Lu-jun

    2004-01-01

    Objective: To study the reliability of a new enucleation method, zona pellucida Dilating (ZPD) assisted enucleation method, for mouse oocyte enucleation.Methods: In ZPD enucleation method, the zona pellucida was dilating with the help of outside pression in enucleation needle after breakthrough by needle tip and the nucleus was aspirated by the needle entering the perivitelline space for enucleation. This method was employed and was compared with three other methods in this experiment. The efficiency of enucleation of mouse oocyte was examed.Result: ZPD assisted enucleation method is better than three others at the survival rate after enucleation and the simplicity of micromanipulation. The rate of forming pronucleus of reconstructed embryo from enucleated oocyte by ZPD methods is as high as 62.8 % and the reconstructed embryo at 4-cell stage was obtained.Conclusion: ZPD assisted method is a highly efficient and simple enucleation method with the advantage of saving manipulating time and it does less damage to the oocyte.

  12. 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.

  13. 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...

  14. 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.

  15. Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations.

    Science.gov (United States)

    Fernandes, Claudia Barbosa; Devito, Liani Gasparini; Martins, Lilian Rigatto; Blanco, Ieda Dala Pria; de Lima Neto, João Ferreria; Tsuribe, Patricia Myakawa; Gonçalves, Camila Gabriela Pereria; da Cruz Landim-Alvarenga, Fernanda

    2014-08-01

    Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

  16. Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

    Institute of Scientific and Technical Information of China (English)

    Xiao-Yu Yang; Jing Wang; Jia-Yin Liu; Yan Gao; Zuo-Min Zhou; Jia-Hao Sha; Wei Zhang; Yu-Gui Cui; Xiao-Qiao Qian

    2012-01-01

    Dear Editor,I am Dr Xiao-Yu Yang,from the Center of Clinical Reproductive Medicine in the First Affiliated Hospital at the NanJing Medical University,Nanjing,China.We present here a case report of a globozoospermic patient whose partner became pregnant after intracytoplasmic sperm injection (ICSI) with assisted oocyte activation (AOA).Globozoospermia is characterized by the presence of 100% round- headed spermatozoa lacking an acrosome.The lack of the acrosome,which renders spermatozoa unable to bind to the zona pellucida or fuse with the oocyte oolemma,is considered to be the cause of infertility in these patients.

  17. EFFECT OF BULL AND SPERM PREPARATION METHOD ON IN VITRO FERTILIZATION OF BUFFALO OOCYTES

    Directory of Open Access Journals (Sweden)

    H. JAMIL, H. A. SAMAD, Z. I. QURESHI, N. REHMAN AND L. A. LODHI

    2007-01-01

    Full Text Available The present study was designed to compare fertilization rates following oocyte exposure to spermatozoa from different buffalo bulls, using three sperm preparation methods i.e. percoll density gradient, swim-up (modified Ca2 free Tyrode’s medium and TALP medium and sodium citrate washing prior to co-incubation with oocytes. Buffalo oocytes were aspirated from ovarian follicles within 1.5 to 2 hours after slaughter. They were matured in TCM-199 supplemented with 20% oestrus buffalo serum drops under mineral oil in CO2 incubator at 39C for 24 hours. Matured oocytes were transferred to the fertilization droplets and inseminated with 1x106 capacitated sperms prepared by different experimental methods. Data collected on recovered sperm motility immediately after treatment and 24 hours after incubation (at 37C and cleavage rate of co-incubated oocytes were subjected to statistical analysis. The percentage of motile spermatozoa was significantly higher (P<0.05 in semen samples prepared by swim-up method in Ca2 free Tyrode’s medium compared to other experimental techniques. Bull wise comparison showed significantly higher (P<0.05 motility in bull B1 (50.50 ± 5.92%, followed by bull B2 (46.46 ± 5.99% and B3 (45.96 ± 5.79%. Fertilization rate was also significantly (P<0.05 higher for spermatozoa prepared by Swim-up method in Ca2 free Tyrode’s medium (63.75 ± 2.81%, followed by sodium citrate (26.70 ± 5.08%, swim-up TALP (29.14 ± 3.74% and Percoll gradient density (23.89 ± 3.88%. Fertilization rate was significantly higher (P<0.05 in oocytes inseminated with semen from bull B1 (43.43 ± 8.59%, followed by B2 (33.38 ± 9.95% and B3 (30.80 ± 9.56%. The results of present study indicate that bulls and sperm preparation methods differ in their contribution to in vitro fertilization rate. Further studies are suggested to ascertain the factors responsible for such specific effects.

  18. Pentose phosphate pathway activity: effect on in vitro maturation and oxidative status of bovine oocytes.

    Science.gov (United States)

    Gutnisky, Cynthia; Dalvit, Gabriel C; Thompson, Jeremy G; Cetica, Pablo D

    2014-08-01

    The relationship between pentose phosphate pathway (PPP) activity in cumulus-oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10mM for 22h) was added to the maturation medium (P<0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P<0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5mM for 22h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5mM (the highest concentration tested; P<0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P<0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P<0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9h maturation (P<0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte.

  19. Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

    Institute of Scientific and Technical Information of China (English)

    杨仲安; 曹丹; 桂建芳

    2000-01-01

    The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

  20. 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.

  1. Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins.

    Science.gov (United States)

    Dorr, Ricardo; Ozu, Marcelo; Parisi, Mario

    2007-04-15

    Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements.

  2. Microfluidic Method of Pig Oocyte Quality Assessment in relation to Different Follicular Size Based on Lab-on-Chip Technology

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    Bartosz Kempisty

    2014-01-01

    Full Text Available Since microfollicular environment and the size of the follicle are important markers influencing oocyte quality, the aim of this study is to present the spectral characterization of oocytes isolated from follicles of various sizes using lab-on-chip (LOC technology and to demonstrate how follicle size may affect oocyte quality. Porcine oocytes (each, n=100 recovered from follicles of different sizes, for example, from large (>5 mm, medium (3–5 mm, and small (<3 mm, were analyzed after preceding in vitro maturation (IVM. The LOC analysis was performed using a silicon-glass sandwich with two glass optical fibers positioned “face-to-face.” Oocytes collected from follicles of different size classes revealed specific and distinguishable spectral characteristics. The absorbance spectra (microspectrometric specificity for oocytes isolated from large, medium, and small follicles differ significantly (P<0.05 and the absorbance wavelengths were between 626 and 628 nm, between 618 and 620 nm, and less than 618 nm, respectively. The present study offers a parametric and objective method of porcine oocyte assessment. However, up to now this study has been used to evidence spectral markers associated with follicular size in pigs, only. Further investigations with functional-biological assays and comparing LOC analyses with fertilization and pregnancy success and the outcome of healthy offspring must be performed.

  3. Microtransplantation of membranes from cultured cells to Xenopus oocytes: A method to study neurotransmitter receptors embedded in native lipids

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    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 cell line (HEK293) stably expressing the rat glutamate receptor 1 were injected into oocytes, and, within a few hours, the oocyte plasma membrane acquired α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors, which had the same properties as those expressed in the original HEK cells. Analogously, oocytes injected with membranes prepared from rat pituitary GH(4)C1 cells, stably expressing homomeric human neuronal α7 nicotinic acetylcholine receptors (α7-AcChoRs), incorporated in their plasma membrane AcChoRs that behaved as those expressed in GH(4)C1 cells. Similar results were obtained with HEK cells stably expressing heteromeric human neuronal α4β2-AcChoRs. All this makes the Xenopus oocyte a powerful tool for detailed investigations of receptors and other proteins expressed in the membrane of cultured cells. PMID:12595576

  4. Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes.

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    Zapata-Martínez, J; Medina, M F; Gramajo-Bühler, M C; Sánchez-Toranzo, G

    2016-08-01

    Rhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.

  5. Phospholipase Czeta mRNA expression and its potency during spermatogenesis for activation of quail oocyte as a sperm factor.

    Science.gov (United States)

    Mizushima, Shusei; Takagi, Soichi; Ono, Tamao; Atsumi, Yusuke; Tsukada, Akira; Saito, Noboru; Shimada, Kiyoshi

    2009-12-01

    This study was conducted to investigate the role of a sperm-borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase Czeta (PLCzeta) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLCzeta cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13 mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca(2+) chelator) before SE injection. On the other hand, when the oocytes were injected with PLCzeta cRNA at 60 microg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCzeta cRNA-induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLCzeta cRNA can induce development. In addition, RT-PCR revealed that PLCzeta mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCzeta is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis.

  6. Relationship between Potential Sperm Factors Involved in Oocyte Activation and Sperm DNA Fragmentation with Intra-Cytoplasmic Sperm Injection Clinical Outcomes

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    Marziyeh Tavalaee

    2016-10-01

    Full Text Available Objective: The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein (PAWP, phospholipase Cζ (PLCζ, and truncated form of the kit receptor (TR-KIT] as candidates of oocyte activation with fertilization rate and early embryonic development. Materials and Methods: In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection (ICSI candidates and analyzed according to World Health Organization criteria (2010. Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation (DGC and the second part was prepared for assessment of sperm morphology (Papanicolaou staining, DNA fragmentation [transferase dUTP nick end labeling (TUNEL], and three Sperm-borne oocyte-activating factor (s (SOAFs-PLCζ, PAWP, and TR-KIT. Results: Significant positive correlations existed between the percentages of PLCζ, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCζ and PAWP. We did not find a relationship between percentages of PLCζ, PAWP, and TR-KIT with embryo quality and pregnancy rate (P>0.05. There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality. Conclusion: Oocyte activation was associated with the studied sperm factors (PAWP, PLCζ, and TR-KIT. These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCζ, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether

  7. Relationship between Potential Sperm Factors Involved in Oocyte Activation and Sperm DNA Fragmentation with Intra-Cytoplasmic Sperm Injection Clinical Outcomes

    Science.gov (United States)

    Tavalaee, Marziyeh; Kiani-Esfahani, Abbas; Nasr-Esfahani, Mohammad Hossein

    2017-01-01

    Objective The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein (PAWP), phospholipase Cζ (PLCζ), and truncated form of the kit receptor (TR-KIT)] as candidates of oocyte activation with fertilization rate and early embryonic development. Materials and Methods In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection (ICSI) candidates and analyzed according to World Health Organization criteria (2010). Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation (DGC) and the second part was prepared for assessment of sperm morphology (Papanicolaou staining), DNA fragmentation [transferase dUTP nick end labeling (TUNEL)], and three Sperm-borne oocyte-activating factor (s) (SOAFs)-PLCζ, PAWP, and TR-KIT. Results Significant positive correlations existed between the percentages of PLCζ, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCζ and PAWP. We did not find a relationship between percentages of PLCζ, PAWP, and TR-KIT with embryo quality and pregnancy rate (P>0.05). There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality. Conclusion Oocyte activation was associated with the studied sperm factors (PAWP, PLCζ, and TR-KIT). These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCζ, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action

  8. Cryopreservation of human failed-matured oocytes followed by in vitro maturation: vitrification is superior to the slow freezing method

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    Zhang ZhiGuo

    2011-12-01

    Full Text Available Abstract Background Oocyte cryopreservation is an important method used in a number of human fertility circumstances. Here, we compared the survival, in vitro maturation, fertilization, and early embryonic development rates of frozen-thawed human immature oocytes using two different cryopreservation methods. Methods A total of 454 failed-matured oocytes [germinal vesicle (GV and metaphase I (MI stages] were collected from 135 patients (mean age 33.84 +/- 5.0 y who underwent intracytoplasmic sperm injection (ICSI cycles between February 2009 and December 2009 and randomly divided into a slow freezing group [1.5 mol/L-1, 2-propanediol (PROH + 0.2 mol/l sucrose] and vitrification group [20% PROH + 20% ethylene glycol (EG + 0.5 mol/l sucrose]. Results The vitrification protocol yielded a better survival rate than the slow freezing protocol at each maturation stage assessed. Regardless of the maturation stage (GV + MI, the slow freezing protocol had a significantly lower survival rate than the vitrification protocol (p in vitro maturation (21.2 vs. 54.0%, respectively; p 0.05. For the GV-matured oocytes, no fertilized eggs were obtained in the slow-freezing group, while a 19.0% (4/21 fertilization rate was observed in the vitrification group. For the MI-matured oocytes, fertilization rates for the slow freezing and vitrified groups were 36% and 61.1%, respectively, but no significant difference was found between the two groups (PIn the Methods section in the MS, all procedures were compliant with ethical guidelines, i.e. approved by the Ethical Committee of our university and Informed Consent signed by each patient. > 0.05. In the GV vitrification group, no embryo formed; however, in the MI slow freezing group, 12 oocytes were fertilized, but only two achieved cleavage and were subsequently blocked at the 2-cell stage. In the MI vitrification group, a total of 22 embryos were obtained, five of which developed to the blastocyst stage. Conclusions

  9. Cryopreservation of human oocytes, zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification methods

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    Tahani Al-Azawi

    2013-12-01

    Full Text Available Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.

  10. Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes.

    Science.gov (United States)

    Bilodeau-Goeseels, Sylvie

    2011-01-01

    Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures.

  11. Method of euthanasia influences the oocyte fertilization rate with fresh mouse sperm.

    Science.gov (United States)

    Hazzard, Karen C; Watkins-Chow, Dawn E; Garrett, Lisa J

    2014-11-01

    In vitro fertilization (IVF) is used to produce mouse embryos for a variety of reasons. We evaluated the effect of the method of euthanasia on the fertilization rate in 2 different IVF protocols. Oocytes collected from C57BL/6J female mice euthanized by CO2 inhalation or cervical dislocation were used in IVF with fresh sperm from either wild-type or genetically engineered C57BL/6J. Compared with CO2 inhalation, cervical dislocation improved the resulting rate of fertilization by 18% in an IVF method using Cook media and by 13% in an IVF method using methyl-B cyclodextrin and reduced glutathione. The lower fertilization rate due to euthanasia by CO2 inhalation was accompanied by changes in blood pH and body temperature despite efforts to minimize temperature drops. In our hands, euthanasia by cervical dislocation improved fertilization rates and consequently reduced the number of egg-donor mice required.

  12. Method of Euthanasia Influences the Oocyte Fertilization Rate with Fresh Mouse Sperm

    Science.gov (United States)

    Hazzard, Karen C; Watkins-Chow, Dawn E; Garrett, Lisa J

    2014-01-01

    In vitro fertilization (IVF) is used to produce mouse embryos for a variety of reasons. We evaluated the effect of the method of euthanasia on the fertilization rate in 2 different IVF protocols. Oocytes collected from C57BL/6J female mice euthanized by CO2 inhalation or cervical dislocation were used in IVF with fresh sperm from either wild-type or genetically engineered C57BL/6J. Compared with CO2 inhalation, cervical dislocation improved the resulting rate of fertilization by 18% in an IVF method using Cook media and by 13% in an IVF method using methyl-B cyclodextrin and reduced glutathione. The lower fertilization rate due to euthanasia by CO2 inhalation was accompanied by changes in blood pH and body temperature despite efforts to minimize temperature drops. In our hands, euthanasia by cervical dislocation improved fertilization rates and consequently reduced the number of egg-donor mice required. PMID:25650969

  13. The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture

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    MORITA, Yasuhiro; TANIGUCHI, Masayasu; TANIHARA, Fuminori; ITO, Aya; NAMULA, Zhao; DO, Lanh Thi Kim; TAKAGI, Mitsuhiro; TAKEMOTO, Tatsuya; OTOI, Takeshige

    2016-01-01

    The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts. PMID:26947170

  14. LIMK1 activity is required for MTOC localization and spindle bipolarity establishment during meiosis in mouse oocytes

    Institute of Scientific and Technical Information of China (English)

    Liu Xiaoyun; Li Xin; Ma Wei

    2015-01-01

    Aneuploid embryo generally leads to infertility, spontaneous abortion and birth defects, mainly resulting from abnormal chromosome segregation during maternal oocytes meiosis. Chromosome division is conducted by bipolar spindle which formed through an acentrosomal way, dependent on a unique microtubule organizing center ( MTOC) in mammalian oocytes, however, the molecular composition and functional regulation of MTOC is still not fully ex-plored. LIM kinases 1 (LIMK1) is a conserved serine/threonine kinase, a major regulator of actin and microtubule dynamics, involved in microtubule stability and spindle positioning during mitosis. So far little is known about LIMK1 protein expression and its roles in oocytes during meiosis. We reported here the protein expression and sub-cellular distribution of LIMK1 in mouse oocytes during meiosis. Western blot procedure detected high and stable expression of LIMK1 in mouse oocytes from germinal vesicle ( GV) stage to metaphase II ( MII) . In contrast, acti-vated LIMK1 ( phosphorylated at Thr508 , pLIMK1 Thr508 ) was only observed after germinal vesicle breakdown ( GVBD) , and gradually increased with peak levels at metaphase I ( MI) and MII. Immunofluorescence analysis showed that LIMK1 was co-localized with microtubules on the whole spindle structure, while pLIMK1Thr508 was con- centrated with key components of MTOC,pericentrin and -Tubulin, on spindle poles in mouse oocytes. Inhibition of LIMK1 activity by BMS3, a specific ATPase competitive inhibitor, distroyed the formation of bipolar spindle structure, disturbed MTOC integrity and MTOC proteins recruitment to spindle poles. Moreover, LIMK1 inhibition caused chromosome misalignment and meiotic progression arrest at MI stage. Therefore, LIMK1 activity is required for formation and maintenance of bipolar spindle in mouse oocytes,importantly, pLIMK1T508 is MTOC-associated protein,involved in establishment and positioning of MTOC.

  15. Studies on Electrical Activation of Porcine Oocytes Matured in vitro and Embryo Culture Systems

    Institute of Scientific and Technical Information of China (English)

    WU Zhong-hong; XING Feng-ying; LIU Guo-shi; ZENG Shen-ming; ZHU Shi-en; ZHANG Zhong-cheng; FU Peng-hui

    2002-01-01

    Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 μs, with a blastocyst development rate of (20.12 ± 8.18)% (P > 0.05). No significant difference was found between treatments of multiple pulses and a single pulse ( P > 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35)% ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) %, P > 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 ± 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79,P < 0.01 ). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2 (5 % CO2:7%O2:88%N2) also showed no significant difference from those in high O2 (5% CO2 in air) [ (20.78 ± 8.80) % and 17.00 ± 6.12 vs. (25.30 ± 7.55) % and 18.01 ± 6.79, P > 0.05 ]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.

  16. An extraovarian protein accumulated in mosquito oocytes is a carboxypeptidase activated in embryos

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    Wenlong Cho; Deitsch, K.W.; Raikhel, A.S. (Michigan State Univ., East Lansing (United States))

    1991-12-01

    The authors report a phenomenon previously unknown for oviparous animals; in Aedes aegypti mosquitoes a serine carboxypeptidase is synthesized extraovarially and then internalized by oocytes. The cDNA encoding mosquito vitellogenic carboxypeptidase (VCP) was cloned and sequenced. The VCP cDNA hybridizes to a 1.5-kilobase mRNA present only in the fat body of vitellogenic females. The deduced amino acid sequence of VCP shares significant homology with members of the serine carboxypeptidase family. Binding assays using a serine protease inhibitor, ({sup 3}H)diisopropyl fluorophosphate, showed that VCP is activated in eggs at the onset of embryonic development. Activation of VCP is associated with the reduction in its size from 53 kDa (inactive proenzyme) to 48 kDa (active enzyme). The active, 48-kDa, form of VCP is maximally present at the middle of embryonic development and disappears by the end.

  17. Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes.

    Science.gov (United States)

    Kim, H Y; Hanley, M R

    1999-06-30

    Acid extracts of thapsigargin-treated Xenopus oocytes revealed Ca2(+)-dependent Cl- currents by microinjection into Xenopus oocytes. These currents were detected in highly purified fractions by carrying out a sequence of purification steps including gel filtration chromatography and high performance thin layer chromatography. The nature of the membrane currents evoked by the highly purified fractions were carried by chloride ions as blockade by the selective chloride channel blocker 1 mM niflumic acid. Injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) eradicated the current activities, indicating that the current responses are completely Ca2(+)-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through plasma membrane calcium entry channels. These results elucidate that the highly purified fractions aquired by thapsigargin-stimulated oocytes is an authentic calcium influx factor (CIF). Thus, the detection of increased CIF production from thapsigargin treatment in Xenopus oocytes would give strong support for the existence of CIF as a diffusible messenger for the activation of capacitative calcium entry pathways in Xenopus oocytes.

  18. Extracts from plants used in Mexican traditional medicine activate Ca(2+)-dependent chloride channels in Xenopus laevis oocytes.

    Science.gov (United States)

    Rojas, A; Mendoza, S; Moreno, J; Arellano, R O

    2003-01-01

    The two-electrode voltage-clamp technique was employed to investigate the effects of chloroform-methanol (1:1) extracts derived from five medicinal plants on Xenopus laevis oocytes. When evaluated at concentrations of 1 to 500 microg/ml, the extracts prepared from the aerial parts of Baccharis heterophylla H.B.K (Asteraceae), Chenopodium murale L. (Chenopodiaceae), Desmodium grahami Gray (Leguminosae) and Solanum rostratum Dun (Solanaceae) produced concentration-dependent oscillatory inward currents in the oocytes, while the extract of Gentiana spathacea did not induce any response. The reversal potential of the currents elicited by the active extracts was -17 +/- 2 mV and was similar to the chloride equilibrium potential in oocytes. These ionic responses were independent of extracellular calcium. However, they were eliminated by overnight incubation with BAPTA-AM (10 microM), suggesting that the currents were dependent on intracellular Ca2+ increase. Thus the plant extracts activate the typical oscillatory Ca(2+)-dependent Cl- currents generated in the Xenopus oocyte membrane more probably via a mechanism that involves release of Ca2+ from intracellular reservoirs. These observations suggest that Xenopus oocyte electrophysiological recording constitutes a suitable assay for the study of the mechanisms of action of herbal medicines.

  19. A live birth of activated one-day-old unfertilized oocyte for a patient who experienced repeatedly near-total fertilization failure after intracytoplasmic sperm injection

    Institute of Scientific and Technical Information of China (English)

    LU Qun; CHEN Xi; LI Yang; ZHANG Xiao-hong; LIANG Rong; ZHAO Yong-ping; WEI Li-hui; SHEN Huan

    2012-01-01

    Total or near-total fertilization failure after intracytoplasmic sperm injection (ICSI) is a rare event,but it occurs repeatedly because of sperm defects in activating oocyte.The case presents a successful pregnancy and live birth after calcium ionophore A23187 (A23187) activation on one-day-old unfertilized oocytes in a patient whose husband suffered oligoasthenoteratozoospermia,and who had experienced repeated near-total fertilization failure after ICSI.In the second ICSI cycle,only one oocyte was fertilized while nine were unfertilized.Oocyte activation with A23187 were performed on the one-day-old unfertilized oocytes after ICSI and resulted in fertilization and embryo transfer.A clinical pregnancy was achieved and a healthy baby was born.To our knowledge,this is the first reported case of a healthy birth after oocyte activation on the one-day-old unfertilized oocyte.This indicates that “rescue oocyte activation” on one-day-old unfertilized oocytes after ICSI may be helpful for preventing total or near-total fertilization failure after ICSI.

  20. Allosteric activation of the Ca2+ receptor expressed in Xenopus laevis oocytes by NPS 467 or NPS 568.

    Science.gov (United States)

    Hammerland, L G; Garrett, J E; Hung, B C; Levinthal, C; Nemeth, E F

    1998-06-01

    The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the concentration of extracellular Ca2+. In this study, two novel phenylalkylamine compounds, NPS 467 and NPS 568, were examined for effects on Xenopus laevis oocytes expressing the bovine or human parathyroid Ca2+ receptors. Increases in chloride current (ICl) were elicited in oocytes expressing the bovine Ca2+ receptor when the extracellular Ca2+ concentration was raised above 1.5 mM, whereas Ca2+ concentrations > 3 mM were generally necessary to elicit responses in oocytes expressing the human Ca2+ receptor. NPS 467 and NPS 568 potentiated the activation of ICl by extracellular Ca2+ in oocytes expressing either Ca2+ receptor homolog, and this resulted in a leftward shift of the Ca2+ concentration-response curve. Neither compound was active in the absence of extracellular Ca2+. Certain inorganic and organic cations known to activate the Ca2+ receptor were substituted for elevated levels of extracellular Ca2+ to increase ICl and the effects of these agonists were also potentiated by NPS 568 or NPS 467. The effects of NPS 568 were stereoselective and the R-enantiomer was about 10-fold more potent than the corresponding S-enantiomer. Neither NPS 467 nor 568 affected ICl in water-injected oocytes or in oocytes expressing the substance K receptor or the metabotropic glutamate receptor 1a. These results provide compelling evidence that NPS 467 and NPS 568 act directly upon the parathyroid Ca2+ receptor to increase its sensitivity to activation by extracellular Ca2+. This activity suggests that these compounds are positive allosteric modulators of the Ca2+ receptor. As such, these compounds define a new class of pharmacological agents with potent and selective actions on the Ca2+ receptor.

  1. 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.

  2. Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

    Science.gov (United States)

    Lu, F; Jiang, J; Li, N; Zhang, S; Sun, H; Luo, C; Wei, Y; Shi, D

    2011-09-15

    The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P fusion (P fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation.

  3. Health of children born through artificial oocyte activation: a pilot study.

    Science.gov (United States)

    Deemeh, Mohammad R; Tavalaee, Marziyeh; Nasr-Esfahani, Mohammad H

    2015-03-01

    Artificial oocyte activation (AOA) has shown to improve fertility in severe male infertility following intracytoplasmic sperm insemination (ICSI). However, the effect of AOA on the health status of children has not been studied. This pilot historical cohort study aims to evaluate physical and mental health of 79 and 89 children from 275 and 406 couples undergoing ICSI-AOA using ionomycin and conventional ICSI, respectively. The outcomes assessed were clinical pregnancy, abortion, type of delivery, and health of children (major birth defect, mental and behavior status). No significant differences were observed between the ICSI-AOA and the ICSI groups for these parameters, and the rate of major birth defects were not significantly different between the 2 groups. In this study, AOA has not imposed a greater risk on physical and mental health of children born through AOA, but for such a solid conclusion, further trails with higher number of cases are required and conclusions drawn are limited to this study.

  4. Reduced oocyte activation and first cleavage rate after ICSI with spermatozoa from a sterile mouse chromosome mutant

    NARCIS (Netherlands)

    Baart, E.B.; Heijden, van der G.W.; Hoeven, van der F.A.; Bakker, R.; Cooper, T.G.; Boer, de P.

    2004-01-01

    BACKGROUND: Male mice, heterozygous for two semi-identical reciprocal translocations T(1;13)70H and T(1;13)1Wa are usually sterile. We have investigated this oligoasthenoteratozoospermic mouse model using ICSI. METHODS: B6D2F1 oocytes were injected with epididymal or testicular sperm from fertile or

  5. Reduced oocyte activation and first cleavage rate after ICSI with spermatozoa from a sterile mouse chromosome mutant

    NARCIS (Netherlands)

    Baart, E.B.; Heijden, van der G.W.; Hoeven, van der F.A.; Bakker, R.; Cooper, T.G.; Boer, de P.

    2004-01-01

    BACKGROUND: Male mice, heterozygous for two semi-identical reciprocal translocations T(1;13)70H and T(1;13)1Wa are usually sterile. We have investigated this oligoasthenoteratozoospermic mouse model using ICSI. METHODS: B6D2F1 oocytes were injected with epididymal or testicular sperm from fertile or

  6. Specific deletion of AMP-activated protein kinase (α1AMPK in murine oocytes alters junctional protein expression and mitochondrial physiology.

    Directory of Open Access Journals (Sweden)

    Michael J Bertoldo

    Full Text Available Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK, an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues. Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.

  7. Active mechanics reveal molecular-scale force kinetics in living oocytes

    CERN Document Server

    Ahmed, Wylie W; Almonacid, Maria; Bussonnier, Matthias; Verlhac, Marie-Helene; Gov, Nir S; Visco, Paolo; van Wijland, Frederic; Betz, Timo

    2015-01-01

    Unlike traditional materials, living cells actively generate forces at the molecular scale that change their structure and mechanical properties. This nonequilibrium activity is essential for cellular function, and drives processes such as cell division. Single molecule studies have uncovered the detailed force kinetics of isolated motor proteins in-vitro, however their behavior in-vivo has been elusive due to the complex environment inside the cell. Here, we quantify active force generation in living oocytes using in-vivo optical trapping and laser interferometry of endogenous vesicles. We integrate an experimental and theoretical framework to connect mesoscopic measurements of nonequilibrium properties to the underlying molecular-scale force kinetics. Our results show that force generation by myosin-V drives the cytoplasmic-skeleton out-of-equilibrium (at frequencies below 300 Hz) and actively softens the environment. In vivo myosin-V activity generates a force of $F \\sim 0.4$ pN, with a power-stroke of len...

  8. T-type Ca2+ current activity during oocyte growth and maturation in the ascidian Styela plicata.

    Directory of Open Access Journals (Sweden)

    Alessandra Gallo

    Full Text Available Voltage-dependent calcium currents play a fundamental role during oocyte maturation, mostly L-type calcium currents, whereas T-type calcium currents are involved in sperm physiology and cell growth. In this paper, using an electrophysiological and pharmacological approach, we demonstrated, for the first time in oocytes, that T-type calcium currents are present with functional consequences on the plasma membrane of growing immature oocytes of the ascidian Styela plicata. We classified three subtypes of immature oocytes at the germinal vesicle stage on the basis of their size, morphology and accessory cellular structures. These stages were clearly associated with an increased activity of T-type calcium currents and hyperpolarization of the plasma membrane. We also observed that T-type calcium currents oscillate in the post-fertilization embryonic stages, with minimal amplitude of the currents in the zygote and maximal at 8-cell stage. In addition, chemical inhibition of T-type calcium currents, obtained by applying specific antagonists, induced a significant reduction in the rate of cleavage and absence of larval formation. We suggest that calcium entry via T-type calcium channels may act as a potential pacemaker in regulating cytosolic calcium involved in fertilization and early developmental events.

  9. Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

    Institute of Scientific and Technical Information of China (English)

    YANGZHONGAN; QUNHUALI; 等

    1999-01-01

    The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp.MPF activity was measured by using histone H1 as phosphorylation substrate.There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes,but there existed some subtle difference between them.The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp.It was suggested that the difference may be related to the spindle behavior changes,such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.

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

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    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.

  11. Abnormally activated one-carbon metabolic pathway is associated with mtDNA hypermethylation and mitochondrial malfunction in the oocytes of polycystic gilt ovaries.

    Science.gov (United States)

    Jia, Longfei; Li, Juan; He, Bin; Jia, Yimin; Niu, Yingjie; Wang, Chenfei; Zhao, Ruqian

    2016-01-13

    Polycystic ovarian syndrome (PCOS) is associated with hyperhomocysteinemia and polycystic ovaries (PCO) usually produce oocytes of poor quality. However, the intracellular mechanism linking hyperhomocysteinemia and oocyte quality remains elusive. In this study, the quality of the oocytes isolated from healthy and polycystic gilt ovaries was evaluated in vitro in association with one-carbon metabolism, mitochondrial DNA (mtDNA) methylation, and mitochondrial function. PCO oocytes demonstrated impaired polar body extrusion, and significantly decreased cleavage and blastocyst rates. The mitochondrial distribution was disrupted in PCO oocytes, together with decreased mitochondrial membrane potential and deformed mitochondrial structure. The mtDNA copy number and the expression of mtDNA-encoded genes were significantly lower in PCO oocytes. Homocysteine concentration in follicular fluid was significantly higher in PCO group, which was associated with significantly up-regulated one-carbon metabolic enzymes betaine homocysteine methyltransferase (BHMT), glycine N-methyltransferase (GNMT) and the DNA methyltransferase DNMT1. Moreover, mtDNA sequences coding for 12S, 16S rRNA and ND4, as well as the D-loop region were significantly hypermethylated in PCO oocytes. These results indicate that an abnormal activation of one-carbon metabolism and hypermethylation of mtDNA may contribute, largely, to the mitochondrial malfunction and decreased quality of PCO-derived oocytes in gilts.

  12. Functions of FZR1 and CDC20, activators of the anaphase-promoting complex, during meiotic maturation of swine oocytes.

    Science.gov (United States)

    Yamamuro, Tadashi; Kano, Kiyoshi; Naito, Kunihiko

    2008-12-01

    Cell division cycle 20 (CDC20) and fizzy/cell division cycle 20 related 1 (FZR1) are activators of the anaphase-promoting complex (APC), which ubiquitinates M-phase regulating proteins, such as cyclin B and securin, and induces their degradation. In the present study, porcine CDC20 and FZR1 were cloned by reverse transcriptase-polymerase chain reaction, and their functions in the meiotic maturation of porcine oocytes were analyzed. FZR1 was readily detected in porcine immature oocytes by immunoblotting, but its levels decreased substantially during maturation. In contrast, CDC20 levels rose during oocyte maturation and were highest by the second meiotic metaphase. The inhibition of CDC20 expression by the injection of CDC20 antisense RNA induced the meiotic arrest at the first meiotic metaphase (M1) and the accumulation of a large amount of cyclin B. On the other hand, the inhibition of FZR1 expression accelerated cyclin B accumulation and the start of germinal vesicle breakdown (GVBD), but did not affect the exit from M1. Conversely, the overexpression of FZR1 by the injection of FZR1 mRNA suppressed the cyclin B accumulation and retarded GVBD. Surprisingly, the injection of CDC20 mRNA into the immature oocytes could not increase CDC20 expression, but increased cyclin B accumulation and accelerated the meiotic progression. As CDC20 is a substrate of APC (FZR1), CDC20 might have competed with cyclin B and inhibited the FZR1 function. These results suggest that porcine FZR1 and CDC20 work on the maintenance of meiotic arrest at the first meiotic prophase and on the exit from M1, respectively, and that their functional phases are strictly distinguished during porcine oocyte maturation.

  13. Clinical benefit of metaphase I oocytes

    Directory of Open Access Journals (Sweden)

    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.

  14. 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.

  15. Jumping the gun: Smoking constituent BaP causes premature primordial follicle activation and impairs oocyte fusibility through oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Sobinoff, A.P.; Pye, V. [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW2308 (Australia); Nixon, B.; Roman, S.D. [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW2308 (Australia); ARC Centre of Excellence in Biotechnology and Development, University of Newcastle, Callaghan, NSW2308 (Australia); McLaughlin, E.A., E-mail: eileen.mclaughlin@newcastle.edu.au [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW2308 (Australia); ARC Centre of Excellence in Biotechnology and Development, University of Newcastle, Callaghan, NSW2308 (Australia)

    2012-04-01

    Benzo(a)pyrene (BaP) is an ovotoxic constituent of cigarette smoke associated with pre-mature ovarian failure and decreased rates of conception in IVF patients. Although the overall effect of BaP on female fertility has been documented, the exact molecular mechanisms behind its ovotoxicity remain elusive. In this study we examined the effects of BaP exposure on the ovarian transcriptome, and observed the effects of in vivo exposure on oocyte dysfunction. Microarray analysis of BaP cultured neonatal ovaries revealed a complex mechanism of ovotoxicity involving a small cohort of genes associated with follicular growth, cell cycle progression, and cell death. Histomorphological and immunohistochemical analysis supported these results, with BaP exposure causing increased primordial follicle activation and developing follicle atresia in vitro and in vivo. Functional analysis of oocytes obtained from adult Swiss mice treated neonatally revealed significantly increased levels of mitochondrial ROS/lipid peroxidation, and severely reduced sperm-egg binding and fusion in both low (1.5 mg/kg/daily) and high (3 mg/kg/daily) dose treatments. Our results reveal a complex mechanism of BaP induced ovotoxicity involving developing follicle atresia and accelerated primordial follicle activation, and suggest short term neonatal BaP exposure causes mitochondrial leakage resulting in reduced oolemma fluidity and impaired fertilisation in adulthood. This study highlights BaP as a key compound which may be partially responsible for the documented effects of cigarette smoke on follicular development and sub-fertility. -- Highlights: ► BaP exposure up-regulates canonical pathways linked with follicular growth/atresia. ► BaP causes primordial follicle activation and developing follicle atresia. ► BaP causes oocyte mitochondrial ROS and lipid peroxidation, impairing fertilisation. ► Short term neonatal BaP exposure compromises adult oocyte quality.

  16. Time-lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C.

    Science.gov (United States)

    Alexandre, H; Mulnard, J

    1988-12-01

    A passive erratic movement of the germinal vesicle (GV), already visible in small incompetent oocytes, is followed by an active scalloping of the nuclear membrane soon before GV breakdown (GVBD) in cultured competent oocytes. Maturation can be inhibited by activators of protein kinase A (PK-A) and protein kinase C (PK-C). Our time-lapse cinematography analysis allowed us to describe an unexpected behaviour of the GV when PK-C, but not PK-A, is activated: GV undergoes a displacement toward the cortex according to the same biological clock which triggers the programmed translocation of the spindle in control oocytes. It is concluded that, when oocytes become committed to undergo maturation, the cytoplasm acquires a PK-A-controlled "centrifugal displacement property" which is not restricted to the spindle.

  17. Clinical pregnancies and livebirths achieved by intracytoplasmic injection of round headed acrosomeless spermatozoa with and without oocyte activation in familial globozoospermia:case report

    Institute of Scientific and Technical Information of China (English)

    Enver K.Dirican; Ahmet Isik; Kubilay Vicdan; Eran Sozen; Zekiye Suludere

    2008-01-01

    We report the successful outcome of intracytoplasmic sperm injection (ICSI) treatment in two siblings with familial globozoospermia. After controlled ovarian hyperstimulation and oocyte pick-up, retrieved oocytes were mechanically activated before ICSI and a fertilization rate of 33.3% was achieved in the first case. The second couple underwent ICSI without oocyte activation and a 9.1% fertilization rate was obtained. The transfer of two grade Ⅰ embryos in the first couple and one grade Ⅰ embryo in the second couple resulted in clinical pregnancies with healthy livebirths. It was concluded that the main problem of cases with globozoospermia is a low fertilization rate, and even though ICSI and oocyte activation can increase this rate it is not necessarily needed to achieve a pregnancy.

  18. Function and interaction of maturation-promoting factor and mitogen-activated protein kinase during meiotic maturation and fertilization of oocyte

    Institute of Scientific and Technical Information of China (English)

    HUO Lijun; FAN Hengyu; CHEN Dayuan; SUN Qingyuan

    2004-01-01

    Mitogen-activated protein kinase (MAP kinase) cascade and maturation-promoting factor (MPF) play very important roles during meiotic maturation and fertilization of oocyte. Interaction between MAP kinase and MPF influences meiotic maturation and fertilization of oocyte throughout the animal kingdom, including stimulation of germinal vesicle breakdown (GVBD), suppression of DNA replication, control of meiotic chromosome segregation, maintenance of metaphase II arrest, and resumption and completion of second meiosis. This review focuses on the function and interaction of MAP kinase and MPF during meiotic maturation and fertilization of oocyte.

  19. Chemical activation in Rhinella arenarum oocytes: effect of dehydroleucodine (DhL) and its hydrogenated derivative (2H-DhL).

    Science.gov (United States)

    Medina, M F; Bühler, M I; Sánchez-Toranzo, G

    2015-12-01

    Mature oocytes are arrested in metaphase II due to the presence of high levels of active maturation promoting factor (MPF). After fertilization, active MPF levels decline abruptly, enabling oocytes to complete meiosis II. One of the first and universal events of oocyte activation is an increase in cytosolic Ca2+ that would be responsible for MPF inactivation. Mature oocytes can also be activated by parthenogenetic activation. The aims of this work are to test the ability of dehydroleucodine (DhL) and its hydrogenated derivative 11,13-dihydro-dehydroleucodine (2H-DhL) to induce chemical activation in amphibian oocytes and to study the participation of calcium in the process. Results indicated that DhL and 2H-DhL induced oocyte activation in a dose-dependent manner. After 90 min of treatment, DhL 36 μM was able to induce 95% activation, while 2H-DhL 36 μM was less active, with only 40% activation. Our results suggest that DhL induced the inhibition of MPF activity, probably by an increase in intracellular Ca2+ concentration. Extracellular Ca2+ would not be significant, although Ca2+ release from intracellular stores is critical. In this sense, IP3Rs and RyRs were involved in the Ca2+ transient induced by lactones. In this species, RyRs appears to be the largest contributor to Ca2+ release in DhL-induced activation. Although more studies are needed on the mechanism of action through which these lactones induce oocyte activation in Rhinella arenarum, the results of this research provide interesting perspectives for the use of these lactones as chemical activators in in vitro fertilization and cloning.

  20. Artificial oocyte activation to improve reproductive outcomes in women with previous fertilization failure: a systematic review and meta-analysis of RCTs.

    Science.gov (United States)

    Sfontouris, Ioannis A; Nastri, Carolina O; Lima, Maria L S; Tahmasbpourmarzouni, Eisa; Raine-Fenning, Nick; Martins, Wellington P

    2015-08-01

    In couples with previous fertilization failure, are reproductive outcomes improved using ICSI followed by artificial oocyte activation (ICSI-AOA) compared with conventional ICSI? There is insufficient evidence available from RCTs to judge the efficacy and safety of ICSI-AOA for couples with previous fertilization failure. In cases with previous low fertilization rates or total fertilization failure using ICSI due to sperm-related, oocyte activation deficiency, several methods of AOA have been described, which employ mechanical, electrical or chemical stimuli. Reported fertilization and pregnancy rates appear to be improved after ICSI-AOA compared with conventional ICSI; however, the small studies performed to date make it difficult to assess the clinical efficacy or safety of AOA. The present systematic review and meta-analysis identified RCTs that compared ICSI-AOA and conventional ICSI. The last electronic search was conducted in August 2014 and there was no limitation regarding language, publication date, or publication status. We included studies that randomized either oocytes or women and included them in two different parts of this review: a women-based review and an oocyte-based review. For the women-based review, the primary outcome of effectiveness was live birth per randomized woman and the primary outcome for safety was congenital anomalies per clinical pregnancy. For the oocyte-based review, the primary outcome was embryo formation per oocyte randomized. Record screening and data extraction were performed independently by two authors and risk of bias was assessed by three authors. The effects of ICSI-AOA compared with conventional ICSI were summarized as risk ratio (RR) and the precision of the estimates was evaluated by the 95% confidence interval (CI). A total of 14 articles were assessed for eligibility and 9 included in the meta-analysis: 2 studies comprised the woman-based review (n = 168 women) and 7 studies the oocyte-based review (n = 4234

  1. Carrying-over effects of GVBD blocking on post-blocking meiotic progression of oocytes: species difference and the signaling pathway leading to MPF activation.

    Directory of Open Access Journals (Sweden)

    Guang-Zhong Jiao

    Full Text Available Efforts to improve the quality of in vitro matured oocytes by blocking germinal vesicle breakdown (GVBD and allowing more time for ooplasmic maturation have achieved little due to a lack of knowledge on the molecular events during GVBD blocking. Such knowledge is also important for studies aimed at regulating gene expression in maturing oocytes prior to GVBD. We studied species difference and signaling pathways leading to the carrying-over effect of GVBD blocking on post-blocking meiotic progression (PBMP. Overall, GVBD-blocking with roscovitine decelerated PBMP of mouse oocytes but accelerated that of pig oocytes. During blocking culture, whereas cyclin B of pig oocytes increased continuously, that of mouse oocytes declined first and then increased slowly. In both species, (a whereas active CDC2A showed a dynamics similar to cyclin B, inactive CDC2A decreased continuously; (b when oocytes were blocked in blocking medium containing cycloheximide, PBMP was decelerated significantly while cyclin B and active CDC2A decreasing to the lowest level; (c whereas sodium vanadate in blocking medium reduced PBMP, epidermal growth factor (EGF in blocking medium accelerated PBMP significantly with no effect on cyclin B levels. In conclusion, the EGF signaling cascade accelerated PBMP by promoting the pre-MPF (M-phase-promoting factor to MPF conversion during GVBD blocking with roscovitine. The significant difference in PBMP observed between mouse and pig oocytes was caused by species difference in cyclin B dynamics during blocking culture as no species difference was observed in either pre-MPF to MPF conversion or the EGF signaling activity.

  2. Carrying-Over Effects of GVBD Blocking on Post-Blocking Meiotic Progression of Oocytes: Species Difference and the Signaling Pathway Leading to MPF Activation

    Science.gov (United States)

    Jiao, Guang-Zhong; Lian, Hua-Yu; Gao, Yan; Sun, Ming-Ju; Gong, Shuai; Zheng, Liang-Liang; Zhang, Chuan-Xin; Tan, Jing-He

    2014-01-01

    Efforts to improve the quality of in vitro matured oocytes by blocking germinal vesicle breakdown (GVBD) and allowing more time for ooplasmic maturation have achieved little due to a lack of knowledge on the molecular events during GVBD blocking. Such knowledge is also important for studies aimed at regulating gene expression in maturing oocytes prior to GVBD. We studied species difference and signaling pathways leading to the carrying-over effect of GVBD blocking on post-blocking meiotic progression (PBMP). Overall, GVBD-blocking with roscovitine decelerated PBMP of mouse oocytes but accelerated that of pig oocytes. During blocking culture, whereas cyclin B of pig oocytes increased continuously, that of mouse oocytes declined first and then increased slowly. In both species, (a) whereas active CDC2A showed a dynamics similar to cyclin B, inactive CDC2A decreased continuously; (b) when oocytes were blocked in blocking medium containing cycloheximide, PBMP was decelerated significantly while cyclin B and active CDC2A decreasing to the lowest level; (c) whereas sodium vanadate in blocking medium reduced PBMP, epidermal growth factor (EGF) in blocking medium accelerated PBMP significantly with no effect on cyclin B levels. In conclusion, the EGF signaling cascade accelerated PBMP by promoting the pre-MPF (M-phase-promoting factor) to MPF conversion during GVBD blocking with roscovitine. The significant difference in PBMP observed between mouse and pig oocytes was caused by species difference in cyclin B dynamics during blocking culture as no species difference was observed in either pre-MPF to MPF conversion or the EGF signaling activity. PMID:25078078

  3. Co-culture with pig membrana granulosa cells modulates the activity of cdc2 and MAP kinase in maturing cattle oocytes.

    Science.gov (United States)

    Motlík, J; Sutovský, P; Kalous, J; Kubelka, M; Moos, J; Schultz, R M

    1996-08-01

    Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8 h of initial culture) or metaphase I (12 h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8 h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6 h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions

  4. 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.

  5. 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).

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

    Science.gov (United States)

    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.

  7. Expression of Heat Shock Protein (HSP A1A and MnSOD Genes Following Vitrification of Mouse MII Oocytes with Cryotop Method

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    Zahra Khodabandeh Jahromi

    2010-01-01

    Full Text Available Objective: The aim of this study was to investigate the effects of two vitrification protocolson mouse metaphase stage II (MII oocytes and evaluate their effects on the expressionof heat shock protein A1A (HSP A1A and MnSOD genes.Materials and Methods: Groups of approximately 15 oocytes without cumulus complexeswere collected and vitrified with 10% (v/v ethylene glycol (EG + 10% (v/v dimethylsulphoxide(DMSO + 0.5M sucrose in group A (VSI and 14.5% (v/v EG + 14.5% PROH+ 0.5M sucrose in group B (VSII, respectively. Thawing after vitrification was performdby placing the oocytes into 1M sucrose for 1 minute and two diluted solutions, each for3 minutes. After thawing, the oocytes were fertilized and cultured in vitro to develop intothe pronuclear stage. The survival rate of vitrified-warmed oocytes and rate of fertilizationwere evaluated. In addition, gene expressions (HSP A1A, MnSOD and β actin of vitrifiedwarmedoocytes were also examined by reverse transcription polymerase chain reaction(RT-PCR.Results: Survival rates of each group were separately compared to the control. The resultshowed significant differences between each experimental group compared to the control(p≤ 0.001. The survival rate of oocytes after warming was higher in VSI (91.2% ± 1.7compared to VSII (89.2% ± 1.5 but there were no significant differences between the twogroups. The rate of fertilization was significantly (p≤0.05 reduced in vitrified-warmed (VSI:39% ± 5.8; VSII: 34% ± 5.7 oocytes compared to the control (88.36% ± 2.3. The expressionof MnSOD increased in the vitrified-warmed oocytes when compared to controloocytes. We also detected HSP A1A only in the control and VSI group.Conclusion: Vitrification of oocytes by cryotop resulted in high survival rate; low developmentalcompetence and fertilization rate of vitrified-warmed oocytes which may be a resultof changing expression of important genes after thawing.

  8. Correlation between oocyte number and follicular fluid concentration of pituitary adenylate cyclase-activating polypeptide (PACAP) in women after superovulation treatment.

    Science.gov (United States)

    Koppan, M; Varnagy, A; Reglodi, D; Brubel, R; Nemeth, J; Tamas, A; Mark, L; Bodis, J

    2012-11-01

    Follicular growth, ovulation, and luteinization are influenced by interactions of peptide and steroid hormone-signaling cascades in the ovary. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the regulation of several endocrine processes and is present in ovarian follicular fluid (FF). However, little is known about PACAP in FF with regard to maturation, ovulation, fertilization, and successful pregnancy. The aim of this pilot study was to investigate whether there is a correlation between PACAP concentration in FF and ovarian response to superovulation treatment in infertile women, performed in volunteers (n = 132; aged between 20 and 35). After treatment, the number of harvested oocytes was recorded and PACAP immunoreactivity in FF was measured by radioimmunoassay. All the corresponding PACAP concentrations were below 290 fmol/ml in cases when the number of harvested oocytes exceeded 14 per patient, while in all cases above 290 fmol/ml, the number of oocytes was below 14. Using these cutoff values, we determined three study groups: high-PACAP concentration, high-oocyte number, and low-PACAP concentration-low-oocyte number groups. Median values of PACAP concentration in these groups were 411.2, 106.5, and 101.0 fmol/ml, respectively, while the median values of harvested oocytes were 5.5, 19.0, and 5.0, respectively. Differences were significant, indicating a correlation between concentration of PACAP in FF and the number of recruited oocytes. Higher concentrations of PACAP in FF might be associated with lower number of developing oocytes, while low concentrations of PACAP might correlate with a markedly higher number of ova retrieved, thus predicting a higher chance for ovarian hyperstimulation. Our present study is among the first few human clinical studies with direct conclusions drawn for possible clinical impact of PACAP.

  9. 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.

  10. Injection of insect membrane in Xenopus oocyte: An original method for the pharmacological characterization of neonicotinoid insecticides.

    Science.gov (United States)

    Crespin, Lucille; Legros, Christian; List, Olivier; Tricoire-Leignel, Hélène; Mattei, César

    2016-01-01

    Insect nicotinic acetylcholine receptors (nAChRs) represent a major target of insecticides, belonging to the neonicotinoid family. However, the pharmacological profile of native nAChRs is poorly documented, mainly because of a lack of knowledge of their subunit stoichiometry, their tissue distribution and the weak access to nAChR-expressing cells. In addition, the expression of insect nAChRs in heterologous systems remains hard to achieve. Therefore, the structure-activity characterization of nAChR-targeting insecticides is made difficult. The objective of the present study was to characterize insect nAChRs by an electrophysiological approach in a heterologous system naturally devoid of these receptors to allow a molecular/cellular investigation of the mode of action of neonicotinoids. Methods To overcome impediments linked to the expression of insect nAChR mRNA or cDNA, we chose to inject insect membranes from the pea aphid (Acyrthosiphon pisum) into Xenopus oocytes. This microtransplantation technique was designed to gain access to native nAChRs embedded in their membrane, through direct stimulation with nicotinic agonists. Results We provide evidence that an enriched-nAChR membrane allows us to characterize native receptors. The presence of such receptors was confirmed with fluorescent α-BgTX labeling. Electrophysiological recordings of nicotine-induced inward currents allowed us to challenge the presence of functional nAChR. We compared the effect of nicotine (NIC) with clothianidin (CLO) and we assessed the effect of thiamethoxam (TMX). Discussion This technique has been recently highlighted with mammalian and human material as a powerful functional approach, but has, to our knowledge, never been used with insect membrane. In addition, the use of the insect membrane microtransplantation opens a new and original way for pharmacological screening of neurotoxic insecticides, including neonicotinoids. Moreover, it might also be a powerful tool to investigate the

  11. Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.

    Directory of Open Access Journals (Sweden)

    Xiaojing Yang

    Full Text Available Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1 in human embryonic kidney 293 (HEK293 cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.

  12. Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.

    Science.gov (United States)

    Yang, Xiaojing; Zhao, Yuewen; Yang, Xiaolong; Kan, Frederick W K

    2015-01-01

    Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.

  13. 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.

  14. A simple and high-throughput method to assess maturation status of bovine oocytes: comparison of anti-lamin A/C-DAPI with an aceto-orcein staining technique.

    Science.gov (United States)

    Prentice-Biensch, J R; Singh, J; Alfoteisy, B; Anzar, M

    2012-10-15

    A precise, accurate, nonambiguous and high-throughput method is required to assess nuclear maturation of mammalian oocytes. The objectives of this study were to compare the efficiency and ease of use of a simplified fluorescence imaging (anti-lamin A/C and 4',6-diamidino-2-phenylindole [DAPI]) technique to the existing technique (aceto-orcein staining) for the evaluation of nuclear maturation of bovine oocytes, and to determine the kinetics of bovine oocyte maturation using an anti-lamin A/C-DAPI technique. In Experiment 1, oocytes were matured in vitro and stained with aceto-orcein and anti-lamin A/C-DAPI staining techniques. The proportions of oocytes lost during procedures and those that could not be classified (because of ambiguous morphology) during evaluation were lower (P 24 h for the aceto-orcein method). Furthermore, > 200 oocytes could be stained in one batch with anti-lamin A/C-DAPI technique. In Experiment 2, nuclear maturation kinetics of bovine oocytes at various time intervals (0, 6, 12, and 22 h) during in vitro maturation (IVM) was evaluated using the anti-lamin A/C-DAPI technique. Germinal vesicle, germinal vesicle breakdown, metaphase I, and metaphase II oocytes were predominant at 0, 6, 12, and 22 h of IVM, respectively. We concluded that the anti-lamin A/C-DAPI was an efficient and simple technique for nonambiguous evaluation of nuclear maturation status of large numbers of oocytes in a short interval.

  15. Effects of Ages of Donors and Conditions of Preserving Ovaries on Porcine Oocytes Maturation in vitro and Efficiency of Parthenogenetic Activation

    Institute of Scientific and Technical Information of China (English)

    XING Feng-ying; WU Zhong-hong; ZENG Shen-ming; LIU Guo-shi; ZHU Shi-en; ZHANG Zhong-cheng; CHEN Xue-jin

    2004-01-01

    Effects of different ages of donors and different conditions of preserving ovaries on porcine oocytes maturation in vitro and efficiency of parthenogenetic activation were studied. The experiments included: 1) effects of different temperatures (22, 30, 37, 38.5and 40℃) of preserving ovaries on porcine oocytes maturation in vitro and developmental potential; 2) effects of periods of preserving ovaries on porcine oocytes maturation in vitro and development in vitro; 3) effects of different ages of donors on porcine oocytes maturation in vitro and developmental potential. The results of the experiment showed:1) There were no statistical differences (p>0.05) of the parthenogenetic cleavage rate (79.64% vs 76.18%) and blastocyst rate (18.11% vs 33.82%) between oocytes from ovaries preserved at 38.5℃ and those preserved at 37℃. When the preserving temperature was increased to 40℃, the cleavage rate (21.68%) and the blastocyst rate (0) were great significantly lower than those at 37℃(p<0.01). The cleavage rate (80.79% vs 76.18%) and blastocyst rate (29.61% vs 33.82%) were not different between 30 and 37℃(p> 0.05). When the preserving temperature was decreased to 22℃, the rate of cleavage was not different,but the rate of blastocyst was significantly lower, compared with that at 37℃; 2) The cleavage and blastocyst rates of the porcine oocytes collected after slaughter 2 or 6h were not different (p>0.05); 3) The cleavage rate of oocytes from gilts and sows after maturation was not different, but the blastocyst rate of the sow group was significantly higher than that of gilt group (p< 0.05). The blastocyst cell number of sows and gilt showed no difference (p>0.05).

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

    Science.gov (United States)

    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.

  17. Maternal diet-induced obesity alters mitochondrial activity and redox status in mouse oocytes and zygotes.

    Directory of Open Access Journals (Sweden)

    Natalia Igosheva

    Full Text Available The negative impact of obesity on reproductive success is well documented but the stages at which development of the conceptus is compromised and the mechanisms responsible for the developmental failure still remain unclear. Recent findings suggest that mitochondria may be a contributing factor. However to date no studies have directly addressed the consequences of maternal obesity on mitochondria in early embryogenesis.Using an established murine model of maternal diet induced obesity and a live cell dynamic fluorescence imaging techniques coupled with molecular biology we have investigated the underlying mechanisms of obesity-induced reduced fertility. Our study is the first to show that maternal obesity prior to conception is associated with altered mitochondria in mouse oocytes and zygotes. Specifically, maternal diet-induced obesity in mice led to an increase in mitochondrial potential, mitochondrial DNA content and biogenesis. Generation of reactive oxygen species (ROS was raised while glutathione was depleted and the redox state became more oxidised, suggestive of oxidative stress. These altered mitochondrial properties were associated with significant developmental impairment as shown by the increased number of obese mothers who failed to support blastocyst formation compared to lean dams. We propose that compromised oocyte and early embryo mitochondrial metabolism, resulting from excessive nutrient exposure prior to and during conception, may underlie poor reproductive outcomes frequently reported in obese women.

  18. 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...

  19. Transducin-like enhancer of split-6 (TLE6) is a substrate of protein kinase A activity during mouse oocyte maturation.

    Science.gov (United States)

    Duncan, Francesca E; Padilla-Banks, Elizabeth; Bernhardt, Miranda L; Ord, Teri S; Jefferson, Wendy N; Moss, Stuart B; Williams, Carmen J

    2014-03-01

    Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)-a maternal effect gene that is part of the subcortical maternal complex-as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation

  20. Comparison of pre- and postimplantation development following the application of three artificial activating stimuli in a mouse model with round-headed sperm cells deficient for oocyte activation

    DEFF Research Database (Denmark)

    Vanden Meerschaut, Frauke; Nikiforaki, D.; De Roo, C.

    2013-01-01

    with fertile controls to assess their fertility. MAIN RESULTS AND THE ROLE OF CHANCE The percentage of oocytes showing calcium rises as well as the number of calcium rises per oscillating oocyte were significantly lower in the wobbler group when compared with the WT group (9.3 versus 96% and 2.1 calcium rises...... was significantly lower at weeks 2, 3 and 4 when compared with female pups originating from WT embryos. However, the latter difference was not observed at later time points, nor in the other artificial activating groups. All offspring mated successfully with fertile controls. LIMITATIONS, REASONS FOR CAUTION...... No gross differences were found between strontium chloride, electrical pulses or ionomycin with respect to the pre- and post-implantation development in the wobbler mouse. WHAT IS KNOWN ALREADY Fertilization failure following intra-cytoplasmic sperm injection (ICSI) occurs in 1–3% of the ICSI cycles...

  1. Supplementation with spermine during in vitro maturation of porcine oocytes improves early embryonic development after parthenogenetic activation and somatic cell nuclear transfer.

    Science.gov (United States)

    Jin, J X; Lee, S; Khoirinaya, C; Oh, A; Kim, G A; Lee, B C

    2016-03-01

    Spermine plays an important role in protection from reactive oxygen species (ROS) in bacteria, yeast, and mammalian cells, but there are few studies on the effects of spermine on porcine oocyte maturation and subsequent embryo development. The aim of this study was to determine the effects of spermine on in vitro maturation (IVM) of porcine oocytes and their developmental competence after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). We evaluated nuclear maturation, intracellular glutathione (GSH), and ROS levels in oocytes, and their subsequent embryonic development, as well as gene expression in mature oocytes, cumulus cells, and PA blastocysts. After treatment with various concentrations of spermine in IVM culture medium, there was no significant difference in nuclear maturation rate. However, spermine treatment groups (10- 500 µM) showed significantly increased intracellular GSH levels and decreased ROS levels compared to the control ( cells ( < 0.05). was increased in spermine-treated oocytes. Levels of transcription for and were significantly increased in PA blastocysts. In conclusion, 10 µM spermine supplementation during IVM improved the development of porcine PA and SCNT embryos by increasing intracellular GSH, scavenging ROS levels, and regulating gene expression.

  2. Effect of oocyte maturation time, sperm selection method and oxygen tension on in vitro embryo development in alpacas.

    Science.gov (United States)

    Ruiz, Jaime; Paulo Santayana, R; José Mendoza, M; Leandra Landeo, J; Huamán, Elizabeth; Ticllacuri, Flamel; Fidel Mujica, L; Silva, Mauricio; Ratto, Marcelo H

    2017-06-01

    We evaluated the effect of in vitro maturation time, sperm selection and oxygen tension on alpaca embryo development. In Experiment I, Cumulus Oocyte- Complexes (COCs) were obtained from abattoir ovaries and in vitro matured in TCM-199 for 24 (n = 217), 28 (215), or 32 h (223) at 38.5 °C, high humidity and 5% CO2 in air. Oocytes from 24 (n = 392), 28 (n = 456) or 32 (n = 368) h groups were in vitro fertilized with epididymal sperm and cultured in SOFaa at 38.5 °C, high humidity and 5% CO2, 5% O2 and 90% N2 for 7 days. Embryo development was evaluated on Day 2, 5 and Day 7 of in vitro culture (Day 0 = in vitro fertilization). In Experiment II, a 2 by 2-factorial design was used to determine the effect of sperm selection (Swim-up vs Percoll) and oxygen tension (20% vs 5%) during embryo culture and their interaction on embryo development. COCs were in vitro matured for 32 h at 38.5 °C and 5% CO2 in air and then in vitro inseminated with epididymal sperm processed by swim-up or Percoll. Zygotes were cultured in SOFaa + cumulus cells at 38.5 °C under 20 or 5% of O2 tension and high humidity for 7 days. A total of 235, 235, 253 and 240 oocytes were assigned to: swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, groups respectively. The proportion of oocytes reaching MII stage was highest after 32 h of in vitro maturation (P < 0.05). Blastocyst rate (29.1 ± 2.7%) was also highest for COCs matured for 32 h (Exp I). In Experiment II, Blastocysts rate (26.03 ± 4.7; 27.7 ± 4.3; 29.7 ± 3.8 and 27.6 ± 4.2% for swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, respectively) was not affected by sperm selection method (P = 0.8), oxygen tension (P = 0.9) or their interaction (P = 0.5). Copyright © 2017 Elsevier Inc. All rights reserved.

  3. The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep.

    Science.gov (United States)

    Dadashpour Davachi, Navid; Kohram, Hamid; Zare Shahneh, Ahmad; Zhandi, Mahdi; Goudarzi, Abbas; Fallahi, Roozbeh; Masoudi, Reza; Yousefi, Ali Reza; Bartlewski, Pawel M

    2017-01-15

    The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in vitro embryo production. In the present study, we examined the influence of conspecific ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout the IVM phase on the developmental competence and maturation-promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this study, namely four groups with and two groups without oviductal epithelial cells added to IVM media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from adult (AAE; G1) or prepubertal donors (prepubertal sheep ampulla oviductal epithelial cells [PAE]; G4), COCs obtained from prepubertal animals cocultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured without oviductal epithelial cells. Coincubation of oocytes retrieved from both adult and sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of metaphase-II (M-II) attainment (G1: 85.1 ± 2.0 and G2: 40.2 ± 1.3) and blastocyst formation (G1: 42.2 ± 1.1 and G2: 21.2 ± 1.0) as well as blastocyst development (total cell count; G1: 130.3 ± 7.8, G2: 70.2 ± 3.5) compared with their respective controls (C1: 94.3 ± 4.1 and C2: 49.7 ± 2.0). Prior to IVM, the activity of MPF was greater (P vitro embryo production efficiency. A significant increase in MPF activity following IVM of G2 oocytes could be responsible, at least partly, for the improved rate of blastocyst formation after IVF of prepubertal sheep oocytes. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  4. Development of Bovine Embryos from Two Different Activated-time Oocytes for Parthenogenesis and Two Kinds of Nucleus Donors for Cloning

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    @@ The aim of Experiment 1 was to compare the effect of two different activating time on parthenogenetic embryonic development. Oocytes which were aspirated, collected ,rinsed and then maturated in maturating microdrops (Sirard et al, Bio Reprod, 1988) for 22-23h.

  5. Jumping the gun: smoking constituent BaP causes premature primordial follicle activation and impairs oocyte fusibility through oxidative stress.

    Science.gov (United States)

    Sobinoff, A P; Pye, V; Nixon, B; Roman, S D; McLaughlin, E A

    2012-04-01

    Benzo(a)pyrene (BaP) is an ovotoxic constituent of cigarette smoke associated with pre-mature ovarian failure and decreased rates of conception in IVF patients. Although the overall effect of BaP on female fertility has been documented, the exact molecular mechanisms behind its ovotoxicity remain elusive. In this study we examined the effects of BaP exposure on the ovarian transcriptome, and observed the effects of in vivo exposure on oocyte dysfunction. Microarray analysis of BaP cultured neonatal ovaries revealed a complex mechanism of ovotoxicity involving a small cohort of genes associated with follicular growth, cell cycle progression, and cell death. Histomorphological and immunohistochemical analysis supported these results, with BaP exposure causing increased primordial follicle activation and developing follicle atresia in vitro and in vivo. Functional analysis of oocytes obtained from adult Swiss mice treated neonatally revealed significantly increased levels of mitochondrial ROS/lipid peroxidation, and severely reduced sperm-egg binding and fusion in both low (1.5mg/kg/daily) and high (3mg/kg/daily) dose treatments. Our results reveal a complex mechanism of BaP induced ovotoxicity involving developing follicle atresia and accelerated primordial follicle activation, and suggest short term neonatal BaP exposure causes mitochondrial leakage resulting in reduced oolemma fluidity and impaired fertilisation in adulthood. This study highlights BaP as a key compound which may be partially responsible for the documented effects of cigarette smoke on follicular development and sub-fertility.

  6. Relationship between Potential Sperm Factors Involved in Oocyte Activation and Sperm DNA Fragmentation with Intra-Cytoplasmic Sperm Injection Clinical Outcomes.

    Science.gov (United States)

    Tavalaee, Marziyeh; Kiani-Esfahani, Abbas; Nasr-Esfahani, Mohammad Hossein

    2017-01-01

    The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein (PAWP), phospholipase Cζ (PLCζ), and truncated form of the kit receptor (TR-KIT)] as candidates of oocyte activation with fertilization rate and early embryonic development. In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection (ICSI) candidates and analyzed according to World Health Organization criteria (2010). Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation (DGC) and the second part was prepared for assessment of sperm morphology (Papanicolaou staining), DNA fragmentation [transferase dUTP nick end labeling (TUNEL)], and three Sperm-borne oocyte-activating factor (s) (SOAFs)-PLCζ, PAWP, and TR-KIT. Significant positive correlations existed between the percentages of PLCζ, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCζ and PAWP. We did not find a relationship between percentages of PLCζ, PAWP, and TR-KIT with embryo quality and pregnancy rate (P>0.05). There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality. Oocyte activation was associated with the studied sperm factors (PAWP, PLCζ, and TR-KIT). These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCζ, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action should take place, and its cost and benefits should

  7. 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.

  8. Mutations of the S4-S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes.

    Science.gov (United States)

    Sanguinetti, M C; Xu, Q P

    1999-02-01

    1. The structural basis for the activation gate of voltage-dependent K+ channels is not known, but indirect evidence has implicated the S4-S5 linker, the cytoplasmic region between the fourth and fifth transmembrane domains of the channel subunit. We have studied the effects of mutations in the S4-S5 linker of HERG (human ether-á-go-go-related gene), a human delayed rectifier K+ channel, in Xenopus oocytes. 2. Mutation of acidic residues (D540, E544) in the S4-S5 linker of HERG channels to neutral (Ala) or basic (Lys) residues accelerated the rate of channel deactivation. Most mutations greatly accelerated the rate of activation. However, E544K HERG channels activated more slowly than wild-type HERG channels. 3. Mutation of residues in the S4-S5 linker had little or no effect on fast inactivation, consistent with independence of HERG channel activation and inactivation 4. In response to large hyperpolarizations, D540K HERG channels can reopen into a state that is distinct from the normal depolarization-induced open state. It is proposed that substitution of a negatively charged Asp with the positively charged Lys disrupts a subunit interaction that normally stabilizes the channel in a closed state at negative transmembrane potentials. 5. The results indicate that the S4-S5 linker is a crucial component of the activation gate of HERG channels.

  9. Study on the mitochondrial activity and membrane potential after exposing later stage oocytes of two gorgonian corals (Junceella juncea and Junceella fragilis) to cryoprotectants.

    Science.gov (United States)

    Tsai, S; Spikings, E; Huang, I-C; Lin, C

    2011-01-01

    Coral reefs provide a valuable habitat for many economically valuable fish and invertebrates. However, they are in serious jeopardy, threatened by increasing over-exploitation, pollution, habitat destruction, disease and global climate change. Here, we examined the effect of cryoprotectant exposure on mitochondrial activity and membrane potential in coral oocytes in order to find suitable cryoprotectants towards their successful cryopreservation. According to the No Observed Effect Concentrations (NOECs), methanol was found to be the least toxic cryoprotectant whilst DMSO was the most toxic cryoprotectant. The results also demonstrated that there were no significant differences (p > 0.05) in ATP concentrations between Junceella juncea and Junceella fragilis after exposure to all concentrations of all cryoprotectants for 30 min. Using confocal microscopy, JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-imidacarbocyanine iodide) staining indicated that the mitochondrial membrane potential of Junceella fragilis oocytes reduced after 1 M and 2 M methanol treatment and a loss of the mitochondrial distribution pattern and poor green fluorescence after 3M methanol treatment. Therefore, even oocytes that show no adverse effect of cryoprotectants on survival might suffer some more subtle impacts. The results obtained from this study will provide a basis for development of protocols to cryopreserve the oocytes of gorgonian corals.

  10. A Simplified Method for Three-Dimensional (3-D Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice.

    Directory of Open Access Journals (Sweden)

    Carolyn M Higuchi

    Full Text Available In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART. Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture. We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-, A (Membrane/activin+, M (Matrigel/activin-, and M+A (Matrigel/activin+. We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A. Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM and in vitro fertilization (IVF. Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A than with those grown in membrane culture (C, A. In particular, activin A treatment further improved 3-D culture (M+A success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian

  11. 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.

  12. 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.

  13. Data on the concentrations of etoposide, PSC833, BAPTA-AM, and cycloheximide that do not compromise the vitality of mature mouse oocytes, parthenogencially activated and fertilized embryos.

    Science.gov (United States)

    Martin, Jacinta H; Bromfield, Elizabeth G; Aitken, R John; Lord, Tessa; Nixon, Brett

    2016-09-01

    These data document the vitality of mature mouse oocytes (Metaphase II (MII)) and early stage embryos (zygotes) following exposure to the genotoxic chemotherapeutic agent, etoposide, in combination with PSC833, a selective inhibitor of permeability glycoprotein. They also illustrate the vitality of parthenogencially activated and fertilized embryos following incubation with the calcium chelator BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester)), cycloheximide (an antibiotic that is capable of inhibiting protein synthesis), and hydrogen peroxide (a potent reactive oxygen species). Finally, they present evidence that permeability glycoprotein is not represented in the proteome of mouse spermatozoa. Our interpretation and discussion of these data feature in the article "Identification of a key role for permeability glycoprotein in enhancing the cellular defense mechanisms of fertilized oocytes" (Martin et al., in press) [1].

  14. 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

  15. 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.

  16. New methods for cooling and storing oocytes and embryos in a clean environment of -196°C.

    Science.gov (United States)

    Arav, Amir; Natan, Yehudit; Levi-Setti, Paolo Emanuele; Menduni, Francesca; Patrizio, Pasquale

    2016-07-01

    It is well documented that oocyte vitrification using open systems provides better results than closed systems. However, its use is limited owing to risks of contamination posed by direct exposure to liquid nitrogen and cross-contamination when stored in liquid nitrogen tanks. A device that produces clean liquid air (CLAir) having similar a temperature as liquid nitrogen and a sterile storage canister device (Esther) that keeps samples sealed in their own compartment while in regular liquid nitrogen tanks were developed. The following experiments were performed: temperature measurements, bioburden tests, vitrification and storage experiments with mice embryos and human oocytes. Results showed similar cooling rates for liquid nitrogen and liquid air. Bioburden tests of CLAir and Esther showed no contamination, while massive contamination was found in "commercial" liquid nitrogen and storage canisters. Mice blastocysts had a survival rate of over 90%, with 80% hatching rate after vitirification in CLAir and 1 week storage in Esther, similar to the fresh (control) results. Human oocytes vitrified in CLAir and in liquid nitrogen for three consecutive vitrification/warming cycles showed 100% survival, seen as re-expansion in both groups. These new systems represent a breakthrough for safe vitrification using open systems and a safe storage process generally.

  17. Apoptosis in mammalian oocytes: a review.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos.

    Science.gov (United States)

    Yao, Li-Juan; Zhong, Zhi-Sheng; Zhang, Li-Sheng; Chen, Da-Yuan; Schatten, Heide; Sun, Qing-Yuan

    2004-05-01

    Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.

  20. Glutathione transferase activity and oocyte development in copepods exposed to toxic phytoplankton

    DEFF Research Database (Denmark)

    Kozlowsky-Suzuki, Betina; Koski, Marja; Hallberg, Eric

    2009-01-01

    Organisms present a series of cellular mechanisms to avoid the effects of toxic compounds. Such mechanisms include the increase in activity of detoxification enzymes [e.g., 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST)I, which could explain the low retention of ingeste...

  1. Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture.

    Science.gov (United States)

    Ganguly, Sujoy; Williams, Lucy S; Palacios, Isabel M; Goldstein, Raymond E

    2012-09-18

    Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA.

  2. 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...

  3. 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.

  4. High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes.

    Science.gov (United States)

    Shishova, Kseniya V; Khodarovich, Yuriy M; Lavrentyeva, Elena A; Zatsepina, Olga V

    2015-10-01

    Nucleolus-like bodies (NLBs) of fully-grown (germinal vesicle, GV) mammalian oocytes are traditionally considered as morphologically distinct entities, which, unlike normal nucleoli, contain transcribed ribosomal genes (rDNA) solely at their surface. In the current study, we for the first time showed that active ribosomal genes are present not only on the surface but also inside NLBs of the NSN-type oocytes. The "internal" rRNA synthesis was evidenced by cytoplasmic microinjections of BrUTP as precursor and by fluorescence in situ hybridization with a probe to the short-lived 5'ETS segment of the 47S pre-rRNA. We further showed that in the NLB mass of NSN-oocytes, distribution of active rDNA, RNA polymerase I (UBF) and rRNA processing (fibrillarin) protein factors, U3 snoRNA, pre-rRNAs and 18S/28S rRNAs is remarkably similar to that in somatic nucleoli capable to make pre-ribosomes. Overall, these observations support the occurrence of rDNA transcription, rRNA processing and pre-ribosome assembly in the NSN-type NLBs and so that their functional similarity to normal nucleoli. Unlike the NSN-type NLBs, the NLBs of more mature SN-oocytes do not contain transcribed rRNA genes, U3 snoRNA, pre-rRNAs, 18S and 28S rRNAs. These results favor the idea that in a process of transformation of NSN-oocytes to SN-oocytes, NLBs cease to produce pre-ribosomes and, moreover, lose their rRNAs. We also concluded that a denaturing fixative 70% ethanol used in the study to fix oocytes could be more appropriate for light microscopy analysis of nucleolar RNAs and proteins in mammalian fully-grown oocytes than a commonly used cross-linking aldehyde fixative, formalin.

  5. [Mitochondrial and oocyte development].

    Science.gov (United States)

    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.

  6. 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.

  7. Abnormally activated one-carbon metabolic pathway is associated with mtDNA hypermethylation and mitochondrial malfunction in the oocytes of polycystic gilt ovaries

    OpenAIRE

    Longfei Jia; Juan Li; Bin He; Yimin Jia; Yingjie Niu; Chenfei Wang; Ruqian Zhao

    2016-01-01

    Polycystic ovarian syndrome (PCOS) is associated with hyperhomocysteinemia and polycystic ovaries (PCO) usually produce oocytes of poor quality. However, the intracellular mechanism linking hyperhomocysteinemia and oocyte quality remains elusive. In this study, the quality of the oocytes isolated from healthy and polycystic gilt ovaries was evaluated in vitro in association with one-carbon metabolism, mitochondrial DNA (mtDNA) methylation, and mitochondrial function. PCO oocytes demonstrated ...

  8. 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.

  9. 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

  10. Epigenetic reprogramming of breast cancer cells with oocyte extracts

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    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.

  11. 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.

  12. Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

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    Masahiro Sato

    2015-08-01

    Full Text Available Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1 encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4, which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP and human Cas9 mRNAs, 65% (24/37 of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24 showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.

  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. Evaluation of Mouse Oocyte In Vitro Maturation Developmental Competency in Dynamic Culture Systems by Design and Construction of A Lab on A Chip Device and Its Comparison with Conventional Culture System

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    Behnaz Sadeghzadeh Oskouei

    2016-07-01

    Full Text Available Objective In conventional assisted reproductive technology (ART, oocytes are cultured in static microdrops within Petri dishes that contain vast amounts of media. However, the in vivo environment is dynamic. This study assesses in vitro oocyte maturation through the use of a new microfluidic device. We evaluate oocyte fertilization to the blastocyct stage and their glutathione (GSH contents in each experimental group. Materials and Methods In this experimental study, we established a dynamic culture condition. Immature oocytes were harvested from ovaries of Naval Medical Research Institute (NMRI mice. Oocytes were randomly placed in static (passive and dynamic (active in vitro maturation (IVM culture medium for 24 hours. In vitro matured oocytes underwent fertilization, after which we placed the pronucleus (PN stage embryos in microdrops and followed their developmental stages to blastocyst formation after 3 days. GSH content of the in vitro matured oocytes was assessed by monochlorobimane (MCB staining. Results We observed significantly higher percentages of mature metaphase II oocytes (MII in the passive and active dynamic culture systems (DCS compared to the static group (P<0.01. There were significantly less mean numbers of germinal vesicle (GV and degenerated oocytes in the passive and active dynamic groups compared to the static group (P<0.01. Fertilization and blastocyst formation rate in the dynamic systems were statistically significant compared to the static cultures (P<0.01. There was significantly higher GSH content in dynamically matured oocytes compared to statically matured oocytes (P<0.01. Conclusion Dynamic culture for in vitro oocyte maturation improves their developmental competency in comparison with static culture conditions.

  15. Evaluation of Mouse Oocyte In Vitro Maturation Developmental Competency in Dynamic Culture Systems by Design and Construction of A Lab on A Chip Device and Its Comparison with Conventional Culture System

    Science.gov (United States)

    Sadeghzadeh Oskouei, Behnaz; Pashaiasl, Maryam; Heidari, Mohammad Hasan; Salehi, Mohammad; Veladi, Hadi; Ghaderi Pakdel, Firuz; Shahabi, Parviz; Novin, Marefat Ghaffari

    2016-01-01

    Objective In conventional assisted reproductive technology (ART), oocytes are cultured in static microdrops within Petri dishes that contain vast amounts of media. However, the in vivo environment is dynamic. This study assesses in vitro oocyte maturation through the use of a new microfluidic device. We evaluate oocyte fertilization to the blastocyct stage and their glutathione (GSH) contents in each experimental group. Materials and Methods In this experimental study, we established a dynamic culture condition. Immature oocytes were harvested from ovaries of Naval Medical Research Institute (NMRI) mice. Oocytes were randomly placed in static (passive) and dynamic (active) in vitro maturation (IVM) culture medium for 24 hours. In vitro matured oocytes underwent fertilization, after which we placed the pronucleus (PN) stage embryos in microdrops and followed their developmental stages to blastocyst formation after 3 days. GSH content of the in vitro matured oocytes was assessed by monochlorobimane (MCB) staining. Results We observed significantly higher percentages of mature metaphase II oocytes (MII) in the passive and active dynamic culture systems (DCS) compared to the static group (P<0.01). There were significantly less mean numbers of germinal vesicle (GV) and degenerated oocytes in the passive and active dynamic groups compared to the static group (P<0.01). Fertilization and blastocyst formation rate in the dynamic systems were statistically significant compared to the static cultures (P<0.01). There was significantly higher GSH content in dynamically matured oocytes compared to statically matured oocytes (P<0.01). Conclusion Dynamic culture for in vitro oocyte maturation improves their developmental competency in comparison with static culture conditions. PMID:27540525

  16. 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.

  17. Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte

    Science.gov (United States)

    Tombes, R. M.; Simerly, C.; Borisy, G. G.; Schatten, G.

    1992-01-01

    During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.

  18. 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.

  19. 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.

  20. 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.

  1. Enhancing survival of mouse oocytes following chemotherapy or aging by targeting Bax and Rad51.

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    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

  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. The testicular and epididymal expression profile of PLCζ in mouse and human does not support its role as a sperm-borne oocyte activating factor.

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    Mahmoud Aarabi

    Full Text Available Phospholipase C zeta (PLCζ is a candidate sperm-borne oocyte activating factor (SOAF which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides.

  5. 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.

  6. 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...

  7. Development of new method and protocol for cryopreservation related to embryo and oocytes freezing in terms of fertilization rate: A comparative study including review of literature

    Science.gov (United States)

    Barik, Mayadhar; Bajpai, Minu; Patnaik, Santosh; Mishra, Pravash; Behera, Priyamadhaba; Dwivedi, Sada Nanda

    2016-01-01

    Background: Cryopreservation is basically related to meritorious thin samples or small clumps of cells that are cooled quickly without loss. Our main objective is to establish and formulate an innovative method and protocol development for cryopreservation as a gold standard for clinical uses in laboratory practice and treatment. The knowledge regarding usefulness of cryopreservation in clinical practice is essential to carry forward the clinical practice and research. Materials and Methods: We are trying to compare different methods of cryopreservation (in two dozen of cells) at the same time we compare the embryo and oocyte freezing interms of fertilization rate according to the International standard protocol. Results: The combination of cryoprotectants and regimes of rapid cooling and rinsing during warming often allows successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include semen, blood, tissue samples like tumors, histological cross-sections, human eggs and human embryos. Although presently many studies have reported that the children born from frozen embryos or “frosties,” show consistently positive results with no increase in birth defects or development abnormalities is quite good enough and similar to our study (50–85%). Conclusions: We ensure that cryopreservation technology provided useful cell survivability, tissue and organ preservation in a proper way. Although it varies according to different laboratory conditions, it is certainly beneficial for patient's treatment and research. Further studies are needed for standardization and development of new protocol. PMID:27512686

  8. Nuclear structures in Tribolium castaneum oocytes.

    Science.gov (United States)

    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.

  9. 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

  10. 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.

  11. Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix.

    Science.gov (United States)

    Briggs, David C; Birchenough, Holly L; Ali, Tariq; Rugg, Marilyn S; Waltho, Jon P; Ievoli, Elena; Jowitt, Thomas A; Enghild, Jan J; Richter, Ralf P; Salustri, Antonietta; Milner, Caroline M; Day, Anthony J

    2015-11-27

    The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. Chromatin dynamics at the hTERT promoter during transcriptional activation and repression by c-Myc and Mnt in Xenopus leavis oocytes.

    Science.gov (United States)

    Wahlström, Therese; Belikov, Sergey; Arsenian Henriksson, Marie

    2013-12-10

    The transcription factors c-Myc and Mnt regulate gene expression through dimerization with Max and binding to E-boxes in target genes. While c-Myc activates gene expression via recruitment of histone modifying complexes, Mnt acts as a transcriptional repressor. Here, we used the Xenopus leavis oocyte system to address the effect of c-Myc and Mnt on transcription and chromatin remodeling over the E-box region in the human telomerase reverse transcriptase (hTERT) promoter. As expected we found elevated and decreased levels of hTERT transcription upon exogenously expressed c-Myc/Max and Mnt/Max, respectively. In addition, we confirmed binding of these heterodimers to both E-boxes already enriched with H3K9ac and H4K16ac. These chromatin marks were further enhanced upon c-Myc/Max binding followed by increased DNA accessibility in the E-box region. In contrast, Mnt/Max inhibited Myc-induced transcription and mediated repression through complete chromatin condensation and deacetylation of H3K9 and H4K16 across the E-box region. Importantly, Mnt was able to counteract c-Myc mediated activation even when expressed at low levels, suggesting Mnt to act as a strong repressor by closing the chromatin structure. Collectively our data demonstrate that the balance between c-Myc and Mnt activity determines the transcriptional outcome of the hTERT promoter by modulation of the chromatin architecture.

  17. 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.

  18. Na+- and Cl−-coupled active transport of carnitine by the amino acid transporter ATB0,+ from mouse colon expressed in HRPE cells and Xenopus oocytes

    Science.gov (United States)

    Nakanishi, Takeo; Hatanaka, Takahiro; Huang, Wei; Prasad, Puttur D; Leibach, Frederick H; Ganapathy, Malliga E; Ganapathy, Vadivel

    2001-01-01

    ATB0,+ is an amino acid transporter energized by transmembrane gradients of Na+ and Cl− and membrane potential. We cloned this transporter from mouse colon and expressed the clone functionally in mammalian (human retinal pigment epithelial, HRPE) cells and Xenopus laevis oocytes to investigate the interaction of carnitine and its acyl esters with the transporter. When expressed in mammalian cells, the cloned ATB0,+ was able to transport carnitine, propionylcarnitine and acetylcarnitine. The transport process was Na+ and Cl− dependent and inhibitable by the amino acid substrates of the transporter. The Michaelis constant for carnitine was 0.83 ± 0.08 mm and the Hill coefficient for Na+ activation was 1.6 ± 0.1. When expressed in Xenopus laevis oocytes, the cloned ATB0,+ was able to induce inward currents in the presence of carnitine and propionylcarnitine under voltage-clamped conditions. There was no detectable current in the presence of acetylcarnitine. Carnitine-induced currents were obligatorily dependent on the presence of Na+ and Cl−. The currents were saturable with carnitine and the Michaelis constant was 1.8 ± 0.4 mm. The analysis of Na+- and Cl−-activation kinetics revealed that 2 Na+ and 1 Cl− were involved in the transport of carnitine via the transporter. These studies describe the identification of a novel function for the amino acid transporter ATB0,+. Since this transporter is expressed in the intestinal tract, lung and mammary gland, it is likely to play a significant role in the handling of carnitine in these tissues. A Na+-dependent transport system for carnitine has already been described. This transporter, known as OCTN2 (novel organic cation transporter 2), is expressed in most tissues and transports carnitine with high affinity. It is energized, however, only by a Na+ gradient and membrane potential. In contrast, ATB0,+ is a low-affinity transporter for carnitine, but exhibits much higher concentrative capacity than OCTN2 because

  19. 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.

  20. Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability.

    Science.gov (United States)

    Kashir, Junaid; Jones, Celine; Mounce, Ginny; Ramadan, Walaa M; Lemmon, Bernadette; Heindryckx, Bjorn; de Sutter, Petra; Parrington, John; Turner, Karen; Child, Tim; McVeigh, Enda; Coward, Kevin

    2013-01-01

    To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. Laboratory study. University laboratory. Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5). Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  1. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABAA receptors expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik S.

    2015-01-01

    The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit...... different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences....... The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABAAR) subtypes α1β2γ2S, α2β2γ2S, α3β2γ2S, α5β2γ2S and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited...

  2. A peroxisome proliferator response elements regulatory system in xenopus oocytes and its application

    Institute of Scientific and Technical Information of China (English)

    YAN Jin; FAN Chun-lei; WO Xing-de; GAO Li-ping

    2005-01-01

    Background Peroxisome proliferator-activated receptor-gamma (PPARγ) is a kind of ligand-activated transcription factors binding to peroxisome proliferator response element (PPRE), a specific recognition site. It is thought to play a critical role in glucose and lipid metabolism and in inflammation control. The aim of this study was to establish a new cellular model for the quick screening of lipid-lowering drugs, which may be effective as PPAR-γ ligands on the PPRE-mediated pathway regulatory system. Methods Two plasmids were constructed: pXOE-PPARγ, in which the human PPARγ gene was in the downstream of TFⅢA gene promoter, and pLXRN-PPRE-d2EGFP, in which the enhanced green fluorescent protein (EGFP) gene was subcloned into PPRE. The xenopus oocytes were injected with these two plamids, and consequently treated with prostaglandin E1, pioglitazone, and different kinds of lipid-lowering drugs. After 3 days, the oocytes were observed under a fluorescence microscope. To confirm the drug action,we injected pXOE-PPARγ plasmid into the oocytes, which then treated with prostaglandin E1and Hawthorn flavonoids. The mass of expressed lipoprotein lipase (LPL) in the cells was determined by enzyme labeling linked immunosorbent assay (ELISA).Conclusions It is possible to establish a PPRE regulatory EGFP reporter system in xenopus oocytes to monitor the activity of PPARγ ligand. Hawthorn flavonoids can increase the expression of gene downsteam of PPRE by effect on the PPRE pathway regulatory system.

  3. Active Learning Methods

    Science.gov (United States)

    Zayapragassarazan, Z.; Kumar, Santosh

    2012-01-01

    Present generation students are primarily active learners with varied learning experiences and lecture courses may not suit all their learning needs. Effective learning involves providing students with a sense of progress and control over their own learning. This requires creating a situation where learners have a chance to try out or test their…

  4. Oocyte cryopreservation in oncological patients.

    Science.gov (United States)

    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.

  5. Effects of acetazolamide and anordiol on osmotic water permeability in AQPI-cRNA injected Xenopus oocyte

    Institute of Scientific and Technical Information of China (English)

    BingMA; YangXIANG; Sheng-meiMU; TaoLI; He-mingYU; Xue-junLI

    2004-01-01

    AIM: To study the effects of acetazolamide and anordiol on osmotic water permeability in aquaporin 1 (AQP1)-cRNA injected Xenopus oocyte and their mechanisms. METHODS: AQP1 gene constructed in pBluescript was transcripted into cRNA in vitro and then the cRNA was injected in Xenopus oocytes. The effects of acetazolamide and anordiol on the water transport function of AQP1 were observed by assaying the osmotic swelling of oocytes.In addition, their effects on protein expression of AQP1 were quantitatively investigated by Western blotting method.RESULTS: After incubation for 15 min or 72 h, acetazolamide, a carbonic anhydrase inhibitor, equally reduced the water permeability of AQPI-cRNA injected oocyte in a dose-dependent manner. After incubation for 72 h, anordiol,an antiestrogen with partial estrogenic activity, reduced the osmotic water permeability dose dependently as well;however, no discernable action was observed after incubation with anordiol for 15 min. The Western blotting analysis showed that acetazolamide did not influence the protein expression of AQP1. However, after incubation for 72 h with anordiol (10 μmol/L), the quantity of AQP1 in the oocyte membrane was decreased dramatically(P<0.05). CONCLUSION: Both acetazolamide and anordiol inhibited the osmotic water permeability of AQP1-cRNA injected oocyte, but their mechanisms were different. Acetazolamide functionally inhibited the osmotic water permeability of AQP1, whereas anordiol primarily decreased the amount of AQP1 protein in the oocyte membrane.

  6. 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....

  7. 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.

  8. 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...

  9. Technical aspects of the piezo, laser-assisted, and conventional methods for nuclear transfer of mouse oocytes and their efficiency and efficacy: Piezo minimizes damage of the ooplasmic membrane at injection.

    Science.gov (United States)

    Chen, Shee-Uan; Chao, Kuang-Han; Chang, Chia-Yi; Hsieh, Fon-Jou; Ho, Hong-Nerng; Yang, Yu-Shih

    2004-04-01

    Assessment of the advantages and disadvantages of the piezo, laser, and conventional methods for nuclear transfer has remained elusive. Furthermore, although the piezo method had been used by some investigators for research of sperm injection and nuclear transfer for several years, many researchers have failed to operate the technique smoothly and achieve reproducible results. The procedures of nuclear transfer using piezo were ascertained and described in detail. Mouse oocytes were enucleated, and injected with cumulus cells using the piezo, laser, or conventional methods. We investigated the time needed and survival of nuclear transfer. Development was compared among the three methods and parthenogenetic control specimens. The average time of nuclear transfer for each oocyte was significantly shorter using the piezo (118 +/- 9 s) and laser methods (120 +/- 11 s) than using the conventional method (170 +/- 11 s). The damage rate was smaller for the piezo group (10%) than the laser (37%) and conventional (40%) groups. The percentages of blastocyst formation (14%, 12%, and 11%) and the number of nuclei of blastocysts (54 +/- 13, 51 +/- 11, and 52 +/- 12) were similar among the piezo, laser, and conventional groups, but significantly lower than for the control group (83%, 105 +/- 14). The piezo technique is more efficient than the conventional method for nuclear transfer. The laser method is easy to operate, but the equipment is expensive. In addition, piezo induced fewer traumas while breaking the membrane than the aspiration techniques used in the laser and conventional methods.

  10. 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.

  11. 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.

  12. 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.

  13. Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C

    DEFF Research Database (Denmark)

    Perdiguero, Eusebio; Pillaire, Marie-Jeanne; Bodart, Jean-Francois

    2003-01-01

    by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38gamma can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38gamma. Our results indicate that phosphorylation of XCdc25C by Xp38gamma/SAPK3 is important...

  14. Methods for testing antioxidant activity.

    Science.gov (United States)

    Antolovich, Michael; Prenzler, Paul D; Patsalides, Emilios; McDonald, Suzanne; Robards, Kevin

    2002-01-01

    Antioxidant activity has been assessed in many ways. The limitation of many newer methods is the frequent lack of an actual substrate in the procedure. The combination of all approaches with the many test methods available explains the large variety of ways in which results of antioxidant testing are reported. The measurement of antioxidant activities, especially of antioxidants that are mixtures, multifunctional or are acting in complex multiphase systems, cannot be evaluated satisfactorily by a simple antioxidant test without due regard to the many variables influencing the results. Several test procedures may be required to evaluate such antioxidant activities. A general method of reporting antioxidant activity independent of the test procedure is proposed.

  15. 补肾调冲法对多囊卵巢综合征患者抗苗勒氏管激素及卵细胞质量的影响%Effects of Shen Invigoration and Chong Regulating Method on Anti-Mūllerlan Hormone and Oocyte Quality in Polycystic Ovarian Syndrone Patients

    Institute of Scientific and Technical Information of China (English)

    连方; 赵帅

    2012-01-01

    Objective To study the effects of Shen Invigorating and Chong-channel Regulating Method (SCRM) on anti-Miillerian hormone (AMH) and its correlation with oocyte quality in the serum and the follicular fluid of infertile patients with polycystic ovarian syndrome (PCOS) who received in vitro fertilization (IVF), thus studying the mechanism of SCRM on the oocyte quality of PCOS patients. Methods Sixty infertile patients with PCOS undergoing in vitro fertilization and embryo transfer (IVF-ET) were randomly assigned to two groups, 30 in each. Erzhi Tiangui Granule combined with Western medicine was given to patients in the treatment group, while Western medicine was given to those in the control group. The single oocyte estradiol (E2) level, the resistive Index (Rl), the pulsatility index (PI) of the follicular membrane, the number of harvested oocytes, the high quality oocyte rate, the fertilization rate, the cleavage rate, the high quality embryo rate, and the difference of AMH in the serum and the follicular fluid were observed between the two groups. The correlation tests were performed between the levels of AMH in the serum and the follicular fluid and the rates of high quality oocyte and high quality embryo. Meanwhile, the correlation analysis of the AMH level between the serum and the follicular fluid was also conducted. Results (1) The single oocyte E2 level, the high quality oocyte rate, the fertilization rate, the cleavage rate, the high quality embryo rate were significantly higher in the treatment group than in the control group (P<0. 05). (2) The Rl and the PI of the foJIicular membrane both significantly more decreased in the treatment group than in the control group (P<0. 05). (3) The levels of AMH in the serum and the follicular fluid were obvi-ously lower in the treatment group than in the control group (P<0. 01). The AMM levels in the serum and the follicle fluid were positively correlated. The level of AMH in the follicular fluid was obviously

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Activity based costing (ABC Method

    Directory of Open Access Journals (Sweden)

    Prof. Ph.D. Saveta Tudorache

    2008-05-01

    Full Text Available In the present paper the need and advantages are presented of using the Activity BasedCosting method, need arising from the need of solving the information pertinence issue. This issue has occurreddue to the limitation of classic methods in this field, limitation also reflected by the disadvantages ofsuch classic methods in establishing complete costs.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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...

  4. Control of gastric H,K-ATPase activity by cations, voltage and intracellular pH analyzed by voltage clamp fluorometry in Xenopus oocytes.

    Directory of Open Access Journals (Sweden)

    Katharina L Dürr

    Full Text Available Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E(1P and E(2P states and measured Rb(+ uptake under various ionic and pH conditions. The steady-state E(1P/E(2P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb(+ uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E(1P/E(2P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V(0.5, the voltage, at which the E(1P/E(2P ratio is 50∶50, by -100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E(1P→E(2P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb(+ uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E(1P→E(2P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na(+ profoundly alters the voltage-dependent E(1P/E(2P distribution indicating that Na(+ ions can act as surrogates for protons regarding the E(2P→E(1P transition. The complexity of the intra- and extracellular cation effects can be

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Cryopreservation of unfertilized human oocytes.

    Science.gov (United States)

    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.

  7. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium f...

  8. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium f...

  9. 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.

  10. 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.

  11. 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...

  12. Effects of Crocin Supplementation during In Vitro Maturation of Mouse Oocytes on Glutathione Synthesis and Cytoplasmic Maturation

    Directory of Open Access Journals (Sweden)

    Elham Mokhber Maleki

    2016-05-01

    Full Text Available Background: Crocin is an active ingredient of saffron (Crocus sativus L. and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. Materials and Methods: In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI mice. Oocytes were subjected to in vitro maturation (IVM in the presence of either crocin (5 or 10 μg/ml, 5 mM buthionine-[S-R]- sulfoximine (BSO, or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631 were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII oocytes after IVM (n=240 were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid (DTNB-GSH reductase recycling assay. Results: Supplementation of IVM media with 10 μg/ml crocin significantly (P<0.05 increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 μg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 μg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. Conclusion: Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential

  13. Effects of Crocin Supplementation during In Vitro Maturation of Mouse Oocytes on Glutathione Synthesis and Cytoplasmic Maturation

    Science.gov (United States)

    Mokhber Maleki, Elham; Eimani, Hussein; Bigdeli, Mohammad Reza; Golkar Narenji, Afsane; Abedi, Reyhane

    2016-01-01

    Background Crocin is an active ingredient of saffron (Crocus sativus L.) and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH) synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. Materials and Methods In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs) from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI) mice. Oocytes were subjected to in vitro maturation (IVM) in the presence of either crocin (5 or 10 μg/ml), 5 mM buthionine-[S-R]- sulfoximine (BSO), or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631) were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII) oocytes after IVM (n=240) were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay. Results Supplementation of IVM media with 10 µg/ml crocin significantly (P<0.05) increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 µg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 µg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. Conclusion Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential of mouse

  14. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A) receptors expressed in Xenopus laevis oocytes.

    Science.gov (United States)

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik Sindal; Jensen, Anders A

    2015-01-01

    The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(A)R) subtypes α1β2γ(2S), α2β2γ(2S), α3β2γ(2S), α5β2γ(2S) and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5)β2γ(2S) GABA(A)Rs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6β2δ GABA(A)R than at the α(1,2,3,5)β2γ(2S) receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation

  15. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A receptors expressed in Xenopus laevis oocytes.

    Directory of Open Access Journals (Sweden)

    Harriet Hammer

    Full Text Available The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(AR subtypes α1β2γ(2S, α2β2γ(2S, α3β2γ(2S, α5β2γ(2S and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5β2γ(2S GABA(ARs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold as positive allosteric modulators at the α6β2δ GABA(AR than at the α(1,2,3,5β2γ(2S receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non

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

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    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.

  18. 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.

  19. 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.

  20. Oocyte development, meiosis and aneuploidy.

    Science.gov (United States)

    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.

  1. 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.

  2. 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.

  3. 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.

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

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    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.

  5. In vitro maturation, fertilization, embryo development & clinical outcome of human metaphase-I oocytes retrieved from stimulated intracytoplasmic sperm injection cycles

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    Cristina Álvarez

    2013-01-01

    Full Text Available Background & objectives: The major cause of fertilisation failure after ICSI is failure of the oocyte to initiate the biochemical processes necessary for activation. This inability could be ascribed to cytoplasmic immaturity of those gametes even if they had reached nuclear maturity. The activation of a mature oocyte is characterised by release from metaphase II (MII arrest and extrusion of the second polar body, followed by pro-nuclear formation. The aim of this study was to evaluate the fate of in vitro matured (IVM metaphase I (MI oocytes subjected to intracytoplasmic sperm injection (ICSI at different time intervals after extrusion of the first polar body (1PB in in vitro fertilization (IVF cycles. Methods: A total of 8030 oocytes were collected from 1400 ICSI cycles, 5504 MII at the time of cumulus retrieval. Four hundred eight metaphase II (MII (27.1% matured to MII after in vitro culture for 2-26 h and 5389 sibling MII in the moment of oocyte denudation were injected. On the other hand, 49 ICSI cycles containing only MI oocytes at retrieval were injected at three different time intervals after reaching the MII. The intervals were as follows: 2-6 h (n=10, 8-11 h (n=4 and 23-26 h (n=10. Fertilization and development potential were evaluated in both studies. Results: Fertilization, embryo cleavage and quality were significantly lower in IVM MI compared to MII at time of denudation. Pregnancy rate was higher in group MII. Pregnancy was achieved in three embryo transfers when ICSI was performed within 2-6 h (group I and 8-11 h (group II after PB extrusion. One pregnancy was obtained in group I and a healthy neonate was born. Interpretation & conclusions: Immature oocytes from women whose ovaries have been stimulated could be matured, fertilized by ICSI, cleaved in vitro and to give rise to a live birth. However, the developmental competence of embryos derived from immature oocytes is reduced, compared with sibling in vivo matured oocytes

  6. 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.

  7. 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.

  8. Functional study of the effect of phosphatase inhibitors on KCNQ4 channels expressed in Xenopus oocytes

    Institute of Scientific and Technical Information of China (English)

    Tzu-rong SU; Cay-huyen CHEN; Shih-jen HUANG; Chun-yi LEE; Mao-chang SU; Gwan-hong CHEN; Shuan-yow LI; Jiann-jou YANG; Min-jon LIN

    2009-01-01

    Aim: KCNQ4 channels play an important part in adjusting the function of cochlear outer hair cells. The aim of this study was to investigate the effects of ser/thr phosphatase inhibitors on human KCNQ4 channels expressed in Xenopus laevis oocytes. Methods: Synthetic cRNA encoding human KCNQ4 channels was injected into Xenopus oocytes. We used a two-electrode voltage clamp to measure the ion currents in the oocytes. Results: Wild-type KCNQ4 expressed in Xenopus oocytes showed the typical properties of slow activation kinetics and low threshold activation. The outward K~+ current was almost completely blocked by a KCNQ4 blocker, linopirdine (0.25 mmol/L). BIMI (a PKC inhibitor) prevented the effects of PMA (a PKC activator) on the KCNQ4 current, indicating that PKC may be involved in the regulation of KCNQ4 expressed in the Xenopus oocyte system. Treatment with the ser/thr phosphatase inhibitors, cyclosporine (2 μmoVL), calyculin A (2 μmol/L) or okadaic acid (1 μmol/L), caused a significant positive shift in V_(1/2) and a decrease in the conductance of KCNQ4 chan-nels. The V_(1/2) was shifted from-14.6±0.5 to-6.4±0.4 mV by cyclosporine, -18.8±0.5 to-9.2±0.4 mV by calyculin A, and-14.1±0.5 to -0.7±0.6 mV by okadaic acid. Moreover, the effects of these phosphatase inhibitors (okadaic acid or calyculin A) on the induction of a positive shift of V_(1/2) were augmented by further addition of PMA. Conclusion: These results indicate that ser/thr phosphatase inhibitors can induce a shift to more positive potentials of the activation curve of the KCNQ4 current. It is highly likely that the phosphatase functions to balance the phosphorylated state of substrate protein and thus has an important role in the regulation of human KCNQ4 channels expressed in Xenopus oocytes.

  9. Novel Cl- currents elicited by follicle stimulating hormone and acetylcholine in follicle-enclosed Xenopus oocytes

    Science.gov (United States)

    1993-01-01

    Voltage-clamp techniques were used to study the membrane currents elicited by follicle stimulating hormone (FSH) and acetylcholine (ACh) in follicle-enclosed oocytes of Xenopus laevis (follicles). Both agonists caused complex responses that were more evident when the follicles were in hypotonic Ringer solution (HR; 190.4 mosM). In this medium, currents activated by FSH regularly showed three phases whereas currents activated by ACh displayed three to six phases. At a holding potential of -60 mV, FSH, and ACh responses involved combinations of inward and outward currents. Both FSH and ACh responses included a slow smooth inward component that was associated with an increase in membrane conductance, mainly to Cl- (S(in)). This current was strongly dependent on the osmolarity of the external solution: an increase in osmolarity of the HR solution of 18-20 mosM caused a 50% decrease in S(in). In contrast, a fast and transient Cl- current (F(in)) specifically elicited by ACh was not dependent on osmolarity. Both, F(in) and S(in) currents required the presence of follicular cells, since defolliculation using three different methods abolished all the response to FSH and at least four components of the ACh responses. The membrane channels carrying F(in) and oscillatory Cl- currents elicited by stimulation of ACh or serum receptors, were much more permeable to I- and Br- than Cl-, whereas S(in) channels were equally permeable to these anions. Unlike the oscillatory Cl- currents generated in the oocyte itself, S(in) and F(in) currents in follicle-enclosed oocytes were not abolished by chelation of intracellular Ca2+, either with EGTA or BAPTA, which suggests that intracellular Ca2+ does not play a critical role in the activation of these currents. Our experiments show that S(in) and F(in) currents are quite distinct from the previously characterized oscillatory Cl- responses of oocytes. Moreover, the results strongly suggest that the FSH and ACh receptors, the Cl- channels

  10. 体外受精取卵日手淫取精失败的取精方法%Research on methods to obtain sperm after failing to obtain sperm by masturbation in oocyte retrieval for in vitro fertilization

    Institute of Scientific and Technical Information of China (English)

    蒲军; 丘彦

    2011-01-01

    目的 研究体外受精取卵日手淫取精失败的取精方法.方法 对120例体外受精取卵日手淫取精失败的临床资料进行回顾分析.结果 120例患者分别采用药物治疗法、性交法、阴茎头震荡刺激法、经直肠输精管壶腹和精囊及前列腺按摩法、经皮穿刺附睾睾丸法5种方法作为手淫取精失败的替代取精法,120例患者全部取精成功.结论 性交法、经皮穿刺附睾睾丸取精法是较满意的两种替代方法.提前冷冻精子是解决体外受精取卵日手淫取精失败的最佳方案.%Objective To research the methods to obtain sperm after failing to obtain sperm by masturbation in oocyte retrieval for in vitro fertilization. Methods To retrospectively analyse the clinical data of 120 patients failing to obtain sperm by masturbation in oocyte retrieval for in vitro fertilization. Results 120 patients adopted 5 methods respectively to obtain sperm for substitution,which including drug treatment,coitus,shaking and stimulating balanus,pressing ampulla of deferent duct and seminal vesicle and prostate per rectum, percutaneous epididymal and testicular sperm aspiration. Conclusion There are two frequently used methods to obtain sperm by coitus and percutaneous epididymal and testicular sperm aspiration for substitution. It is the optimal method to freeze sperm in advance to deal with one patient fail to obtain sperm by masturbation in oocyte retrieval for in vitro fertilization.

  11. Activity – based costing method

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    Èuchranová Katarína

    2001-06-01

    Full Text Available Activity based costing is a method of identifying and tracking the operating costs directly associated with processing items. It is the practice of focusing on some unit of output, such as a purchase order or an assembled automobile and attempting to determine its total as precisely as poccible based on the fixed and variable costs of the inputs.You use ABC to identify, quantify and analyze the various cost drivers (such as labor, materials, administrative overhead, rework. and to determine which ones are candidates for reduction.A processes any activity that accepts inputs, adds value to these inputs for customers and produces outputs for these customers. The customer may be either internal or external to the organization. Every activity within an organization comprimes one or more processes. Inputs, controls and resources are all supplied to the process.A process owner is the person responsible for performing and or controlling the activity.The direction of cost through their contact to partial activity and processes is a new modern theme today. Beginning of this method is connected with very important changes in the firm processes.ABC method is a instrument , that bring a competitive advantages for the firm.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics

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    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.

  17. 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.

  18. Four-dimensional visualization and quantitative analysis of meiotic spindle movements in live mouse oocytes.

    Science.gov (United States)

    Tian, N; Zhang, L; Liu, B; Wang, P; Li, Y; Ma, W

    2012-09-01

    This paper made a different attempt of real-time observation of the meiotic spindle movements in living mouse oocyte using a convenient method. This method was based on an experimental phenomenon discovered in our work. In living mouse oocytes, a high concentration of calcium ions (Ca(2+)) was observed throughout the region occupied by the initial meiotic spindle. After Ca(2+) labelling with Fura-2, a weakly fluorescent area (WFA) appeared on each side of the chromosomes. The activities of the WFAs changed during spindle development. By real-time tracking of WFAs, we were able to indirectly observe the meiotic spindle movements. Occasionally, it was observed that the first meiotic spindle rotated from an orientation parallel to the cortex to become perpendicular, instead of migrating from the oocyte centre to the cortex along its axis. Moreover, we analysed this uncommon rotation of the first meiotic spindle and found that the whole rotation process can be divided into two phases: the early slow-speed rotation and the subsequent rapid-speed rotation. We further characterized this rotation with respect to rotational speed and acceleration at all the stages of development. By using a two-photon laser-scanning microscope in combination with Fura-2 dye that is nondamaging to oocytes, we provide a convenient method for indirect visualization and quantitative analysis of spindle movements by real-time tracking of WFAs. This method is easy to operate and master, and economical with time and effort. © 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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. 

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. Evolutionary conservation of the mature oocyte proteome

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 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

  13. 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...

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Human oocyte maturation in vitro.

    Science.gov (United States)

    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.

  3. 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

    the deterioration of oocyte quality in terms of competence and of the risk of transmitting mitochondrial abnormalities to the offspring. In contrast, some mtDNA haplogroups are protective against the decline of ovarian reserve. Quantitatively, mitochondrial biogenesis is crucial during oogenesis for constituting a mitochondrial pool sufficiently large to allow normal early embryonic development and to avoid the untimely activation of mitochondrial biogenesis. Ovarian ageing also seriously affects the dynamic nature of mitochondrial biogenesis in the surrounding granulosa cells that may provide interesting alternative biomarkers of oocyte quality. A fuller understanding of the involvement of mitochondria in cases of infertility linked to ovarian ageing would contribute to a better management of the disorder in the future. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  4. Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation.

    Directory of Open Access Journals (Sweden)

    Kenji Ezoe

    Full Text Available Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK or 3-isobutyl-1-methylxanthine (IBMX to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

  5. Recent Progress in Cryopreservation of Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

    Science.gov (United States)

    Nakajima, Keisuke; Yaoita, Yoshio

    2015-01-16

    Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  11. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Keisuke Nakajima

    2015-01-01

    Full Text Available Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf. In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  12. 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.

  13. 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.

  14. 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.

  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. 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.

  17. Activating teaching methods in french language teaching

    OpenAIRE

    Kulhánková, Anna

    2009-01-01

    The subject of this diploma thesis is activating teaching methods in french language teaching. This thesis outlines the issues acitvating teaching methods in the concept of other teaching methods. There is a definition of teaching method, classification of teaching methods and characteristics of each activating method. In the practical part of this work are given concrete forms of activating teaching methods appropriate for teaching of french language.

  18. Oocyte maturation and fertilization in marine nemertean worms: using similar sorts of signaling pathways as in mammals, but often with differing results.

    Science.gov (United States)

    Stricker, Stephen A; Cline, Cory; Goodrich, David

    2013-08-01

    In marine worms belonging to the phylum Nemertea, oocyte maturation and fertilization are regulated by the same general kinds of signals that control such processes in mammals. However, unlike mammalian oocytes that develop within follicles, nemertean oocytes characteristically lack a surrounding sheath of follicle cells and often respond differently to maturation-related cues than do mammalian oocytes. For example, elevators of cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) levels promote the resumption of meiotic maturation (=germinal vesicle breakdown, GVBD) in nemertean oocytes, whereas increasing intraoocytic cAMP and cGMP typically blocks GVBD in mammals. Similarly, AMP-activated kinase (AMPK) signaling keeps nemertean oocytes from maturing, but in mouse oocytes, AMPK activation triggers GVBD. In addition, protein kinase C (PKC) activity is required for seawater-induced GVBD in nemerteans, whereas some PKCs have been shown to inhibit GVBD in mammals. Furthermore, although fertilization causes both types of oocytes to reorganize their endoplasmic reticulum and generate calcium oscillations that can involve soluble sperm factor activity and inositol 1,4,5-trisphosphate signaling, some discrepancies in the spatiotemporal patterns and underlying mechanisms of fertilization are also evident in nemerteans versus mammals. Thus, to characterize differences and similarities in gamete biology more fully, aspects of oocyte maturation and fertilization in marine nemertean worms are reviewed and briefly compared with related findings that have been published for mammalian oocytes. In addition, possible causes of the alternative responses displayed by oocytes in these two animal groups are addressed.

  19. 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.

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

    Science.gov (United States)

    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.

  1. 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.

  2. 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.

  3. 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.

  4. 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...

  5. 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...

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Establishment of conditions for ovum pick up and IVM of jennies oocytes toward the setting up of efficient IVF and in vitro embryos culture procedures in donkey (Equus asinus).

    Science.gov (United States)

    Goudet, Ghylène; Douet, Cécile; Kaabouba-Escurier, Aurore; Couty, Isabelle; Moros-Nicolás, Carla; Barrière, Philippe; Blard, Thierry; Reigner, Fabrice; Deleuze, Stefan; Magistrini, Michèle

    2016-07-15

    Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryos cryopreservation allows the preservation of genetics from both male and female and is the fastest method to restore a breed. Because embryo production in vivo is limited in equids, our objective was to establish conditions for in vitro production of embryos in donkey using ovum pick up (OPU), IVM, IVF, and in vitro culture of zygotes. Donkey cumulus-oocyte complexes (COCs) were collected by transvaginal ultrasound-guided aspirations OPU in adult cyclic jennies and in vitro matured in tissue culture medium 199 supplemented with fetal calf serum and epidermal growth factor for 24, 30, 34, or 38 hours. They were preincubated with oviductal fluid for 30 minutes, coincubated with frozen-thawed donkey semen treated with procaine for 18 hours, and cultured for 30 hours in Dulbecco's Modified Eagle Medium-F12 supplemented with NaHCO3, fetal calf serum, and gentamycin. From the five OPU sessions, we collected 92 COCs in 193 follicles (48%) with an average of 4.2 COCs per jenny. All COCs were expanded after more than 24-hour IVM. At collection, jennies oocytes contained a germinal vesicle. Metaphase 1 oocytes were observed after 30-hour IVM and 44% were in metaphase 2 after 34-hour IVM. In our conditions, IVM of donkey oocytes was slower than IVM of equine oocytes and optimal duration for donkey oocytes IVM may be 34 hours. Only 15% of jennies oocytes contained two pronuclei after coincubation with donkey spermatozoa and none of them developed further after 48 hours post-IVF. Moreover, some parthenogenetic activation occurred. Thus, the treatment of donkey sperm with procaine may not be efficient for IVF. In conclusion, we established for the first time conditions for OPU in jennies with high recovery rates. We reported that IVM of jennies oocytes can produce 44% of metaphase 2 oocytes after 34 hours in culture

  9. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes

    Science.gov (United States)

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S.; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming. PMID:27472658

  10. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.

    Science.gov (United States)

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji; Kimura, Akatsuki

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.

  11. Potential targets of transforming growth factor-beta1 during inhibition of oocyte maturation in zebrafish

    Directory of Open Access Journals (Sweden)

    Clelland Eric

    2005-09-01

    Full Text Available Abstract Background TGF-beta is a multifunctional growth factor involved in regulating a variety of cellular activities. Unlike mammals, the function of TGF-beta in the reproduction of lower vertebrates, such as fish, is not clear. Recently, we showed that TGF-beta1 inhibits gonadotropin- and 17alpha, 20beta-dihydroxyprogesterone (DHP-induced maturation in zebrafish. The aim of the present study was to investigate the mechanisms underlying this action. Method To determine if the effect of TGF-beta1 on oocyte maturation involves transcription and/or translation, ovarian follicles were pre-treated with actinomycin D, a blocker of transcription, and cyclohexamide, an inhibitor of translation, and incubated with hCG or DHP, either alone or in combination with TGF-beta1 and oocyte maturation scored. To determine the effect of TGF-beta1 on mRNA levels of several key effectors of oocyte maturation, three sets of experiments were performed. First, follicles were treated with control medium or TGF-beta1 for 2, 6, 12, and 24 h. Second, follicles were treated with different concentrations of TGF-beta1 (0 to 10 ng/ml for 18 h. Third, follicles were incubated with hCG in the absence or presence of TGF-beta1 for 18 h. At the end of each experiment, total RNA was extracted and reverse transcribed. PCR using primers specific for 20beta-hydroxysteroid dehydrogenase (20beta-HSD which is involved in DHP production, follicle stimulating hormone receptor (FSHR, luteinizing hormone receptor (LHR, the two forms of membrane progestin receptor: mPR-alpha and mPR-beta, as well as GAPDH (control, were performed. Results Treatment with actinomycin D, a blocker of transcription, reduced the inhibitory effect of TGF-beta1 on DHP-induced oocyte maturation, indicating that the inhibitory action of TGF-beta1 is in part due to regulation of gene transcription. Treatment with TGF-beta1 caused a dose and time-dependent decrease in mRNA levels of 20beta-HSD, LHR and mPR-beta in

  12. 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

  13. [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.

  14. 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.

  15. 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.

  16. 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

  17. 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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Effect of sera of normal cycling, pregnant and repeat breeding buffaloes (Bubalus bubalis) on in vitro maturation of buffalo, sheep and goat oocytes

    Institute of Scientific and Technical Information of China (English)

    Anthony Sabasthin; Sumanta Nandi; Venkataswamy Girish Kumar

    2013-01-01

    Objective: To examine the oocytes maturation capacity of buffalo, sheep and goat using media containing sera of three different groups of buffaloes (regularly cycling, pregnant and repeat breeding). Methods: The buffalo, sheep and goat oocytes were matured under suitable conditions in medium containing sera of regularly cycling, pregnant and repeat breeding buffaloes. Results:The oocytes maturation rate containing buffalo oocytes cultured in media containing sera of the control group and regularly cycling group were not significantly different. However when oocytes cultured in the media containing sera of pregnant buffaloes the maturation rate were significantly declined. Further significant declined in maturation rate were observed when oocytes cultured in media containing sera of repeat breeding buffaloes. When sheep and goat oocytes cultured in the media containing control, pregnant and regularly cycling animals sera the oocytes maturation rate were not significantly different. A significant decline in maturation rate of sheep and goat oocytes were observed, when oocytes cultured in media containing sera of repeat breeding buffaloes. Conclusion: We may conclude that serum collected from normal cycling buffaloes can be used for oocytes maturation in both homogeneous and heterogeneous species.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. 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.

  7. 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.

  8. Activation of Students with Various Teaching Methods

    DEFF Research Database (Denmark)

    Andersen, Shuang Ma

    2011-01-01

    A group of teaching methodes to active engineer students have been tried out. The methodes are developed based on the Pedagogical Cyclic Workflow (PCW). Comparing with earlier evaluation, positive feedback is achieved among the students.......A group of teaching methodes to active engineer students have been tried out. The methodes are developed based on the Pedagogical Cyclic Workflow (PCW). Comparing with earlier evaluation, positive feedback is achieved among the students....

  9. 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

  10. Active teaching methods, studying responses and learning

    DEFF Research Database (Denmark)

    Christensen, Hans Peter; Vigild, Martin Etchells; Thomsen, Erik Vilain;

    2010-01-01

    Students’ study strategies when exposed to activating teaching methods are measured, analysed and compared to study strategies in more traditional lecture-based teaching. The resulting learning outcome is discussed.......Students’ study strategies when exposed to activating teaching methods are measured, analysed and compared to study strategies in more traditional lecture-based teaching. The resulting learning outcome is discussed....

  11. Active teaching methods, studying responses and learning

    DEFF Research Database (Denmark)

    Christensen, Hans Peter; Vigild, Martin Etchells; Thomsen, Erik Vilain;

    Students’ study strategies when exposed to activating teaching methods are measured, analysed and compared to study strategies in more traditional lecture-based teaching.......Students’ study strategies when exposed to activating teaching methods are measured, analysed and compared to study strategies in more traditional lecture-based teaching....

  12. Active teaching methods, studying responses and learning

    DEFF Research Database (Denmark)

    Christensen, Hans Peter; Vigild, Martin Etchells; Thomsen, Erik Vilain

    Students’ study strategies when exposed to activating teaching methods are measured, analysed and compared to study strategies in more traditional lecture-based teaching.......Students’ study strategies when exposed to activating teaching methods are measured, analysed and compared to study strategies in more traditional lecture-based teaching....

  13. Active teaching methods, studying responses and learning

    DEFF Research Database (Denmark)

    Christensen, Hans Peter; Vigild, Martin Etchells; Thomsen, Erik Vilain

    2010-01-01

    Students’ study strategies when exposed to activating teaching methods are measured, analysed and compared to study strategies in more traditional lecture-based teaching. The resulting learning outcome is discussed.......Students’ study strategies when exposed to activating teaching methods are measured, analysed and compared to study strategies in more traditional lecture-based teaching. The resulting learning outcome is discussed....

  14. The DNA damage response in mammalian oocytes

    Directory of Open Access Journals (Sweden)

    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.

  15. The impact of different time intervals between hCG priming and oocyte retrieval on ART outcomes

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    Mohammad Hadi Bahadori

    2013-01-01

    Full Text Available Background: Abnormal oocyte morphology has been associated with the hormonal environment to which the gametes are exposed. Objective: In this study, we evaluated the oocytes morphology, fertilization rate, embryos quality, and implantation rate resulted of retrieved oocytes in different times after human chorionic gonadotrophin (HCG administration. Materials and Methods: A total of 985 metaphase II oocytes were retrieved 35, 36, 37 and 38 h after the injection of HCG as groups 1, 2, 3, and 4 respectively. Oocyte morphology was divided into (I normal morphology, (II extracytoplasmic abnormalities, (III cytoplasmic abnormalities and (IV intracytoplasmic vacuoles and in each group, oocytes were evaluated according to this classification. Results: Extracytoplasmic abnormalities were encountered in 17.76% and 31.1% of these oocytes (groups 3 and 4 respectively, p=0.007 in comparison with 12.23% group 2. Cytoplasmic abnormalities in group 4 were higher than other groups. 23.88% (p=0.039 and 43.25% (p=0.089 of resulted 2PN (two pronucleus from groups 3 and 4 showed grade Z3 respectively in comparison to group 2 (16.44%. Normal and various categories of abnormal oocytes did not differ regarding fertilization and cleavage rates (p=0.061. However, group 4 showed significant difference in the rate of embryos fragmentation (grade III and IV embryo in comparison with group 2 (40.96% vs. 24.93%, p=0.078. The pregnancy rate was higher in G2 and G3 groups (28.5 and 24.13% respectively. Conclusion: Oocyte retrieval time following HCG priming affected on oocyte morphology, 2PN pattern and embryos qualities subsequently. Both good quality embryo formation and pregnancy outcomes were noticeably higher when oocytes were retrieved 36 h after HCG priming in ART program.

  16. Artificial fertilization of oocytes and sperm activation in pacu: effects of the spermatozoa:oocyte ratio, water volume, and in natura semen preservation Fertilização artificial de ovócitos e ativação espermática em pacus: efeito da razão espermatozoide:ovócito, volume de água e preservação do sêmen in natura

    Directory of Open Access Journals (Sweden)

    Eduardo Antônio Sanches

    2011-01-01

    Full Text Available The objective of this work was to investigate artificial fertilization and the duration of sperm motility in pacu with different insemination doses, water volume, and in natura semen preservation. It was carried out four experimentsfor evaluation of insemination doses (7x10³, 7x10(4, 7x10(5, 7x10(6, and 7x10(7 spermatozoa oocytes-1 on the artificial fertilization of oocytes; the effect of water volume (0.5, 15.0, 30.0, 45.0, and 60.0 mL water mL-1 of oocyte with insemination doses of 105,481 and 210,963 spermatozoa oocytes-1; the effect of semen dilutions (0.005, 0.05, 0.5, and 5.0 µL semen mL-1 of water on sperm motility duration; and the effect of storage at 15ºC for 9h on sperm motility duration and sperm survival ratio. The highest results obtained were: insemination doses from 7x10³ to 7x10(7 spermatozoa oocytes-1; from 15 to 60mL water mL-1 of oocytes; semen dilution of 0.005 µL semen/mL water and 98.65% sperm survival until 2h45min 36s preservation time. Preservation at 15ºC for 9h does not influence sperm motility duration. The highest fertilization rates can be observed by using 0.27 to 270 µL semen mL-1 of oocytes with 15 at 60 mL water for activation.Objetivou-se foi avaliar a fertilização artificial e a duração da motilidade espermática em pacus com diferentes doses inseminantes, volumes de água e preservação do sêmen in natura. Foram realizados quatro experimentos para avaliação do efeito de doses inseminantes (7x10³, 7x10(4, 7x10(5, 7x10(6 e 7x10(7 espermatozoides ovócito-1 sobre a fertilização artificial dos ovócitos; do efeito do volume de água (0,5; 15,0; 30,0; 45,0 e 60,0 mL de água mL-1 de ovócitos com doses inseminantes de 105.481 e 210.963 espermatozoides ovócito-1; do efeito de diluição do sêmen (0,005; 0,05; 0,5 e 5,0 µL de sêmen mL-1 de água sobre a duração da motilidade espermática; e do efeito do armazenamento a 15 ºC por 9 h sobre a duração da motilidade espermática e o

  17. 间接免疫荧光法在小鼠卵子sPLA2GIID蛋白表达检测中的应用%Establishment of indirect immunofluorescenct analysis as an assay method for CFTR protein expression in mouse oocytes

    Institute of Scientific and Technical Information of China (English)

    李红阁; 吕建新; 陈文颖; 石其贤; 倪崖

    2011-01-01

    目的:建立间接免疫荧光检测小鼠卵子上未知蛋白表达的方法,为实验室开展卵子上未知蛋白表达的研究提供参考.方法:参考间接免疫荧光检测未知蛋白的常用方法,优化实验步骤中各个环节,通过系统性的回顾和分析本实验室近一年来几个阶段对小鼠卵子sPLA2 GIID 型蛋白表达的探索,建立间接免疫荧光检测小鼠卵子上未知蛋白表达的新方法;并用免疫印迹法(Western blott)验证结果的准确性.结果:证明小鼠卵子上有很丰富的sPLA2 GIID型蛋白的表达;免疫印迹法证实小鼠卵子存在特异的分子质量为16 000蛋白条带.结论:本方法测定结果准确可靠、操作简便、重复性好,适宜于实验室推广.%Objective To establish indirect immunofluorescence analytic technique to assay mouse oocyte unknown protein expression, and provide a useful reference for laboratory research on oocyte unknown protein expression. Methods Reference to frequent indirect immunofluorescence method to detect unknown protein,optimize steps in the various links, and through systematic survey and analysis of our laboratory work over the past year several stages on mouse oocyte sPLA2 GIID protein expression, thus establish indirect immunofluorescent new way to assay mouse oocyte unknown protein expression. Results We have proved that a strong sPLA2 GIID protein expression is present on mouse oocyte plasma membrane and detectes a hand of 16 kD protein. Conclusions The results demonstrated that the method is accurate , reliable, easy to operate,repetitive and suitable for laboratory purposes.

  18. 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.

  19. 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

  20. Reduced developmental competence of immature, in-vitro matured and postovulatory aged mouse oocytes following IVF and ICSI

    Directory of Open Access Journals (Sweden)

    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

  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. 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.

  3. 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.

  4. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

  7. Multiple requirements of PLK1 during mouse oocyte maturation.

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    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.

  8. 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

  9. 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.

  10. Regulation of fatty acid oxidation in mouse cumulus-oocyte complexes during maturation and modulation by PPAR agonists.

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    Kylie R Dunning

    Full Text Available Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of ³H₂O from [³H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further

  11. The effect of hepatocyte growth factor on mouse oocyte in vitro maturation and subsequent fertilization and embryo development

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    Mohammad H. Bahadori

    2011-05-01

    Full Text Available Background: Oocyte invitro maturation is an enormously promising technology for the treatment of infertility, yet its clinical application remains limited owing to poor success rates. Therefore, this study was devised to evaluate the effect of hepatocyte growth factor (HGF on in vitro maturation of immature mouse oocytes and resulting embryos development. Materials and Method: Cumulus – oocyte complex and germinal vesicle were obtained from eighteen 6-8 weeks-old female NMRI mice 46-48 hours after administration of an injection of 5 IU PMSG (Pregnant Mares’ Serum Gonadotrophin. Oocytes were culture in TCM199 (Tissue culture medium-199 supplemented with dosages of 0, 10, 20, 50 and 100 ng/ml of HGF. After 24 hours, metaphase ІІ oocytes were co-incubated with sperms for 4-6 hours in T6 medium. Following isolation of two pronucleus embryos, cleavage of embryos was assessed in the same medium till blastocyst stage. The number of oocytes and embryos was recorded under an invert microscope and the rate of oocyte maturation, fertilization and embryos cleavage until blastocyst stage compared using of student χ2 test. Results: In all compared groups, oocytes growth and embryos development rate in the 20 ng/ml of HGF treatment group was significantly higher (p<0.05 than the control group (p<0.05.Conclusion: 20 ng/ml of HGF improved the nuclear maturation and embryo development up to blastocyst stage during culture condition

  12. Impact of stage III-IV endometriosis on recipients of sibling oocytes: matched case-control study.

    Science.gov (United States)

    Díaz, I; Navarro, J; Blasco, L; Simón, C; Pellicer, A; Remohí, J

    2000-07-01

    To evaluate the impact of severe endometriosis on IVF-ET outcome in women receiving oocytes from the-same donor. A matched case-control study. Oocyte donation program at the Instituto Valenciano de Infertilidad. Fifty-eight recipients were included in a matched case-control study of IVF-ET in our oocyte donation program. Twenty-five patients were diagnosed by laparoscopy with stage III-IV endometriosis (group I), while the remaining 33 were free of the disease (group II). On the day of retrieval, oocytes from a single donor were donated to recipients from both groups. Some of the donors supplied oocytes for more than 2 patients. Recipients received steroid replacement therapy for endometrial preparation. Ovarian stimulation and oocyte retrieval in donors. Uterine embryo transfer (ET) in recipients after appropriate exogenous hormone replacement therapy (HRT). Pregnancy, implantation, miscarriage, and live birth rates. The number of oocytes donated and fertilized, as well as the number of available and transferred embryos, was not statistically different between the two groups. Pregnancy, implantation, and miscarriage rates were not affected by stage III-IV endometriosis when compared with the control group. The live birth rate was 28.0% in the group with endometriosis and 27.2% in the control group. These results show that implantation is not affected by stage III-IV endometriosis. Given the contemporary methods of endometrial preparation for transfer of embryos derived from donor oocytes, any potential negative effect of severe endometriosis on the uterine environment is undetectable.

  13. Microsensors and image processing for single oocyte qualification: toward multiparametric determination of the best time for fertilization

    Science.gov (United States)

    Wacogne, B.; Ivascu, I.; Zeggari, R.; Pieralli, C.; Amiot, C.; Pazart, L.; Roux, C.

    2013-10-01

    During intracytoplasmic sperm injection (ICSI) attempts, oocytes reaching metaphase II are microinjected. A morphological examination under a microscope is the usual method for determining oocyte maturity. The level of oocyte maturity is based on the meiotic status (Germinal Vesicle, metaphase I and metaphase II) of the oocytes with respect to their increasing maturity. In this letter, we summarize the studies conducted to analyze cytoplasm maturity using various microsystems and image processing. Optical microsystems are used to measure the transmission spectra and refractive index of the oocytes. We compared the transmission spectra measurements to the transmission electron microscopy results. Karhunen-Loeve transform is also used to evaluate the maturity of the oocytes. To summarize, optical analysis techniques are a minimally invasive technology allowing cytoplasm maturity to be assessed. Oocytes should not only be qualified in terms of GV, MI or MII, but also regarding their temporal evolution over the course of these maturation stages. The ultimate aim of this work is to describe the maturation of the oocytes by a trajectory in a multidimensional space and to determine when would be the best time for successful fertilization.

  14. Fractal organization of feline oocyte cytoplasm.

    Science.gov (United States)

    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.

  15. Fractal organization of feline oocyte cytoplasm

    Directory of Open Access Journals (Sweden)

    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.

  16. Age-dependent radiosensitivity of mouse oocytes

    Energy Technology Data Exchange (ETDEWEB)

    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)

  17. 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

  18. Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

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    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. 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.

  20. 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.

  1. 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.

  2. 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....

  3. Metabolic Determinants of Mitochondrial Function in Oocytes.

    Science.gov (United States)

    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.

  4. 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.

  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. ERK3 is required for metaphase-anaphase transition in mouse oocyte meiosis.

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    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.

  7. Maternal death after oocyte donation at high maternal age: case report

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    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.

  8. Obstetric outcome in donor oocyte pregnancies: a matched-pair analysis

    Directory of Open Access Journals (Sweden)

    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. IL-6 and mouse oocyte spindle.

    Directory of Open Access Journals (Sweden)

    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.

  10. Activating teaching methods, studying responses and learning

    OpenAIRE

    Christensen, Hans Peter; Vigild, Martin E.; Thomsen, Erik; Szabo, Peter; Horsewell, Andy

    2009-01-01

    Students’ study strategies when exposed to activating teaching methods are measured, analysed and compared to study strategies in more traditional lecture-based teaching. The resulting learning outcome is discussed. Peer Reviewed

  11. 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.

  12. Inhibition of Raf/MAPK signaling in Xenopus oocyte extracts by Raf-1-specific peptides.

    Science.gov (United States)

    Radziwill, G; Steinhusen, U; Aitken, A; Moelling, K

    1996-10-01

    Raf-1 is an upstream element of the mitogen-activated protein kinase (MAPK) pathway which leads to cell proliferation and differentiation. In this study Raf-1 derived peptides comprising the conserved amino acid residues Arg89 and Ser259, involved in binding of activated Ras and 14-3-3 proteins, respectively, were shown to interfere with MAPK activation in extracts from immature Xenopus oocytes. Lipids prepared from oocyte extracts can stimulate MAPK in a Ras- and protein kinase C-independent manner. This lipid-induced MAPK activation is blocked by a Raf-1 derived peptide comprising Ser259.

  13. Optimization methods for activities selection problems

    Science.gov (United States)

    Mahad, Nor Faradilah; Alias, Suriana; Yaakop, Siti Zulaika; Arshad, Norul Amanina Mohd; Mazni, Elis Sofia

    2017-08-01

    Co-curriculum activities must be joined by every student in Malaysia and these activities bring a lot of benefits to the students. By joining these activities, the students can learn about the time management and they can developing many useful skills. This project focuses on the selection of co-curriculum activities in secondary school using the optimization methods which are the Analytic Hierarchy Process (AHP) and Zero-One Goal Programming (ZOGP). A secondary school in Negeri Sembilan, Malaysia was chosen as a case study. A set of questionnaires were distributed randomly to calculate the weighted for each activity based on the 3 chosen criteria which are soft skills, interesting activities and performances. The weighted was calculated by using AHP and the results showed that the most important criteria is soft skills. Then, the ZOGP model will be analyzed by using LINGO Software version 15.0. There are two priorities to be considered. The first priority which is to minimize the budget for the activities is achieved since the total budget can be reduced by RM233.00. Therefore, the total budget to implement the selected activities is RM11,195.00. The second priority which is to select the co-curriculum activities is also achieved. The results showed that 9 out of 15 activities were selected. Thus, it can concluded that AHP and ZOGP approach can be used as the optimization methods for activities selection problem.

  14. 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.

  15. Slow oocyte freezing and thawing in couples with no sperm or an insufficient number of sperm on the day of in vitro fertilization

    Directory of Open Access Journals (Sweden)

    Tomazevic Tomaz

    2011-02-01

    Full Text Available Abstract Background The clinical results of in vitro fertilization of slowly frozen-thawed oocytes are known to be significantly worse than those obtained by fresh oocytes. Little is known about the factors affecting the clinical outcome of frozen-thawed oocytes. The aim of this retrospective study was to explore the role of oocyte cryopreservation in the group of patients with no available sperm on the day of in vitro fertilization. Additionally, the effects of the female serum FSH level and sperm quality on the clinical outcome of frozen-thawed oocytes were evaluated. Methods Oocytes were slowly frozen and thawed in 22 infertile couples with no sperm or insufficient number of sperm on the day of in vitro fertilization (IVF. In 9 couples with severe azoospermia or oligoasthenoteratozoospermia frozen-thawed oocytes were fertilized by autologous sperm of bad quality when available (Group 1. In 13 couples with non-ejaculation due to psychological stress on the day of classical IVF or severe azoospermia frozen-thawed oocytes were fertilized by autologous or donated sperm of normal quality (Group 2. Oocytes were thawed in 23 cycles and microinjected by the autologous or donated sperm, when available. The clinical outcome of intracytoplasmic sperm injection - ICSI (fertilization, blastocyst, and pregnancy rates was compared to the outcome of fresh oocytes of the same group of patients; additionally, the female serum FSH level and the sperm quality were compared. Results In all couples, 70.5% of oocytes survived the freeze-thaw procedure. After ICSI, 61.5% of thawed oocytes were fertilized. Twenty one% of embryos developed to the blastocyst stage. The pregnancy rates per embryo transfer and freeze-thaw cycle were 33.3% and 17.4%, respectively. All pregnancies ended in the birth of a baby without congenital anomalies. In patients with severe azoospermia or oligoasthenoteratozoospermia there was no statistically significant difference in pregnancy rates

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

    Science.gov (United States)

    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.

  17. Prooxidant Effects of Verbascoside, a Bioactive Compound from Olive Oil Mill Wastewater, on In Vitro Developmental Potential of Ovine Prepubertal Oocytes and Bioenergetic/Oxidative Stress Parameters of Fresh and Vitrified Oocytes

    Directory of Open Access Journals (Sweden)

    M. E. Dell'Aquila

    2014-01-01

    Full Text Available Verbascoside (VB is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART. Juvenile ART is a promising topic because, in farm animals, it reduces the generation gap and, in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs.

  18. Vitrification of in vitro matured oocytes collected from antral follicles at the time of ovarian tissue cryopreservation

    Directory of Open Access Journals (Sweden)

    Fasano Giovanna

    2011-11-01

    Full Text Available Abstract Background In the past few years, cryopreservation of ovarian tissue has become an established procedure proposed in many centers around the world and transplantation has successfully resulted in full-term pregnancies and deliveries in human. This prospective study aims to evaluate the feasibility of vitrifying in vitro matured oocytes (IVM isolated at the time of ovarian tissue cryopreservation to improve the efficiency of fertility preservation programs. Methods Oocyte-cumulus complexes were retrieved from freshly collected ovarian cortex by aspirating antral follicular fluid, and were matured in vitro for 24-48 h prior to vitrification. Oocytes were matured in an IVM commercial medium (Copper Surgical, USA supplemented with 75 mIU/ml FSH and 75 mIU/ml LH and vitrified using a commercial vitrification kit (Irvine Scientific, California in high security vitrification straws (CryoBioSystem, France. Oocyte collection and IVM rates were evaluated according to the age, the cycle period and the amount of tissue collected. Results Immature oocyte retrieval from ovarian tissue was carried out in 57 patients between 8 and 35 years of age, undergoing ovarian tissue cryopreservation. A total of 266 oocytes were isolated, 28 of them were degenerated, 200 were at germinal vesicle stage (GV, 35 were in metaphase I (MI and 3 displayed a visible polar body (MII. The number of oocytes collected was positively correlated with the amount of tissue cryopreserved (p p = 0.005. Oocytes were obtained regardless of menstrual cycle period or contraception. A total maturation rate of 31% was achieved, leading to the vitrification of at least one mature oocyte for half of the cohort. Conclusions The study showed that a significant number of immature oocytes can be collected from excised ovarian tissue whatever the menstrual cycle phases and the age of the patients, even for prepubertal girls.

  19. Effect of vitrification at the germinal vesicle stage on the global methylation status in mouse oocytes subsequently matured in vitro

    Institute of Scientific and Technical Information of China (English)

    Yan Jie; Zhang Lu; Wang Tianren; Li Rong; Liu Ping; Yan Liying; Qiao Jie

    2014-01-01

    Background It is still unclear whether the vitrification procedure itself is associated with the incidence of abnormal DNA methylation during oocytes vitrification.The purpose of this study was to evaluate the epigenetic profile of mouse oocytes,which went through vitrification either at a mature stage or at an immature stage following in vitro maturation (IVM) by analyzing the global DNA methylation.Methods Metaphase Ⅱ (M Ⅱ) stage and germinal vesicle (GV) stage oocytes were collected from adult female mice and were vitrified respectively.The M Ⅱ oocytes were assessed for cryo-survival and global DNA methylation.The GV oocytes were assessed for cryo-survival and only the surviving GV oocytes were cultured in vitro for subsequent assessment of global DNA methylation in mature oocytes.In vivo matured fresh M Ⅱ oocytes without undergoing vitrification were used as control.The level of global DNA methylation in the M Ⅱ oocytes was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG under a laser scanning confocal microscope.Results In terms of the effect of vitrification on global DNA methylation status in matured oocytes,in the M Ⅱ-v group,all the examined oocytes (90/90) were found with hypermethylation,including 63.3% (57/90) of them displaying DNA methylation of a very high level,25.6% (23/90) with a high level,and 11.1% (10/90) with an intermediate level,whereas in the GV-v group,all the matured oocytes (129/129) were also examined with hypermethylation,including 67.4% (87/129) of them displaying DNA methylation of a very high level,23.3% (30/129) with a high level,and 9.3% (12/129) with an intermediate level.Statistically,it was similar between both groups,which were similar to the control:68.6% (83/121) of fresh M Ⅱ oocytes displayed DNA methylation of a very high level,21.5% (26/121) with a high level,and 9.9%(12

  20. 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...

  1. Neem (Azadirachta indica L.) leaf extract deteriorates oocyte quality by inducing ROS-mediated apoptosis in mammals.

    Science.gov (United States)

    Chaube, Shail K; Shrivastav, Tulsidas G; Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ajai K

    2014-01-01

    Neem (Azadirachta indica L.) leaf has been widely used in ayurvedic system of medicine for fertility regulation for a long time. The molecular mechanism by which neem leaf regulates female fertility remains poorly understood. Animal studies suggest that aqueous neem leaf extract (NLE) induces reactive oxygen species (ROS) - mediated granulosa cell apoptosis. Granulosa cell apoptosis deprives oocytes from nutrients, survival factors and cell cycle proteins required for the achievement of meiotic competency of follicular oocytes prior to ovulation. Under this situation, follicular oocyte becomes more susceptible towards apoptosis after ovulation. The increased level of hydrogen peroxide (H2O2) inside the follicular fluid results in the transfer of H2O2 from follicular fluid to the oocyte. The increased level of H2O2 induces p53 activation and over expression of Bax protein that modulates mitochondrial membrane potential and trigger cytochrome c release. The increased cytosolic cytochrome c level induces caspase-9 and caspase-3 activities that trigger destruction of structural and specific proteins leading to DNA fragmentation and thereby oocyte apoptosis. Based on these animal studies, we propose that NLE induces generation of ROS and mitochondria-mediated apoptosis both in granulosa cells as well as in follicular oocyte. The induction of apoptosis deteriorates oocyte quality and thereby limits reproductive outcome in mammals.

  2. Sacred activity by the Method of Excavation

    Directory of Open Access Journals (Sweden)

    Eliana Anjos Furtado

    2014-04-01

    Full Text Available Excavation is a clinical and teaching methodology created by the first author of this article. It constitutes a way of helping subjects think, make and speak. This process occurs through the xcavation of making, unblinding and/or unveiling elements in search for the understanding of sacred activity. Through xcavation, subjects understand their thoughts, values and the society in which they live in as they excavate their life stories and their relations with making, including their daily, social and work activities. We applied a thoughtful analysis to approach the role of excavation and the concept of sacred activity. Sacred activity is the core activity of every human being, which is not necessarily the activity we perform in our everyday or work life. Sacred activity is that in which subjects are inserted and experience integrally, genuinely and with no masking. Through sacred activities, subjects surrender, not only to the activity itself, but also to the other, to himself and into himself, by quest. Seeking sacred activity is, therefore, a process of questioning, excavating the routine of subjects, watching and understanding the content of this excavation, replacing and adjusting what is being unveiled, and thus making this activity sacred. In this analysis, we concluded that, by the method of excavation, the sacralization of this action should allow subjects to have tender relationships, and thus be cured.

  3. Regulatory role of kit ligand-c-kit interaction and oocyte factors in steroidogenesis by rat granulosa cells.

    Science.gov (United States)

    Miyoshi, Tomoko; Otsuka, Fumio; Nakamura, Eri; Inagaki, Kenichi; Ogura-Ochi, Kanako; Tsukamoto, Naoko; Takeda, Masaya; Makino, Hirofumi

    2012-07-06

    Although kit ligand (KL)-c-kit interaction is known to be critical for oogenesis and folliculogenesis, its role in ovarian steroidogenesis has yet to be elucidated. We studied the impact of KL-c-kit interaction in regulation of steroidogenesis using rat oocyte/granulosa cell co-culture. In the presence of oocytes, soluble KL suppressed FSH-induced estradiol production and aromatase mRNA expression without affecting FSH-induced progesterone production. The KL effect on steroidogenesis was interrupted by an anti-c-kit neutralizing antibody, suggesting that KL-c-kit interaction is involved in suppression of estrogen by granulosa cells through oocyte c-kit action. The cAMP-PKA pathway activity was not directly involved in the estrogen regulation by KL-c-kit action. It was of note that KL treatment increased the expression levels of oocyte-derived FGF-8, GDF-9 and BMP-6, while it reduced the expression levels of oocyte-derived BMP-15 in the oocyte-granulosa cell co-culture. Given the findings that FGF-8, but not GDF-9, BMP-6 or -15, suppressed FSH-induced estrogen production by granulosa cells, oocyte-derived FGF-8 is linked to suppression of FSH-induced estrogen production through the KL-c-kit interaction. Furthermore, the suppression of FSH-induced estrogen production by KL in the co-culture was reversed by a FGF receptor kinase inhibitor and the effect of the inhibitor was enhanced in combination with extracellular-domain protein of BMPRII, which interferes with BMP-15 and GDF-9 activities. Thus, the actions of endogenous oocyte factors including FGF-8 and BMP-15/GDF-9 were involved in the KL activity that inhibited FSH-induced estradiol production. Collectively, the results indicate that KL-c-kit interaction plays a role in estrogenic regulation through oocyte-granulosa cell communication.

  4. Development of Cryopreservation Techniques for Gorgonian (Junceella juncea Oocytes through Vitrification.

    Directory of Open Access Journals (Sweden)

    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.

  5. 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

  6. 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.

  7. Ultrasonographic-guided retrieval of cumulus oocyte complexes after super-stimulation in dromedary camel (Camelus dromedarius).

    Science.gov (United States)

    Wani, N A; Skidmore, J A

    2010-08-01

    In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 microg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal). Overall an average of 12.12 +/- 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28-29 h (91.2 +/- 4.1) and 26-27 h (82.1 +/- 3.4) were higher (P dromedary camels 26-28 h after GnRH administration. The developmental response, to chemical activation, of in vivo

  8. 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.

  9. 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.

  10. Identification of PDE9 as a cGMP-specific phosphodiesterase in germinal vesicle oocytes: A proposed role in the resumption of meiosis

    Science.gov (United States)

    Hanna, Carol B.; Yao, Shan; Wu, Xuemei; Jensen, Jeffrey T.

    2012-01-01

    Objective To identify a cGMP-specific phosphodiesterase (PDE) in non-human primate germinal vesicle (GV) oocytes and establish a proposed effect on oocyte maturation through preliminary experiments in mouse GV oocytes. Design Controlled non-human primate and rodent experiments. Setting Academic research institution. Animals Rhesus macaques and B6/129F1 mice. Interventions Rhesus macaques were stimulated with FSH to collect GV oocytes and cumulus for gene expression analysis. Female mice were stimulated with PMSG to collect GV oocytes. Main Outcome Measures PDE transcript expression in primate GV oocytes and cumulus cells. Fluorescence polarization (FP) measurements of PDE3A activity. Spontaneous resumption of meiosis in mouse GV oocytes. Results Five PDE transcripts were detected in Rhesus GV oocytes, only PDE9A was cGMP-specific. FP assays indicated cGMP has an inhibitory effect on PDE3A while the PDE9 inhibitor, BAY73-6691, did not. Similarly, BAY73-6691, had little effect on preventing spontaneous maturation in oocytes, but did augment the inhibitory effects of cGMP. Inclusion of 0µM (control), 10µM, 100µM, and 1 mM BAY73-6691 significantly increased the proportion of mouse oocytes maintaining GV arrest in the presence of the cGMP analog 8-Br-cGMP at: 100µM (8.8%, 11.4%, 18.8%, and 28%), 500µM (21.1%, 38.1%, 74.5%,and 66.5%), and 1 mM (57.8%, 74.5%, 93.9%, and 94.0%) respectively, when P<0.05. Conclusions PDE9 is a cGMP-specific hydrolyzing enzyme present in primate oocytes, and PDE9 antagonists augment the inhibitory effect of cGMP during spontaneous in vitro maturation of GV mouse oocytes. PMID:22704629

  11. Prophase I Mouse Oocytes Are Deficient in the Ability to Respond to Fertilization by Decreasing Membrane Receptivity to Sperm and Establishing a Membrane Block to Polyspermy1

    Science.gov (United States)

    Kryzak, Cassie A.; Moraine, Maia M.; Kyle, Diane D.; Lee, Hyo J.; Cubeñas-Potts, Caelin; Robinson, Douglas N.; Evans, Janice P.

    2013-01-01

    ABSTRACT Changes occurring as the prophase I oocyte matures to metaphase II are critical for the acquisition of competence for normal egg activation and early embryogenesis. A prophase I oocyte cannot respond to a fertilizing sperm as a metaphase II egg does, including the ability to prevent polyspermic fertilization. Studies here demonstrate that the competence for the membrane block to polyspermy is deficient in prophase I mouse oocytes. In vitro fertilization experiments using identical insemination conditions result in monospermy in 87% of zona pellucida (ZP)-free metaphase II eggs, while 92% of ZP-free prophase I oocytes have four or more fused sperm. The membrane block is associated with a postfertilization reduction in the capacity to support sperm binding, but this reduction in sperm-binding capacity is both less robust and slower to develop in fertilized prophase I oocytes. Fertilization of oocytes is dependent on the tetraspanin CD9, but little to no release of CD9 from the oocyte membrane is detected, suggesting that release of CD9-containing vesicles is not essential for fertilization. The deficiency in membrane block establishment in prophase I oocytes correlates with abnormalities in two postfertilization cytoskeletal changes: sperm-induced cortical remodeling that results in fertilization cone formation and a postfertilization increase in effective cortical tension. These data indicate that cortical maturation is a component of cytoplasmic maturation during the oocyte-to-egg transition and that the egg cortex has to be appropriately primed and tuned to be responsive to a fertilizing sperm. PMID:23863404

  12. Attempt at intracytoplasmic sperm injection of in vitro matured oocytes in common minke whales (Balaenoptera acutorostrata) captured during the Kushiro Coast Survey.

    Science.gov (United States)

    Fukui, Yutaka; Iwayama, Hiroshi; Matsuoka, Taiki; Nagai, Hiroki; Koma, Noriko; Mogoe, Toshihiro; Ishikawa, Hajime; Fujise, Yoshihiro; Hirabayashi, Masumi; Hochi, Shinichi; Kato, Hidehiro; Ohsumi, Seiji

    2007-08-01

    The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; Pwhales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prepubertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.

  13. The nuclear mitotic apparatus (NuMA) protein: localization and dynamics in human oocytes, fertilization and early embryos.

    Science.gov (United States)

    Alvarez Sedó, Cristian; Schatten, Heide; Combelles, Catherine M; Rawe, Vanesa Y

    2011-06-01

    The oocyte's meiotic spindle is a dynamic structure that relies on microtubule organization and regulation by centrosomes. Disorganization of centrosomal proteins, including the nuclear mitotic apparatus (NuMA) protein and the molecular motor complex dynein/dynactin, can lead to chromosomal instability and developmental abnormalities. The present study reports the distribution and function of these proteins in human oocytes, zygotes and early embryos. A total of 239 oocytes, 90 zygotes and discarded embryos were fixed and analyzed with confocal microscopy for NuMA and dynactin distribution together with microtubules and chromatin. Microtubule-associated dynein-dependent transport functions were explored by inhibiting phosphatase and ATPase activity with sodium-orthovanadate (SOV). At germinal vesicle (GV) stages, NuMA was dispersed across the nucleoplasm. After GV breaks down, NuMA became cytoplasmic before localizing at the spindle poles in metaphase I and II oocytes. Aberrant NuMA localization patterns were found during oocyte in vitro maturation. After fertilization, normal and abnormal pronuclear stage zygotes and embryos displayed translocation of NuMA to interphase nuclei. SOV treatment for up to 2 h induced lower maturation rates with chromosomal scattering and ectopic localization of NuMA. Accurate distribution of NuMA is important for oocyte maturation, zygote and embryo development in humans. Proper assembly of NuMA is likely necessary for bipolar spindle organization and human oocyte developmental competence.

  14. Methods for using polypeptides having cellobiohydrolase activity

    Energy Technology Data Exchange (ETDEWEB)

    Morant, Marc D; Harris, Paul

    2016-08-23

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. 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.

  16. 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.

  17. High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification

    DEFF Research Database (Denmark)

    Du, Y; Pribenszky, C S; Molnár, M

    2008-01-01

    The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcine in vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of 1668 porcine IVM oocytes were used in our...

  18. 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

  19. 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.

  20. 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.

  1. Review: Lamin A/C, caspase-6, and chromatin configuration during meiosis resumption in the mouse oocyte.

    Science.gov (United States)

    Arnault, Emilie; Doussau, Mireille; Pesty, Arlette; Lefèvre, Brigitte; Courtot, Anne-Marie

    2010-02-01

    After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific caspase-6 inhibitor to determine the presence of lamin A/C and caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of caspase-6 in the oocyte apoptotic pathway. Lamin A/C and caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.

  2. Aurora kinase A is not involved in CPEB1 phosphorylation and cyclin B1 mRNA polyadenylation during meiotic maturation of porcine oocytes.

    Science.gov (United States)

    Komrskova, Pavla; Susor, Andrej; Malik, Radek; Prochazkova, Barbora; Liskova, Lucie; Supolikova, Jaroslava; Hladky, Stepan; Kubelka, Michal

    2014-01-01

    Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.

  3. Aurora kinase A is not involved in CPEB1 phosphorylation and cyclin B1 mRNA polyadenylation during meiotic maturation of porcine oocytes.

    Directory of Open Access Journals (Sweden)

    Pavla Komrskova

    Full Text Available Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1. The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.

  4. 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.

  5. 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

  6. 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.

  7. Evaluation of methods to assess physical activity

    Science.gov (United States)

    Leenders, Nicole Y. J. M.

    Epidemiological evidence has accumulated that demonstrates that the amount of physical activity-related energy expenditure during a week reduces the incidence of cardiovascular disease, diabetes, obesity, and all-cause mortality. To further understand the amount of daily physical activity and related energy expenditure that are necessary to maintain or improve the functional health status and quality of life, instruments that estimate total (TDEE) and physical activity-related energy expenditure (PAEE) under free-living conditions should be determined to be valid and reliable. Without evaluation of the various methods that estimate TDEE and PAEE with the doubly labeled water (DLW) method in females there will be eventual significant limitations on assessing the efficacy of physical activity interventions on health status in this population. A triaxial accelerometer (Tritrac-R3D, (TT)), an uniaxial (Computer Science and Applications Inc., (CSA)) activity monitor, a Yamax-Digiwalker-500sp°ler , (YX-stepcounter), by measuring heart rate responses (HR method) and a 7-d Physical Activity Recall questionnaire (7-d PAR) were compared with the "criterion method" of DLW during a 7-d period in female adults. The DLW-TDEE was underestimated on average 9, 11 and 15% using 7-d PAR, HR method and TT. The underestimation of DLW-PAEE by 7-d PAR was 21% compared to 47% and 67% for TT and YX-stepcounter. Approximately 56% of the variance in DLW-PAEE*kgsp{-1} is explained by the registration of body movement with accelerometry. A larger proportion of the variance in DLW-PAEE*kgsp{-1} was explained by jointly incorporating information from the vertical and horizontal movement measured with the CSA and Tritrac-R3D (rsp2 = 0.87). Although only a small amount of variance in DLW-PAEE*kgsp{-1} is explained by the number of steps taken per day, because of its low cost and ease of use, the Yamax-stepcounter is useful in studies promoting daily walking. Thus, studies involving the

  8. Optimization Methods for Supply Chain Activities

    Directory of Open Access Journals (Sweden)

    Balasescu S.

    2014-12-01

    Full Text Available This paper approach the theme of supply chain activities for medium and large companies which run many operations and need many facilities. The first goal is to analyse the influence of optimisation methods of supply chain activities on the success rate for a business. The second goal is to compare some logistic strategies applied by companies with the same profile to see which is the most effective. The final goal is to show which is the necessity of strategic optimum for a company and how can be achieved the considering the demand uncertainty.

  9. 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.

  10. Effects of Vitrification on Outcomes of In VivoMature, In Vitro-Mature and Immature Human Oocytes

    Directory of Open Access Journals (Sweden)

    Wen-yan Song

    2016-05-01

    Full Text Available Background/Aims: To observe the effects of vitrification on spindle, zona pellucida, embryonic aneuploidy and DNA injury in in vivo-maruted, in vitro-mature and immature human oocytes. Methods: Between January 2009 and February 2015, 223 immature oocytes from 450 infertile patients, and 31 in vivo-mature oocytes from 3 infertile couples were collected. Of the 223 immature oocytes, 113 were used for in vitro culture before vitrification. Some oocytes were randomly divided into in vivo-mature group (group A, n = 15, in vitro-mature group (group B, n = 88 and immature group (group C, n = 85, and then the oocytes with spindle in these three groups after freezing-thawing were selected to use for Polscope imaging, embryonic aneuploidy screening and embryo development evaluation. Other oocytes were randomly divided into group A (n = 16, group B (n = 25 and group C (n = 25 for detecting DNA injury. Results: After thawing, spindle occurrence rate, spindle Retardance value, and cleavage rate were significantly higher in groups A and B than in group C (all P P > 0.05. Zona pellucida density (ZPD was significantly lower in group A than in groups B and C both before and after vitrification (all P P P > 0.05. Rate of comet cells was significantly lower in group A than in groups B and C (all P P Conclusion: In vivo- and in vitro-mature human oocytes are more suitable to vitrification than immature human oocytes. Spindle Retardance value has more predictive value for embryonic development potential than ZPD and ZPT.

  11. A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish

    Science.gov (United States)

    Xie, Shao-Lin; Bian, Wan-Ping; Wang, Chao; Junaid, Muhammad; Zou, Ji-Xing; Pei, De-Sheng

    2016-01-01

    Contemporary improvements in the type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system offer a convenient way for genome editing in zebrafish. However, the low efficiencies of genome editing and germline transmission require a time-intensive and laborious screening work. Here, we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization (IVF) in zebrafish. Compared to conventional methods, the prior micro-injection of zebrafish oocytes improved the efficiency of genome editing, especially for the sgRNAs with low targeting efficiency. Due to high throughputs, simplicity and flexible design, this novel strategy will provide an efficient alternative to increase the speed of generating heritable mutants in zebrafish by using CRISPR/Cas9 system. PMID:27680290

  12. Vitrification of in vitro matured oocytes collected from surplus ovarian medulla tissue resulting from fertility preservation of ovarian cortex tissue

    DEFF Research Database (Denmark)

    Yin, Huiqun; Jiang, Hong; Kristensen, Stine Gry

    2016-01-01

    PURPOSE: The aim of the study was to investigate the maturation rate of immature oocytes collected from ovarian medulla tissue normally discarded during preparation of ovarian cortical tissue for fertility preservation. Further we evaluated survival of derived MII oocytes following vitrification...... and warming. METHODS: 36 patients aged from 8 to 41 years who had one ovary excised for fertility preservation were included. Oocytes were collected from the medulla tissue and matured in vitro 44-48 h followed by vitrification. Number of oocytes collected, the rates of maturation and post-warming survival...... of cortical tissue may pose a risk of relapse, but the IVM approach is currently too inefficient to be the only method used for fertility preservation....

  13. Experimental Study on Meiotic Spindles and Chromosomes of Mouse Mature (MⅡ) Stage Oocytes under Laser Scanning Confocal Microscopy

    Institute of Scientific and Technical Information of China (English)

    WANG Yanlin; ZHU Guijin; LI Xia; LI Xiaolan

    2006-01-01

    Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope (LSCM) was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional (3D) image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM.The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of M Ⅱ oocytes.

  14. Analysis of Precision of Activation Analysis Method

    DEFF Research Database (Denmark)

    Heydorn, Kaj; Nørgaard, K.

    1973-01-01

    The precision of an activation-analysis method prescribes the estimation of the precision of a single analytical result. The adequacy of these estimates to account for the observed variation between duplicate results from the analysis of different samples and materials, is tested by the statistic T......, which is shown to be approximated by a χ2 distribution. Application of this test to the results of determinations of manganese in human serum by a method of established precision, led to the detection of airborne pollution of the serum during the sampling process. The subsequent improvement in sampling...... conditions was shown to give not only increased precision, but also improved accuracy of the results....

  15. [Bioluminescent method of determining antiprotease activity].

    Science.gov (United States)

    Gitel'zon, I I; Rykov, S A; Kratasiuk, G A; Petushkov, V N; Shvetskiĭ, A G

    1985-11-01

    A method for antiprotease activity measurement based on the use of luminous bacteria luciferase as protein substrate of proteases is suggested. Antiprotease is incubated with protease for 1 to 2 min at 30 degrees C and then it is added to the reaction mixture containing luciferase, NADH: FMN-oxidoreductase and their substrates--myristic aldehyde, FMN and NADH. Biofluorescence is measured in a temperature-controlled cuvette for 1 min. The total time of the measurement is 3 min. The method can be applied both in fine biochemical assays and in medical rapid diagnosis.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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....

  6. Method to produce catalytically active nanocomposite coatings

    Energy Technology Data Exchange (ETDEWEB)

    Erdemir, Ali; Eryilmaz, Osman Levent; Urgen, Mustafa; Kazmanli, Kursat

    2016-02-09

    A nanocomposite coating and method of making and using the coating. The nanocomposite coating is disposed on a base material, such as a metal or ceramic; and the nanocomposite consists essentially of a matrix of an alloy selected from the group of Cu, Ni, Pd, Pt and Re which are catalytically active for cracking of carbon bonds in oils and greases and a grain structure selected from the group of borides, carbides and nitrides.

  7. 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.

  8. [Cajal bodies in insect oocytes. II. New data on the molecular composition of cajal bodies in oocytes of the house cricket Acheta domesticus with special reference to interactions between cajal bodies and interchromatin granule clusters].

    Science.gov (United States)

    Stepanova, I S; Bogoliubov, D S; Parfenov, V N

    2007-01-01

    Cajal bodies (CBs) in oocytes of the house cricket Acheta domesticus are large, perfectly spherical nuclear organelles with a complex internal structure. These consist of a fibrillar coilin-containing matrix and a central cavity with a prominent fibrogranular body inside; the latter has been referred to as an "internal" interchromatin granule cluster (IGC). Within the matrix of CBs we detected transcriptional co-activators CBP/p300 and TATA-box binding protein (TBP). No RNA polymerase II was revealed in CBs of both normal and actynomycin D treated oocytes. In the nucleoplasm of A. domesticus oocytes, besides CBs, free IGCs were observed. In oocytes treated with actynomycin D, the amount of "free" IGCs in the nucleoplasm increase significantly, granular and fibrillar components of IGCs were seen segregated, and RNA polymerase II and CBP/p300 were found to be accumulated in fibrillar zones of IGCs.

  9. Development of rat oocytes following intracytoplasmic injection of sperm heads isolated from testicular and epididymal spermatozoa.

    Science.gov (United States)

    Said, S; Han, M-S; Niwa, K

    2003-07-01

    The possibility of obtaining normal development of rat oocytes following intracytoplasmic injection of rat sperm heads, obtained by sonicating spermatozoa from testes and epididymides, was evaluated. Irrespective of the source of spermatozoa, sperm heads were successfully injected into approximately 45% of oocytes used; after 9-12h of culture, approximately 55% of injected oocytes still had normal morphology. Of the oocytes injected with testicular sperm heads 45% were activated, with a female pronucleus and a second polar body, but significantly more oocytes (approximately 68%) injected with caput and cauda epididymal sperm heads were activated. Male pronuclear formation was observed in 67-84% of the activated oocytes, with no difference in the proportions among the different sources of sperm heads. When zygotes showing two pronuclei and a second polar body at 10h after injection were cultured in conditions that support development of 1-cell embryos produced in vivo, no embryos derived from testicular sperm heads developed to blastocysts after 120 h of culture. Development of embryos derived from cauda sperm heads was significantly higher at all points of assessment, while embryos from caput sperm showed an intermediate degree of development, compared with embryos from testicular spermatozoa. However, similar proportions (2-4%) of 1-cell embryos derived from all three groups of sperm heads developed into normal offspring after transfer to foster mothers; of the limited number of offspring tested, all were fertile. These results demonstrate that sperm heads from all sources tested are similar in their ability to contribute to full development of normal, fertile offspring.

  10. 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.

  11. Nitric oxide acts through different signaling pathways in maturation of cumulus cell-enclosed mouse oocytes

    Directory of Open Access Journals (Sweden)

    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

  12. 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.

  13. 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...

  14. [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.

  15. 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.

  16. Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte.

    Science.gov (United States)

    Dahlgaard, Katja; Raposo, Alexandre A S F; Niccoli, Teresa; St Johnston, Daniel

    2007-10-01

    Mutants in the actin nucleators Cappuccino and Spire disrupt the polarized microtubule network in the Drosophila oocyte that defines the anterior-posterior axis, suggesting that microtubule organization depends on actin. Here, we show that Cappuccino and Spire organize an isotropic mesh of actin filaments in the oocyte cytoplasm. capu and spire mutants lack this mesh, whereas overexpressed truncated Cappuccino stabilizes the mesh in the presence of Latrunculin A and partially rescues spire mutants. Spire overexpression cannot rescue capu mutants, but prevents actin mesh disassembly at stage 10B and blocks late cytoplasmic streaming. We also show that the actin mesh regulates microtubules indirectly, by inhibiting kinesin-dependent cytoplasmic flows. Thus, the Capu pathway controls alternative states of the oocyte cytoplasm: when active, it assembles an actin mesh that suppresses kinesin motility to maintain a polarized microtubule cytoskeleton. When inactive, unrestrained kinesin movement generates flows that wash microtubules to the cortex.

  17. 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

  18. Effects of MEK inhibitor U0126 on meiotic progression in mouse oocytes:microtuble organization, asymmetric division and metaphase Ⅱ arrest

    Institute of Scientific and Technical Information of China (English)

    JIAN WEN DONG; HAl FENG ZHU; WEI ZHONG ZHU; HAI LEI DING; TIE MIN MA; ZHAO NIAN ZHOU

    2003-01-01

    In this study we used U0126, a potent and specific inhibitor of MEK, to study the roles of MEK/ERK/p90rsk signaling pathway in the meiotic cell cycle of mouse oocytes. The phosphorylation of MAP kinase and p90rsk in the oocytes treated with 1.5 μMU0126 was the same as that in oocytes cultured in drug-free medium. With 1.5 μM U0126 treatment, the spindles appeared normal as they formed in oocytes, but failed to maintain its structure.Instead, the spindle lost one pole or elongated extraordinarily. After further culture, some oocytes extruded gigantic polar bodies (>30 μm) that later divided into two small ones. Some oocytes underwent symmetric division and produced two equal-size daughter cells in which normal spindles formed. In oocytes with different division patterns,MAP kinase was normally phosphorylated. When the concentration of U0126 was increased to 15 mM, the phosphorylation of both MAPK and p90rsk were inhibited, while symmetric division was decreased. When incubating in medium containing 15 μM U0126 for 14 h, oocytes were activated, but part of them failed to emit polar bodies. MII oocytes were also activated by 15 μM U0126, at the same time the dephosphorylation of MAP kinase and p90rsk was observed. Our results indicate that 1) MEK plays important but not indispensable roles in microtubule organization;2) MEK keeps normal meiotic spindle morphology, targets peripheral spindle positioning and regulates asymmetric division by activating some unknown substrates other than MAP kinase/p90rsk; and 3) activation of MEK/ERK/p90rsk cascade maintains MII arrest in mouse oocytes.

  19. Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

    Directory of Open Access Journals (Sweden)

    Halkier Barbara A

    2006-10-01

    Full Text Available Abstract Background We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector. Results Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized. Conclusion Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.

  20. Elevated NaCl concentration improves cryotolerance and developmental competence of porcine oocytes

    DEFF Research Database (Denmark)

    Lin, L; Du, Y; Liu, Y

    2009-01-01

    High hydrostatic pressure has been reported to improve the fertilizing or developmental ability of mammalian spermatozoa, oocytes and embryos. This study investigated the effect of another stress, temporarily increased NaCl concentration, on cryotolerance and developmental competence of porcine...... in untreated controls, but blastocyst rates increased in both NaCl-treated groups. In conclusion, treatment of porcine oocytes with elevated NaCl concentrations improved their developmental competence after vitrification and parthenogenetic activation or SCNT. Further experiments are required to investigate in...

  1. Expression of the CTCF gene in bovine oocytes and preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Álvaro F.L. Rios

    2007-01-01

    Full Text Available The CCCTC - binding factor (CTCF is a protein involved in repression, activation, hormone-inducible gene silencing, functional reading of imprinted genes and X-chromosome inactivation. We analyzed CTCF gene expression in bovine peripheral blood, oocytes and in different cellular stages (2-4 cells, 8-16 cells, 16-32 cells, morulae, and blastocysts of in vitro fertilized embryos. This is the first report of CTCF expression in oocytes and preimplantation bovine embryos and has implications for the production of embryonic stem cells and the development of novel medical technologies for humans.

  2. 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.

  3. A new method for FMRI activation detection

    Science.gov (United States)

    Wei, Jianing; Talavage, Thomas M.; Pollak, Ilya

    2009-02-01

    The objective of fMRI data analysis is to detect the region of the brain that gets activated in response to a specific stimulus presented to the subject. We develop a new algorithm for activation detection in event-related fMRI data. We utilize a forward model for fMRI data acquisition which explicitly incorporates physiological noise, scanner noise and the spatial blurring introduced by the scanner. After slice-by-slice image restoration procedure that independently restores each data slice corresponding to each time index, we estimate the parameters of the hemodynamic response function (HRF) model for each pixel of the restored data. In order to enforce spatial regularity in our estimates, we model the prior distribution of the HRF parameters as a generalized Gaussian Markov random field (GGMRF) model. We develop an algorithm to compute the maximum a posteriori (MAP) estimates of the parameters. We then threshold the amplitude parameters to obtain the final activation map. We illustrate our algorithm by comparing it with the widely used general linear model (GLM) method. In synthetic data experiments, under the same probability of false alarm, the probability of correct detection for our method is up to 15% higher than GLM. In real data experiments, through anatomical analysis and benchmark testing using block paradigm results, we demonstrate that our algorithm produces fewer false alarms than GLM.

  4. Children born after cryopreservation of embryos or oocytes: a systematic review of outcome data

    DEFF Research Database (Denmark)

    Wennerholm, U-B; Söderström-Anttila, V; Bergh, C;

    2009-01-01

    embryos, blastocysts and oocytes. METHODS: A systematic review was performed. We searched the PubMed, Cochrane and Embase databases from 1984 to September 2008. Inclusion criteria for slow freezing of early cleavage stage embryos were controlled studies reporting perinatal or child outcomes. For slow...... freezing and vitrification of blastocysts and oocytes, and vitrification of early cleavage stage embryos, case reports on perinatal or child outcomes were also included. Three reviewers independently read and evaluated all selected studies. RESULTS: For early cleavage embryos, data from controlled studies...... of blastocysts and for vitrification of early cleavage stage embryos, blastocysts and oocytes, limited neonatal data was reported. We found no long-term child follow-up data for any cryopreservation technique. CONCLUSION: Data concerning infant outcome after slow freezing of embryos was reassuring. Properly...

  5. BMP-15 m-RNA expression of mouse oocytes in vitro maturation in different droplet medium volume

    Institute of Scientific and Technical Information of China (English)

    Sri Rahayu; Nashi Widodo; Yumi Hoshino; Eimei Sato

    2015-01-01

    Objective:To investigate droplet medium volume effect on the BMP-15 mRNA expression. Methods:Oocytes are collected from mice ovaries by puncturing with a sterile 26-G needle. The droplet medium volumes are using 50 µL, 100 µL and 200 µL. The BMP-15 mRNA expression is determined in each group.Results:The results indicated that BMP-15 mRNA expression did not significantly differ when oocyte were cultured in 50 and 100 µL/droplet medium volume, but significant difference (P < 0.05) was found when oocytes were cultured in 200 µL/droplet medium volume.Conclusions:The highest BMP-15 m-RNA expression occur when oocytes are cultured in 200 µL/droplet medium volume.

  6. Active and passive cooling methods for dwellings

    DEFF Research Database (Denmark)

    Oropeza-Perez, Ivan; Østergaard, Poul Alberg

    2018-01-01

    they might be applied, and what their energy needs are. Secondly, what scientific analysis has been carried out and what the major findings are. Thirdly, what their economic and technical feasibility of use at the stage of implementation and operation within a dwelling are. Then, either with the reviewed......In this document a review of three active as well as ten passive cooling methods suitable for residential buildings is carried out. The review firstly addresses how the various technologies cool the space according to the terms of the building heat balance, under what technical conditions...... studies or with a simulation modelling, an assessment of temperature drop of each cooling method within a standard-size dwelling is carried out. Also, a comparison of initial investment, energy consumption, maintenance, retrofitting and required space is done. Thereafter, with this information, a decision...

  7. Inhibitory effects of coronary vasodilator papaverine on heterologously-expressed HERG currents in Xenopus oocytes

    Institute of Scientific and Technical Information of China (English)

    Cuk-seong KIM; Jin-bong PARK; Nam LEE; Sook-jin SON; Kyu-seung LEE; Hyo-shin KIM; Yong-geun KWAK; Soo-wan CHAE; Sang-do LEE; Byeong-hwa JEON

    2007-01-01

    Aim: To characterize the effects of papaverine on HERG channels expressed in Xenopus oocytes as well as cardiac action potential in rabbit ventricular myocytes.Methods: Conventional microelectrodes were used to record action potential in rabbit ventricular myocytes. HERG currents were recorded by 2-electrode voltage clamp technique in Xenopus oocytes injected with HERG cRNA. Results: Papa-verine increased the cardiac action potential duration in rabbit ventricular myocytes.It blocked heterologously-expressed HERG currents in a concentration-depen-dent manner (IC50 71.03±4.75 μmol/L, NH 0.80, n=6), whereas another phosphodi-esterase inhibitor, theophyUine (500 μmol/L), did not. The blockade of papaverine on HERG currents was not voltage-dependent. The slope conductance measured as a slope of the fully activated HERG current-voltage curves decreased from 78.03±4.25 μS of the control to 56.84±5.33, 36.06±6.53, and 27.09±5.50 μS (n=4) by 30, 100, and 300 μmol/L of papaverine, respectively. Papaverine (100 μmol/L)caused a 9 mV hyperpolarizing shift in the voltage-dependence of steady-state inactivation, but there were no changes in the voltage-dependence of HERG cur-rent activation. Papaverine blocked HERG channels in the closed, open, and inactivated states. Conclusion: These results showed that papaverine blocked HERG channels in a voltage- and state-independent manner, which may most likely be the major mechanism of papaverine-induced cardiac arrhythmia reported in humans.

  8. Functional analysis of human Na~+/K~+-ATPase familial or sporadic hemiplegic migraine mutations expressed in Xenopus oocytes

    Institute of Scientific and Technical Information of China (English)

    Susan; Spiller; Thomas; Friedrich

    2014-01-01

    AIM: Functional characterization of ATP1A2 mutations that are related to familial or sporadic hemiplegic migraine(FHM2, SHM). METHODS: cRNA of human Na+/K+-ATPase α2- and β1-subunits were injected in Xenopus laevis oocytes. FHM2 or SHM mutations of residues located in putative α/β interaction sites or in the α2-subunit’s C-terminal region were investigated. Mutants were analyzed by the twoelectrode voltage-clamp(TEVC) technique on Xenopus oocytes. Stationary K+-induced Na+/K+ pump currents were measured, and the voltage dependence of apparent K+ affinity was investigated. Transient currents were recorded as ouabain-sensitive currents in Na+ buffers to analyze kinetics and voltage-dependent presteady state charge translocations. The expression of constructs was verified by preparation of plasma membrane and total membrane fractions of cRNA-injected oocytes. RESULTS: Compared to the wild-type enzyme, the mutants G900R and E902K showed no significant dif-ferences in the voltage dependence of K+-induced currents, and analysis of the transient currents indicated that the extracellular Na+ affinity was not affected. Mutant G855R showed no pump activity detectable by TEVC. Also for L994del and Y1009X, pump currents could not be recorded. Analysis of the plasma and total membrane fractions showed that the expressed proteins were not or only minimally targeted to the plasma membrane. Whereas the mutation K1003E had no impact on K+ interaction, D999H affected the voltage dependence of K+-induced currents. Furthermore, kinetics of the transient currents was altered compared to the wild-type enzyme, and the apparent affinity for extracellular Na+ was reduced. CONCLUSION: The investigated FHM2/SHM mutations influence protein function differently depending on the structural impact of the mutated residue.

  9. 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

  10. Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes.

    Science.gov (United States)

    Gui, Liming; Homer, Hayden

    2012-06-01

    The spindle assembly checkpoint (SAC) averts aneuploidy by coordinating proper bipolar chromosomal attachment with anaphase-promoting complex/cyclosome (APC/C)-mediated securin and cyclin B1 destruction required for anaphase onset. The generation of a Mad2-based signal at kinetochores is central to current models of SAC-based APC/C inhibition. During mitosis, kinetochores of polar-displaced chromosomes, which are at greatest risk of mis-segregating, recruit the highest levels of Mad2, thereby ensuring that SAC activation is proportionate to aneuploidy risk. Paradoxically, although an SAC operates in mammalian oocytes, meiosis I (MI) is notoriously error prone and polar-displaced chromosomes do not prevent anaphase onset. Here we find that Mad2 is not preferentially recruited to the kinetochores of polar chromosomes of wild-type mouse oocytes, in which polar chromosomes are rare, or of oocytes depleted of the kinesin-7 motor CENP-E, in which polar chromosomes are more abundant. Furthermore, in CENP-E-depleted oocytes, although polar chromosomal displacement intensified during MI and the capacity to form stable end-on attachments was severely compromised, all kinetochores nevertheless became devoid of Mad2. Thus, it is possible that the ability of the SAC to robustly discriminate chromosomal position might be compromised by the propensity of oocyte kinetochores to become saturated with unproductive attachments, thereby predisposing to aneuploidy. Our data also reveal novel functions for CENP-E in oocytes: first, CENP-E stabilises BubR1, thereby impacting MI progression; and second, CENP-E mediates bi-orientation by promoting kinetochore reorientation and preventing chromosomal drift towards the poles.

  11. Rejuvenation of meiotic cohesion in oocytes during prophase I is required for chiasma maintenance and accurate chromosome segregation.

    Science.gov (United States)

    Weng, Katherine A; Jeffreys, Charlotte A; Bickel, Sharon E

    2014-09-01

    Chromosome segregation errors in human oocytes are the leading cause of birth defects, and the risk of aneuploid pregnancy increases dramatically as women age. Accurate segregation demands that sister chromatid cohesion remain intact for decades in human oocytes, and gradual loss of the original cohesive linkages established in fetal oocytes is proposed to be a major cause of age-dependent segregation errors. Here we demonstrate that maintenance of meiotic cohesion in Drosophila oocytes during prophase I requires an active rejuvenation program, and provide mechanistic insight into the molecular events that underlie rejuvenation. Gal4/UAS inducible knockdown of the cohesion establishment factor Eco after meiotic S phase, but before oocyte maturation, causes premature loss of meiotic cohesion, resulting in destabilization of chiasmata and subsequent missegregation of recombinant homologs. Reduction of individual cohesin subunits or the cohesin loader Nipped B during prophase I leads to similar defects. These data indicate that loading of newly synthesized replacement cohesin rings by Nipped B and establishment of new cohesive linkages by the acetyltransferase Eco must occur during prophase I to maintain cohesion in oocytes. Moreover, we show that rejuvenation of meiotic cohesion does not depend on the programmed induction of meiotic double strand breaks that occurs during early prophase I, and is therefore mechanistically distinct from the DNA damage cohesion re-establishment pathway identified in G2 vegetative yeast cells. Our work provides the first evidence that new cohesive linkages are established in Drosophila oocytes after meiotic S phase, and that these are required for accurate chromosome segregation. If such a pathway also operates in human oocytes, meiotic cohesion defects may become pronounced in a woman's thirties, not because the original cohesive linkages finally give out, but because the rejuvenation program can no longer supply new cohesive linkages

  12. Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development.

    Science.gov (United States)

    Nogueira, D; Ron-El, R; Friedler, S; Schachter, M; Raziel, A; Cortvrindt, R; Smitz, J

    2006-01-01

    Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6-12 mm) were retrieved 34-36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

  13. Methods of marketing and advertising activity evaluation

    Directory of Open Access Journals (Sweden)

    A.I. Yakovlev

    2016-09-01

    Full Text Available The result of the business entities’ activities is associated with the development of instruments of the economic processes efficiency determination, including marketing activities. It has determined the purpose of the article. The methodological principles in this area are developed. It is proved that the increase in sales of the profit margin is only partly dependent on the implementation of advertising measures. The methodical approaches for estimation of exhibition and advertising activity and promotion of its employees are specified. The results of work involve evaluation of the advertising effect value on the basis of share of the advertising impact on the increase of sales and revenue from the sale of products. The corresponding proportion of such impact is determined based on the consumer inquiry. The index of trade fair works, its calculation based on two components: how many times a specific company participated in such events; and how well the company was presented at relevant trade fairs. The indices of the cost on advertising and promotion of certain products manufacturer are provided. The scientific innovation of the research is as follows. It is proved that the sales increase effect should not be assigned to advertising only. The compositions that influence the consumer preferences and their share in the total value effect are determined. The new is the proposed index of influence of the trade fair work results depending on the selected factors. The practical importance of the research results involve more accurate calculation of the effect of the activities made and, consequently, increase efficiency of the business entities.

  14. 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.

  15. Anomalias meióticas de oócitos de pacientes com endometriose submetidas à estimulação ovariana Meiotic abnormalities of oocytes from patients with endometriosis submitted to ovarian stimulation

    Directory of Open Access Journals (Sweden)

    Ionara Diniz Evangelista Santos Barcelos

    2008-08-01

    Full Text Available OBJETIVO: avaliar o fuso meiótico e a distribuição cromossômica de oócitos maturados in vitro, obtidos de ciclos estimulados de mulheres inférteis com endometriose e fatores masculino e/ou tubário de infertilidade (Grupo Controle, comparando as taxas de maturação in vitro (MIV entre os dois grupos avaliados. MÉTODOS: quatorze pacientes com endometriose e oito com fator tubário ou masculino, submetidas à estimulação ovariana para injeção intracitoplasmática de espermatozóide, foram selecionadas, prospectiva e consecutivamente, e constituíram os Grupos de Estudo e Controle, respectivamente. Oócitos imaturos (46 e 22, respectivamente, dos Grupos Endometriose e Controle foram submetidos à MIV. Oócitos que apresentaram a extrusão do primeiro corpúsculo polar foram fixados e corados para avaliação dos microtúbulos e cromatina por técnica de imunofluorescência. A análise estatística foi realizada utilizando o teste exato de Fisher, com significância estatística quando pPURPOSE: to evaluate the meiotic spindle and the chromosome distribution of in vitro mature oocytes from stimulated cycles of infertile women with endometriosis, and with male and/or tubal infertility factors (Control Group, comparing the rates of in vitro maturation (IVM between the two groups evaluated. METHODS: fourteen patients with endometriosis and eight with male and/or tubal infertility factors, submitted to ovarian stimulation for intracytoplasmatic sperm injection have been prospectively and consecutively selected, and formed a Study and Control Group, respectively. Immature oocytes (46 and 22, respectively, from the Endometriosis and Control Groups were submitted to IVM. Oocytes presenting extrusion of the first polar corpuscle were fixed and stained for microtubules and chromatin evaluation through immunofluorescence technique. Statistical analysis has been done by the Fisher's exact test, with statistical significance at p<0.05. RESULTS

  16. LSD1 is essential for oocyte meiotic progression by regulating CDC25B expression in mice.

    Science.gov (United States)

    Kim, Jeesun; Singh, Anup Kumar; Takata, Yoko; Lin, Kevin; Shen, Jianjun; Lu, Yue; Kerenyi, Marc A; Orkin, Stuart H; Chen, Taiping

    2015-12-02

    Mammalian oocytes are arrested at prophase I until puberty when hormonal signals induce the resumption of meiosis I and progression to meiosis II. Meiotic progression is controlled by CDK1 activity and is accompanied by dynamic epigenetic changes. Although the signalling pathways regulating CDK1 activity are well defined, the functional significance of epigenetic changes remains largely unknown. Here we show that LSD1, a lysine demethylase, regulates histone H3 lysine 4 di-methylation (H3K4me2) in mouse oocytes and is essential for meiotic progression. Conditional deletion of Lsd1 in growing oocytes results in precocious resumption of meiosis and spindle and chromosomal abnormalities. Consequently, most Lsd1-null oocytes fail to complete meiosis I and undergo apoptosis. Mechanistically, upregulation of CDC25B, a phosphatase that activates CDK1, is responsible for precocious meiotic resumption and also contributes to subsequent spindle and chromosomal defects. Our findings uncover a functional link between LSD1 and the major signalling pathway governing meiotic progression.

  17. Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies.

    Science.gov (United States)

    Selesniemi, Kaisa; Lee, Ho-Joon; Muhlhauser, Ailene; Tilly, Jonathan L

    2011-07-26

    Increased meiotic spindle abnormalities and aneuploidy in oocytes of women of advanced maternal ages lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Despite the significance of the problem, strategies to sustain oocyte quality with age have remained elusive. Here we report that adult female mice maintained under 40% caloric restriction (CR) did not exhibit aging-related increases in oocyte aneuploidy, chromosomal misalignment on the metaphase plate, meiotic spindle abnormalities, or mitochondrial dysfunction (aggregation, impaired ATP production), all of which occurred in oocytes of age-matched ad libitum-fed controls. The effects of CR on oocyte quality in aging females were reproduced by deletion of the metabolic regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Thus, CR during adulthood or loss of PGC-1α function maintains female germline chromosomal stability and its proper segregation during meiosis, such that ovulated oocytes of aged female mice previously maintained on CR or lacking PGC-1α are comparable to those of young females during prime reproductive life.

  18. Survival, Fertilization and Developmental Rates of Cryotop-Vitrified Oocyte and Embryo Using Low Concentrated Cryoprotectants

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    A Roozbehi

    2012-10-01

    Full Text Available Background & Aim: The preserving embryos, the risk of multiple pregnancies, the existence of factors in stimulated uterine cycle, are important forces in perfecting embryo cryopreservation. The aim of current study was to assess Survival, Fertilization and Developmental Rates (SRs, FRs, DRs of the mouse oocytes and embryos using cryotop and low concentrated cryoprotectants solutions. Methods: Mouse C57BL/6 oocytes and embryos were collected. Oocytes SRs, FRs, DRs were recorded after cryotop-vitrification/ warming. As well as comparing fresh oocytes and embryos, the data obtained from experimental groups (exp. applying 1.25, 1.0, and 0.75 Molar (M CPAs were analyzed in comparison to those of exp. adopting 1.5 M CPAs (largely-used concentration of EthylenGlycol (EG and Dimethylsulphoxide (DMSO. Results: The data of oocytes exposed to 1.25 M CPAs were in consistency with those exposed to 1.5 M and control group in terms of SR, FR and DR. As fewer concentrations were applied, the more decreased SRs, FRs and DRs were obtained from other experimental groups. The results of embryos were exposed to 1.25 M and 1.0 M was close to those vitrified with 1.5 M and fresh embryos. The results of 0.75 M concentrated CPAs solutions were significantly lower than those of control, 1.5 M and 1.0 M treated groups. Conclusion: CPAs limited reduction to 1.25 M and 1.0 M instead of using 1.5 M, for oocyte and embryo cryotop-vitrification procedure may be a slight adjustment.

  19. Effect of Basic Fibroblast Growth Factor on in Vitro Maturation of Oocytes of Mouse at the Stage of Germinal Vesicle

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    R Khanbabaee

    2014-10-01

    Full Text Available Introduction: In vitro maturation (IVM of oocytes, providing oocytes maturation out of normal conditions, is an appropriate infertility treatment system, though the clinical use of IVM is limited due to low rate of success. Accordingly, this study aimed to analyze the effect of fibroblast growth factor on in vitro maturation of immature oocytes. Methods: Immature oocytes of 20 female mice of NMRI strain aged 8-10 weeks were obtained 46-48 hours after intraperitoneal injection of 10 units of Pregnant Mare`s Serum Gonadotrophin (PMSG. The oocytes were treated within Modified Essential Medium (MEM-α supplemented with 0 ng/ml, 10 ng/ml, 20 ng/ml and 40 ng/ml doses of fibroblast growth factor respectively. After 24 hours, Oocyte maturation stage was scrutinized by an invert microscope and its growth rate was analyzed via SPSS software utilizing ANOVA test. Results: The resumption percentage of meiosis was reported as 23 in the first control group, while it was 25.7, 26.2, 27.3 % respectively for the second, third and fourth experimental groups; thus, no significant differences was observed among control groups and experimental groups. Yet in vitro maturation of the control group, a significant difference was observed compared to those of the second and third experimental groups (p<0.01. In fact, the rate of vitro metaphase matured oocytes were reported as 45, 60.8, 62.6 and 45.2 % respectively in the control group and the second, third, and fourth experimental groups. Conclusion: The obtained results of study illustrated that 10 ng/ml and 20 ng/ml concentrations of fibroblast growth factor have a major impact on resumption of meiosis, nucleus break down and extrusion of the first polar body, whereas the effect of 40 mg/ml concentration on improvement of oocyte maturation was trivial.

  20. Conforming with current regulation in Turkey regarding the freezing of oocytes: A case report of the first pregnancy in Turkey achieved through oocyte vitrification

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    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

  1. 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

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

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    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.

  3. [Polarized light microscopy for evaluation of oocytes as a prognostic factor in the evolution of a cycle in assisted reproduction].

    Science.gov (United States)

    González-Ortega, C; Cancino-Villarreal, P; Alonzo-Torres, V E; Martínez-Robles, I; Pérez-Peña, E; Gutiérrez-Gutiérrez, A M

    2016-04-01

    Identification of the best embryos to transfer is a key element for success in assisted reproduction. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems has allowed the visualization of the meiotic spindle and the different layers of the zona pellucida in human oocytes on the basis of birefringence in a non-destructive way. Conflicting results have been reported regarding the predictive value in ICSI cycles. To assess the predictive ability of meiotic spindle and zona pellucida of human oocytes to implant by polarized microscopy in ICSI cycles. Prospective and observational clinical study. 903 oocytes from 94 ICSI cycles were analyzed with polarized microscopy. Meiotic spindle visualization and zona pellucida birefringence values by polarized microscopy were correlated with ICSI cycles results. Meiotic spindle visualization and birefringence values of zona pellucida decreased in a direct basis with increasing age. In patients aged over the 35 years, the percentage of a visible spindle and mean zona pellucida birefringence was lower than in younger patients. Fertilization rate were higher in oocytes with visible meiotic spindle (81.3% vs. 64%; p Polarized light microscopy improves oocyte selection, which significantly impacts in the development of embryos with greater implantation potential. The use of polarized light microscopy with sperm selection methods, blastocyst culture and deferred embryo transfers will contribute to transfer fewer embryos without diminishing rates of live birth and single embryo transfer will be more feasible.

  4. The predictive value of serum concentrations of anti-Müllerian hormone for oocyte quality, fertilization, and implantation

    Science.gov (United States)

    Borges, Edson; Braga, Daniela P. A. F.; Setti, Amanda; Figueira, Rita de Cássia; Iaconelli Jr, Assumpto

    2017-01-01

    Objective This study aimed to identify a possible correlation between serum levels of anti-Müllerian hormone (AMH) and oocyte quality, embryo developmental competence, and implantation potential. Methods 4488 oocytes obtained from 408 patients undergoing ICSI cycles were evaluated. Oocyte dimorphisms, embryo quality on days two and three, blastocyst formation competence, fertilization rates, implantation rates, and pregnancy rates were correlated with serum levels of AMH using Pearson's correlation coefficient and regression analysis. Results A positive correlation was observed between serum levels of AMH and number of retrieved oocytes (CC: 0.600, p<0.001), fertilization rate (CC:0.595, p=0.048), and number of obtained embryos (CC:0.495, p<0.001). AMH did not affect the quality of cleavage stage embryos or the chance of blastocyst formation. However, AMH levels affected oocyte quality (OR:0.75, CI 0.44-0.96, p<0.001), and implantation (CC:0,116, p=0.031) and pregnancy (OR:1.22, CI:1.03-1.53, p<0.001) rates. Conclusion Serum levels of AMH are a useful predictor of ovarian response to COS, oocyte quality, and fertilization. However, AMH levels may also compromise clinical outcomes; lower AMH levels did not impair embryo development. PMID:28837024

  5. ACTIVE AND PARTICIPATORY METHODS IN BIOLOGY: MODELING

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    Brînduşa-Antonela SBÎRCEA

    2011-01-01

    Full Text Available By using active and participatory methods it is hoped that pupils will not only come to a deeper understanding of the issues involved, but also that their motivation will be heightened. Pupil involvement in their learning is essential. Moreover, by using a variety of teaching techniques, we can help students make sense of the world in different ways, increasing the likelihood that they will develop a conceptual understanding. The teacher must be a good facilitator, monitoring and supporting group dynamics. Modeling is an instructional strategy in which the teacher demonstrates a new concept or approach to learning and pupils learn by observing. In the teaching of biology the didactic materials are fundamental tools in the teaching-learning process. Reading about scientific concepts or having a teacher explain them is not enough. Research has shown that modeling can be used across disciplines and in all grade and ability level classrooms. Using this type of instruction, teachers encourage learning.

  6. 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...

  7. 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.

  8. Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases Ativação oocitária e desenvolvimento pré-implantação de embriões bovinos obtidos com o uso de inibidores específicos das quinases dependentes de ciclina

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    F. Perecin

    2007-04-01

    Full Text Available The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100µM and different exposure periods (2, 4 or 6 hours to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs showed better results for activation rates (77.3% and initial embryonic development (35.2% than the single ionomycin treatment (69.4% for activation and 21.9% for development; and also lead to a more uniform activation (nearly 90% single pronucleus development. The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.Realizaram-se dois experimentos para avaliar a eficiência da bohemina e roscovitina associadas à ionomicina para ativação partenogenética e desenvolvimento embrionário inicial de bovinos. No primeiro, foram testadas diferentes concentrações (0, 50, 75 ou 100µM e diferentes tempos de exposição (2, 4 ou 6 horas à bohemina ou à roscovitina na ativação de oócitos bovinos maturados in vitro (MIV pré-expostos à ionomicina. Os melhores tratamentos, bohemina 75µM e roscovitina 50µM, ambos por seis horas, foram utilizados no segundo experimento, no qual oócitos bovinos

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

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    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.

  10. 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

  11. 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.

  12. Dynamic changes of connexin-43, gap junctional protein, in outer layers of cumulus cells are regulated by PKC and PI 3-kinase during meiotic resumption in porcine oocytes.

    Science.gov (United States)

    Shimada, M; Maeda, T; Terada, T

    2001-04-01

    Mammalian oocytes are surrounded by numerous layers of cumulus cells, and the loss of gap junctional communication in the outer layers of cumulus cells induces meiotic resumption in oocytes. In this study, we investigated the dynamic changes in the gap junctional protein connexin-43 in cumulus cells during the meiotic resumption of porcine oocytes. The amount of connexin-43 in all layers of cumulus cells recovered from cumulus-oocyte complexes was increased after 4-h cultivation. However, at 12-h cultivation, the positive signal for connexin-43 immunoreactivity was markedly reduced in the outer layers of cumulus cells. When these reductions of connexin-43 were blocked by protein kinase C (PKC) or phosphatidylinositol (PI) 3-kinase inhibitor, networks of filamentous bivalents (i.e., advanced chromosomal status) were undetectable in the germinal vesicle of the oocyte. After 28-h cultivation, when the majority of oocytes were reaching the metaphase I (MI) stage, the connexin-43 in the inner layers of cumulus cells was phosphorylated, regardless of mitogen-activated protein (MAP) kinase activation. These results suggest that the initiation of meiotic resumption, namely, the formation of networks of filamentous bivalents in germinal vesicle, is associated with the reduction of gap junctional protein connexin-43 in the outer layers of cumulus cells via the PKC and/or PI 3-kinase pathway. Moreover, the connexin-43 in the inner layers of cumulus cells is phosphorylated during meiotic progression beyond the MI stage, regardless of MAP kinase activation in cumulus cells surrounding the oocyte.

  13. Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion.

    Science.gov (United States)

    Balboula, Ahmed Z; Blengini, Cecilia S; Gentilello, Amanda S; Takahashi, Masashi; Schindler, Karen

    2017-06-01

    During oocyte meiotic maturation, Aurora kinase C (AURKC) is required to accomplish many critical functions including destabilizing erroneous kinetochore-microtubule (K-MT)attachments and regulating bipolar spindle assembly. How localized activity of AURKC is regulated in mammalian oocytes, however, is not fully understood. Female gametes from many species, including mouse, contain stores of maternal transcripts that are required for downstream developmental events. We show here that depletion of maternal RNA in mouse oocytes resulted in impaired meiotic progression, increased incidence of chromosome misalignment and abnormal spindle formation at metaphase I (Met I), and cytokinesis defects. Im