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Sample records for bovine oocytes fertilized

  1. Tetraspanin CD9 in bovine oocytes and its role in fertilization.

    Science.gov (United States)

    Zhou, Guang-Bin; Liu, Guo-Shi; Meng, Qing-Gang; Liu, Ying; Hou, Yun-Peng; Wang, Xiao-Xu; Li, Ning; Zhu, Shi-En

    2009-06-01

    This study was conducted in bovine to investigate whether CD9 (a member of the tetraspanin superfamily of proteins) is present on oocytes and whether it functions in sperm-oocyte binding and fusion. First, the presence of CD9 in bovine matured oocytes was examined by immunofluorescence with the anti-CD9 monoclonal antibody (mAb) and fluorescein isothiocyanate-conjugated goat anti-mouse antibody, and the results showed that CD9 was expressed on the plasma membrane of matured oocytes. Sperm binding and fusion with oocytes was then examined by in vitro fertilization. When the zona pellucida-free matured oocytes were fertilized, both sperm binding to ooplasma and sperm penetrating into oocytes were significantly (PmAb potentially inhibits sperm-oocyte binding and fusion. These results demonstrated that the CD9 present on bovine matured oocytes is involved in sperm-oocyte interaction during fertilization. PMID:19293563

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

    Science.gov (United States)

    Makita, Miho; Ueda, Mayuko; Miyano, Takashi

    2016-08-25

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

  3. Sericin Accelerates the Production of Hyaluronan and Decreases the Incidence of Polyspermy Fertilization in Bovine Oocytes During In Vitro Maturation

    OpenAIRE

    Hosoe, Misa; Yoshida, Nao; Hashiyada, Yutaka; TERAMOTO, Hidetoshi; Takahashi, Toru; Niimura, Sueo

    2014-01-01

    Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15...

  4. Simulated Microgravity Influences Bovine Oocyte In Vitro Fertilization and Preimplantation Embryo Development

    Science.gov (United States)

    The aim of this study was to investigate whether in vitro fertilization and preimplantation embryos exposed to a simulated microgravity environment in vitro would improve, or be deleterious to, their fertilization and embryonic development. A Rotating Cell Culture System™ (RCCS) bioreactor with a Hi...

  5. Recent Progress in Cryopreservation of Bovine Oocytes

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    In-Sul Hwang

    2014-01-01

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

  6. Oocyte-secreted factors in oocyte maturation media enhance subsequent development of bovine cloned embryos.

    Science.gov (United States)

    Su, Jianmin; Wang, Yongsheng; Zhang, Lei; Wang, Bo; Liu, Jun; Luo, Yan; Guo, Zekun; Quan, Fusheng; Zhang, Yong

    2014-04-01

    Successful in vitro maturation (IVM) and oocyte quality both affect the subsequent development of cloned embryos derived from somatic-cell nuclear transfer (SCNT). Developmental competence is usually lower in oocytes matured in vitro compared with those that matured in vivo, possibly due to insufficient levels of oocyte-secreted factors (OSFs) and disrupted oocyte-cumulus communication. This study investigated the effects of OSFs secreted by denuded oocytes (DOs) during IVM on the subsequent developmental competence of cloned bovine embryos. Cumulus-oocyte complexes (COCs) from antral follicles of slaughtered-cow ovaries collected from an abattoir were divided into four groups: COCs co-cultured with and without DOs in maturation media used for SCNT, as well as COCs co-cultured with and without DOs in maturation media used for in vitro fertilization (IVF). Based on the developmental competence and embryo quality of bovine embryos generated from these four groups, we found that co-culturing the COCs with DOs enhanced the in vitro development of IVF and cloned bovine embryos, and potentially generated more high-quality cloned blastocysts that possessed locus-specific histone modifications at levels similar to in vitro-fertilized embryos. These results strongly suggest that co-culturing COCs with DOs enhances subsequent developmental competence of cloned bovine embryo. PMID:24420374

  7. 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).

  8. Cryopreservation of Human Unfertilized and Fertilized Oocytes

    OpenAIRE

    Vanderzwalmen P; Zech NH; Nagy ZP; Stecher A; Papatheodorou P; Schuff M; Wirleitner B

    2013-01-01

    Cryopreservation of human fertilized oocytes and embryos are nowadays well established in IVF practice with a wide range of clinical applications. However, freezing of unfertilized MII oocytes turned out to be one of the greatest challenges in the field of human reproductive cryobiology since the protocols remained ineffective for over 25 years. Only in the last 10 years the efficiency and safety of oocyte cryopreservation tremendously improved with the realization that zona hardening occurs...

  9. Assessment of meiotic spindle configuration and post-warming bovine oocyte viability using polarized light microscopy.

    Science.gov (United States)

    Caamaño, J N; Díez, C; Trigal, B; Muñoz, M; Morató, R; Martín, D; Carrocera, S; Mogas, T; Gómez, E

    2013-06-01

    The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule-polymerized protein in in vitro-matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro-matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule-polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n = 115), which strongly correlated (r = 1; p PLM for 10 min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(-) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post-warming viability in vitrified bovine oocytes.

  10. Synergistic Effect of Insulin on in vitro Development of Immature Bovine Oocytes

    OpenAIRE

    Mojtaba Dashtizad; Abd W. Haron; Rosnina Yusoff; Morteza Daliri; Hadi Hajarian; Mehdi Najari; Yap K. Chee; Abas M. Othman

    2010-01-01

    Problem statement: Development of efficient culture system to support embryonic development would be valuable when quality of produced embryos was important. However, the rate of bovine embryo production in vitro was still lower than expected. Present study, including of three experiments, was carried out to investigate the effect of insulin on nuclear maturation and subsequent development of immature bovine oocytes and in vitro fertilized embryos. Approach: Grade one cumulus-oocyte-complexes...

  11. [In Vitro Fertilization: beware of oocyte retrieval without oocyte!].

    Science.gov (United States)

    Bringer-Deutsch, S; Mayenga, J-M; Grefenstette, I; Grzegorczyk, V; Kulski, O; Belaisch-Allart, J

    2010-11-01

    A 30-year-old woman undergoing an In Vitro Fertilization (IVF) treatment for tubal infertility and for whom no oocyte was retrieved at the puncture ("white puncture") presented an ectopic pregnancy. The patient was asymptomatic except some bleeding events reported for several days prior to the puncture. The ovulation monitoring was normal throughout the stimulation by gonadotrophin and hCG was administered for the final oocyte maturation on the twelfth day of stimulation at a rate of 2771 pg/ml of estradiol with a perfect ultrasound follicular growth. This case demonstrates that we have to beware of a "white puncture" and that the hCG measurement the day of the oocyte retrieval is necessary because of the possibility of an ectopic pregnancy. PMID:21115382

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

    OpenAIRE

    R.G Sianturi; Thein, M; H. Wahid; Yono C Rosnina

    2002-01-01

    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 cumulu...

  13. The presence of corpus luteum may have a negative impact on in vitro developmental competency of bovine oocytes.

    Science.gov (United States)

    Hajarian, Hadi; Shahsavari, Mohammad H; Karami-shabankareh, Hamed; Dashtizad, Mojtaba

    2016-03-01

    The aim of the current study was to investigate the effects of the presence or absence of corpus luteum (CL) on in vitro developmental competence of bovine oocytes. In experiment 1, cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and divided according to the presence (CL(+) oocytes) or absence (CL(-) oocytes) of a CL in the ovary. Control oocytes (C group) were obtained from ovaries which were not selected toward the presence or absence of CL. All oocytes were submitted to in vitro maturation, fertilization and culture. In experiment 2, the oocytes from the CL(+) and CL(-) ovaries were divided into grown (BCB(+)) and growing (BCB(-)) categories by means of the brilliant cresyl blue (BCB) test. The oocytes from all groups (CL(+)/BCB(+), CL(-)/BCB(+), CL(+)/BCB(-), CL(-)/BCB(-) and control oocytes) were subjected to in vitro embryo production. In experiment 1, the cleavage and blastocyst rates of CL(-) oocytes were higher than those of CL(+) oocytes (83.9% and 43% vs. 69.3% and 22.5%, respectively). In experiment 2, there was less BCB(+) oocytes (more competent oocytes) in the group of CL(+) oocytes than in the group of CL(-) oocytes. Furthermore, developmental competence of all CL(+) oocytes (CL(+)/BCB(+) and CL(+)/BCB(-)) was lower than that of all CL(-) oocytes (CL(-)/BCB(+) and CL(-)/BCB(-)). Thus, the presence of a corpus luteum in the ovary may have negative effects on developmental competence of ipsilateral oocytes. PMID:26952753

  14. Viabilidade e fertilização in vitro de oócitos bovinos após vitrificação Viability and in vitro fertilization of bovine oocytes after vitrification

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    Sérgio Galbinski

    2003-09-01

    ários para proteção da zona pelúcida e do oolema.PURPOSE: to verify vitrification techniques using 6 M DMSO to cryopreserve in vitro matured bovine oocytes, and to assess the effects of the time of exposure to vitrification solutions (VS. METHODS: dilutions of VS were prepared from the stock VS (VS 100% consisting of 6 M DMSO to give 25 and 65% DMSO solutions. Bovine oocytes were in vitro matured for 18-22 h. Matured oocytes were placed first into 25% VS, at room temperature for 5 min, then transferred to 65% VS, before being pipetted into the 100% VS in plastic straws. Three experimental groups were formed: in the first group, time of pipetting through 65% VS and loading the straw took up to 60 s, in the second group it did not exceed 30 s. For thawing, straws were held in air for 10 s and then in a water bath for 10 s. The contents of each straw were expelled in sucrose solution and held for 5 min. In the third experimental group, oocytes went through all VS, but were not vitrified. All retrieved oocytes were inseminated. For control, fresh, in vitro matured oocytes were inseminated. RESULTS: after vitrification, 69.1 and 59.8% of the oocytes were retrieved from the 30 s and 60 s groups, respectively, and 93 and 89% of these oocytes appeared morphologically normal 24 h after insemination, respectively. In the group of oocytes exposed without vitrification, 75.6% were retrieved and 84.7% were morphologically viable, 24 h after insemination. No fertilization was observed in the experimental groups. Among controls, 65.4% were fertilized. CONCLUSIONS: the vitrification technique using 6 M DMSO is not a feasible approach to cryopreserve in vitro matured bovine oocytes. Decreasing the time of exposure to VS did not overcome deleterious effects of the procedure on the fertilizability of oocytes. Improvements in the technique are needed to protect the zona pellucida and oolemma.

  15. Replication of somatic micronuclei in bovine enucleated oocytes

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    Canel Natalia

    2012-11-01

    Full Text Available Abstract Background Microcell-mediated chromosome transfer (MMCT was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes. Methods Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+] or not [Micronucleus- injected (−] to a transgene (50 ng/μl pCX-EGFP during 5 min. Enucleated oocytes [Enucleated (+] and parthenogenetic [Parthenogenetic (+] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (−] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (−, Parthenogenetic (− and in vitro fertilized (IVF embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05. Results All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had

  16. 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.

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

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    Robert F. Casper

    2010-01-01

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

  18. 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.

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

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    Reza Mahmodi

    2009-01-01

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

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

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    R.G Sianturi

    2002-10-01

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

  1. 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...

  2. Morphology of preovulatory bovine follicles as related to oocyte maturation.

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    de Loos, F A; Bevers, M M; Dieleman, S J; Kruip, T A

    1991-03-01

    Thirty-three preovulatory bovine oocytes and their follicles were collected during the period of final maturation in normally cyclic cows. Cell density of the membrana granulosa, mitotic index of the membrana granulosa, and the occurrence of eosinophilic granulocytes around the basal membrane as well as the maturational stage of the oocyte were determined. Cell density decreased during the period of final maturation. Mitotic indices also decreased after an initial high level in the first hours of the final maturation. Eosinophilic granulocytes were only seen during the last hours of final maturation. The maturational stages of the oocytes were related to distinct maturational stages of the follicular wall as determined by morphological characteristics. We propose a scoring system for the maturity of the follicular wall based on cell density, presence of mitotic figures and the presence of eosinophilic granulocytes outside the vascular compartment. PMID:16726922

  3. Effect of antifreeze glycoprotein 8 supplementation during vitrification on the developmental competence of bovine oocytes.

    Science.gov (United States)

    Liang, Shuang; Yuan, Bao; Kwon, Jeong-Woo; Ahn, Mija; Cui, Xiang-Shun; Bang, Jeong Kyu; Kim, Nam-Hyung

    2016-07-15

    The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury. PMID:26948296

  4. Effect of oocyte quality and activation protocols on bovine embryo development following intracytoplasmic sperm injection

    OpenAIRE

    KORKMAZ, Ömer; KÜPLÜLÜ, Şükrü; AĞCA, Yüksel; POLAT, İbrahim Mert

    2013-01-01

    The purpose of this study was to investigate the effects of oocyte quality and activation protocols on the in vitro developmental competence of bovine embryos after intracytoplasmic sperm injection (ICSI). Bovine oocytes were grouped as being of excellent, good, and poor quality. All of the oocytes were activated using a calcium ionophore only, ethanol only, and 6-dimethylaminopurine (6-DMAP) following calcium ionophore. For the excellent quality oocytes, cleavage rates after ICSI were 70% in...

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

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    Jorgelina Buschiazzo

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

  6. Prolonged exposure to hyaluronidase decreases the fertilization and development rates of fresh and cryopreserved mouse oocytes

    OpenAIRE

    ISHIZUKA, Yuta; Takeo, Toru; NAKAO, Satohiro; YOSHIMOTO, Hidetaka; Hirose, Yumiko; Sakai, Yuki; HORIKOSHI, Yuka; TAKEUJI, Shiori; Tsuchiyama, Shuuji; Nakagata, Naomi

    2014-01-01

    Hyaluronidase is generally used to remove cumulus cells from mouse oocytes before oocyte cryopreservation, intracytoplasmic sperm injection or DNA injection. In general, use of cumulus-free mouse oocytes decreases in vitro fertilizing ability compared with cumulus-surrounded oocytes. The effect of hyaluronidase exposure on the quality of mouse oocytes is not fully understood. Here, we investigated the effect of hyaluronidase exposure time on the fertilization rate of fresh and vitrified mouse...

  7. Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

    OpenAIRE

    Quetglas M.D.; Coelho L.A.; Garcia J.M.; Oliveira Filho E.B.; Esper C.R.

    2001-01-01

    The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) and on in vitro embryo development (experiment II) of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR), blastocyst (BR) and hatching (HR) rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium) and gonadotropins (14 U/ml PMSG and 7 U/ml hCG)....

  8. Synergistic Effect of Insulin on in vitro Development of Immature Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    Mojtaba Dashtizad

    2010-01-01

    Full Text Available Problem statement: Development of efficient culture system to support embryonic development would be valuable when quality of produced embryos was important. However, the rate of bovine embryo production in vitro was still lower than expected. Present study, including of three experiments, was carried out to investigate the effect of insulin on nuclear maturation and subsequent development of immature bovine oocytes and in vitro fertilized embryos. Approach: Grade one cumulus-oocyte-complexes harvested from slaughterhouse ovaries were selected and randomly allocated in each treatment groups. In experiment 1, in vitro maturation medium (Hepes-buffered medium 199 + fetal calf serum + gonadotrophins + antibiotics supplemented with 0 (control, 1, 10, 20 and 100 µg mL-1 of insulin. In experiment 2, to eliminate the effect of serum and hormones, Hepesbuffered medium 199 was supplemented with 1 mg mL-1 polyvinyl alcohols (PVA and same levels of insulin. In experiment 3, the effect of insulin on bovine in vitro embryo development was assessed. Presumptive zygotes were randomly cultured in synthetic oviductal fluid added with 0 (control, 1, 10, 20 and 100 ìg mL-1 of insulin. Results: In experiment 1, nuclear maturation and embryo development rates were significantly higher in 1 and 10 µg mL-1 compared with other groups (P-1 insulin. The only treatment resulted in higher hatchability was 10 ìg mL-1 insulin (17.1±2.34% compared with control (11.34±3.94. In experiment 3, cleavage and morula rates were significantly greater in 1 and 10 µg mL-1 insulin compared with other groups; although the highest rates resulted by using 10 µg mL-1. Conclusion: Obtained results show that inclusion of 10 µg mL-1 insulin in maturation and culture medium exerted beneficial effects on nuclear maturation of bovine oocytes and in vitro embryo development till morula stage.

  9. Pregnancies and improved early embryonic development with bovine oocytes matured in vitro with 9-cis-retinoic acid

    OpenAIRE

    Hidalgo, C.O. (Carlos); Díez, Carmen; Duque, Paloma; Facal, Nieves; Gómez, Enrique

    2010-01-01

    Retinoids have an important role in cell growth, morphogenesis and differentiation. In the present study the developmental potential of bovine oocytes was examined after in vitro maturation in the presence of 9-cis-retinoic acid, a vitamin A metabolite, at 5 nmol l(-1) in chemically defined conditions. Experiments studied early in vitro development, blastocyst differential cell counts and the capacity of embryos to establish pregnancy after transfer to recipients. After in vitro fertilization...

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  11. 牛性机能发育阶段与卵泡、黄体因素对卵母细胞%Effects of sexual developmental stages, follicular and? Luteal factors on in Vitro maturation and in vitro fertilization of bovine oocytes

    Institute of Scientific and Technical Information of China (English)

    王艳军; 梁冠生; 刘健; 张嘉保

    2000-01-01

    In order to improve the clevage rate and the blastocysst rate, we have studied several elements including stage of ? sexual development, follicles and Luteal factors that influnce in vitro maturation and in vitro fertilization of bovine oocytes. The results are described as follows: There was no difference in the cleavage rate(74.7% and 81.5%)and the blastocyst rate (23.0 and 26.8%) between ovarien oocytes ? from pubertal heifers and from matured cows, but all of them were significantly higher than those of oocytes from prepubetal heifers (P? <0.05). The development competence after in maturation of oocytes from ovaries with Corpus Luteal was inferior to that of oocytes from ? ovaries without Corput Luteal (the cleavage rate was 58.0% and 79.9%, the blastocyst rate was 13.4% and 27.1%, P<0.05). There was no ? difference between the oocytes from ovaries with dominent follicle and from ovaries without dominent follicles.%利用屠宰母牛卵巢,对来自不同性机能发育阶段母牛的卵巢卵母细胞,不同状态卵巢卵泡的卵母细胞的体外成熟(IVM)、体外受精(IVF? )进行了系列研究。结果表明,来自初情期、性成熟母牛卵巢的卵母细胞受精后的卵裂率(74.7%和81.5%)、囊胚率(23.0%和26.8%)显著高于来自初情期前? 母牛卵巢卵母细胞的卵裂率(18.8%)和囊胚率(1.8%),P<0.05;有黄体卵巢的卵母细胞体外受精后的发育能力显著低于无黄体卵巢卵母细胞? (卵裂率58.0%∶79.9%,囊胚率13.%∶27.1%, P<0.05);优势卵泡的有无对卵母细胞的卵裂率和囊胚率无明显影响

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

    Directory of Open Access Journals (Sweden)

    Hamed Karami Shabankareh

    2015-10-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    AR Rafati

    2007-04-01

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

  14. DNA methylation pattern in mouse oocytes and their in vitro fertilized early embryos: effect of oocyte vitrification.

    Science.gov (United States)

    Liang, Ying; Fu, Xiang-Wei; Li, Jun-Jie; Yuan, Dian-Shuai; Zhu, Shi-En

    2014-05-01

    This study was conducted to investigate the pattern of DNA methylation in vitrified-thawed mouse oocytes and their in vitro fertilized early embryos. Firstly, mouse oocytes at metaphase II (MII) stage of meiosis were allocated randomly into three groups: (1) untreated (control); (2) exposed to vitrification solution without being plunged into liquid nitrogen (toxicity); or (3) vitrified by open-pulled straw (OPS) method (vitrification). Oocytes from all three groups were fertilized subsequently in vitro. The level of DNA methylation in the MII oocytes and their early embryos was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Developmental rates to 2-cell embryos (62.28%) and blastocysts (43.68%) of the vitrified-thawed oocytes were lower (P < 0.01) than those of fresh oocytes (81.47%, 61.99%) and vitrification solution treated (79.20%, 60.04%) oocytes. DNA methylation (as reflected by 5-MeC fluorescence intensity) in the vitrification group was less (P < 0.01) for MII oocyte and 2- to 8-cell stages compared with that in the control and toxicity groups. Accordingly, a reduction in global genomic methylation due to vitrification of MII oocytes may result in compromised in vitro developmental potential in early mouse embryos. PMID:23174120

  15. Enhancement of developmental capacity of meiotically inhibited bovine oocytes by retinoic acid

    OpenAIRE

    Duque, Paloma; Díez, C; Royo, L.J. (Luis); Lorenzo, P.L. (Pedro); Carneiro, G.; Hidalgo, C.O. (Carlos); Facal, Nieves; Gómez, E.

    2012-01-01

    BACKGROUND: Although high vitamin A may be teratogenic to the embryo, retinol has been shown to support oocyte developmental potential in vivo. Similarly, addition of retinol metabolite 9-cis-retinoic acid to in-vitro cultured oocytes could promote cytoplasmic maturation and subsequent early embryonic development. The objective of this study was to evaluate the effects of 5 nmol/l retinoic acid during in-vitro pre-maturation and maturation of bovine oocyte-cumulus complexes. METHODS AND...

  16. Influence of antral follicle size on oocyte characteristics and embryo development in the bovine

    DEFF Research Database (Denmark)

    Lequarre, Anne Sophie; Vigneron, Céline; Ribaucour, Fabrice;

    2005-01-01

    The developmental competence of bovine oocytes isolated from antral follicles of different sizes was assessed in three European laboratories (Belgium, UCL; Denmark, DIAS; France, INRA). Using the same protocol for in vitro production of embryos, the oocytes isolated from follicles with a diameter...

  17. Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

    DEFF Research Database (Denmark)

    Hou, Y -p; Liu, Ying; Dai, Y -p;

    2009-01-01

    evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4 h...

  18. Influence of corpus luteum and ovarian volume on the number and quality of bovine oocytes.

    Science.gov (United States)

    Penitente-Filho, Jurandy Mauro; Jimenez, Carolina Rodrigues; Zolini, Adriana Moreira; Carrascal, Erly; Azevedo, Jovana Luiza; Silveira, Camila Oliveira; Oliveira, Fabrício Albani; Torres, Ciro Alexandre Alves

    2015-02-01

    In order to evaluate whether ovarian volume, presence and diameter of the corpus luteum (CL) have effects on the number and quality of bovine recovered oocytes, 110 ovaries were obtained from the slaughterhouse. Cumulus oocytes complex were aspirated and evaluated under stereomicroscope. Oocytes were counted and classified according to their quality (Grades I, II, III and IV). Ovarian volume was weakly correlated to the number of good quality oocytes (P Ovaries with CL showed greater numbers of good quality oocytes than ovaries without CL (P quality oocytes (P < 0.05). In conclusion, ovarian volume is not a good parameter itself to predict important ovarian characteristics; moreover, analysis of CL, its presence and diameter, may be a good tool to improve efficiency on in vitro embryo production programs. PMID:25187422

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

    Institute of Scientific and Technical Information of China (English)

    SunQingyuan; FengHuailiang; 等

    1994-01-01

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

  20. The bovine oocyte in vitro maturation model: A potential tool for reproductive toxicology screening

    NARCIS (Netherlands)

    Beker-van Woudenberg, A.C.R.L.; Gröllers-Mulderij, M.; Snel, C.; Jeurissen, N.; Stierum, R.; Wolterbeek, A.

    2012-01-01

    Reproductive toxicity testing according to the present guidelines requires a high number of animals. Therefore, the development of alternative in vitro methods is urgently required. The aim of the present study was to investigate the applicability domain of the bovine oocyte in vitro maturation assa

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Looft C

    2011-01-01

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

  3. The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries.

    Science.gov (United States)

    Tessaro, I; Luciano, A M; Franciosi, F; Lodde, V; Corbani, D; Modina, S C

    2011-08-01

    In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with ovaries; Lo) show less developmental competence than oocytes collected from ovaries with >10 medium antral follicles (high ovaries; Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to 1) immunolocalize eNOS protein during folliculogenesis; 2) quantify eNOS protein/vasculature in the follicle wall; and 3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial nitric oxide synthase protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P bovine ovaries with small mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may affect oocyte quality, thus inducing a premature decline of fertility. PMID:21421835

  4. Effect of the bovine oviductal fluid on in vitro fertilization, development and gene expression of in vitro-produced bovine blastocysts.

    Science.gov (United States)

    Cebrian-Serrano, A; Salvador, I; García-Roselló, E; Pericuesta, E; Pérez-Cerezales, S; Gutierrez-Adán, A; Coy, P; Silvestre, M A

    2013-04-01

    Oviductal microenvironment generally provides better conditions for early embryo development than the conventional in vitro system. In an attempt to simulate the oviduct conditions or the main potentially influencing factors, the effect was studied of a bovine oviductal fluid (bOF) treatment applied prior to IVF on (i) IVF parameters, (ii) cleavage rate, (iii) blastocyst yield and (iv) blastocyst quality. Embryo quality was assessed by morphological embryo quality and relative transcript abundance of several developmental genes in bovine blastocysts. Furthermore, to study the effect of bOF without the male effect and zona-sperm interaction, artificially activated metaphase II oocytes were also treated with bOF. In vitro-matured bovine oocytes from abattoir ovaries were treated or untreated with bOF for 30 min and then washed prior to IVF or activation. Subsequently, in vitro-fertilized and parthenogenetic embryos were in vitro cultured for 7 to 8 days. The bOF treatment had no effect on fertilization parameters, cleavage, blastocyst rates both on parthenogenetic and IVF bovine embryos and neither on morphological quality of IVF blastocysts. G6PD and SOD2 genes from IVF blastocysts showed significant changes in their expression after a bOF treatment. Significant differences were found for the expression of SCL2A1, GPX1, BAX, AKR1B1 and PLAC8 genes between excellent or good blastocysts (Grade 1) and fair blastocysts (Grade 2). To our knowledge, this is the first study that evaluates the effect of bOF oocyte treatment on fertilization parameters, development and quality of bovine embryos. PMID:22908847

  5. 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.

  6. 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

    2016-08-01

    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. PMID:27260975

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    . Follicular characteristics were measured and oocyte quality was assessed by morphology, mRNA expression of eight marker genes or developmental ability after in vitro/in vivo maturation and subsequent in vitro fertilization and culture. Approaching ovulation, expected increases in follicular size and cumulus...

  8. Xenogenous fertilization of equine oocytes following recovery from slaughterhouse ovaries and in vitro maturation.

    Science.gov (United States)

    Wirtu, G; Bailey, T L; Chauhan, M S; Parker, N A; Dascanio, J J; Gwazdauskas, F C; Ley, W B

    2004-01-15

    The in vitro production (IVP) of equine embryos using currently available protocols has met limited success; therefore investigations into alternative approaches to IVP are justified. The objective of this study was to evaluate the feasibility of xenogenous fertilization and early embryo development of in vitro matured (IVM) equine oocytes. Follicular aspirations followed by slicing of ovarian tissue were performed on 202 equine ovaries obtained from an abattoir. A total of 667 oocytes (3.3 per ovary) were recovered from 1023 follicles (recovery rate, 65%). Oocytes underwent IVM for 41 +/- 2 h (mean +/- S.D.), before being subjected to xenogenous gamete intrafallopian transfer (XGIFT). An average of 13 +/- 0.8 oocytes and 40x10(3) spermatozoa per oocyte were transferred into 20 oviducts of ewes. Fourteen percent of transferred oocytes (36/259) were recovered between 2 and 7 days post-XGIFT and 36% of those recovered displayed embryonic development ranging from the 2-cell to the blastocyst stage. Fertilization following XGIFT was also demonstrated by the detection of zinc finger protein Y (ZFY) loci. Ligation of the uterotubal junction (UTJ), ovarian structures, or the duration of oviductal incubation did not significantly affect the frequency of embryonic development or recovery of oocytes/embryos after XGIFT. In conclusion, equine embryos can be produced in a smaller non-equine species that is easier for handling. PMID:14662137

  9. Genomic DNA methylation patterns in bovine preimplantation embryos derived from in vitro fertilization

    Institute of Scientific and Technical Information of China (English)

    HOU Jian; LIU Lei; LEI TingHua; CUI XiuHong; AN XiaoRong; CHEN YongFu

    2007-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplantation embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methylation level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.

  10. Genomic DNA methylation patterns in bovine preim-plantation embryos derived from in vitro fertilization

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplanta-tion embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methyla-tion level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.

  11. Effect of melatonin on maturation capacity and fertilization of Nili-Ravi buffalo (Bubalus bubalis oocytes

    Directory of Open Access Journals (Sweden)

    G. Nagina

    2016-08-01

    Full Text Available This study evaluated the effect of melatonin supplementation of in vitro maturation media on in vitro maturation (IVM and in vitro fertilization (IVF rate of buffalo oocytes. Cumulus oocytes complexes (COCs were aspirated from follicles of 2-8 mm diameter. In experiment I, COCs were matured in IVM medium supplemented with 0 (control, 250, 500, and 1000 μM melatonin for 22-24 hours in CO2 incubator at 38.5°C with 5% CO2 and at 95% relative humidity. The maturation rate did not differ in media supplemented with melatonin at 250 μM, 500 μM, 1000 μM and control (0 μM. In experiment II, the matured oocytes were fertilized in 50 μl droplets of Tyrode’s Albumin Lactate Pyruvate (TALP medium having 10 ug/ml heparin for sperm (2 million/ml capacitation. The fertilization droplets were then kept for incubation at 5% CO2, 39°C and at 95% relative humidity for 18 hours. The fertilization rate was assessed by sperm penetration and pronuclear formation. Fertilization rate was improved when maturation medium was supplemented with 250 μM melatonin compared to control. In conclusion, melatonin supplementation to serum free maturation media at 250 μM improved the fertilization rate of buffalo oocytes.

  12. Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

    Directory of Open Access Journals (Sweden)

    Jaqueline Sudiman

    Full Text Available Developmental competence of in vitro matured (IVM oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15 or growth differentiation factor (GDF9 to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(PH, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2% compared to controls (43.3±2.4%, 28.9±3.7% and to mature GDF9+FSH (36.1±3.0%. The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(PH, and reduced glutathione (GSH levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

  13. Effective Oocyte Vitrification and Survival Techniques for Bovine Somatic Cell Nuclear Transfer.

    Science.gov (United States)

    Park, Min Jee; Lee, Seung Eun; Kim, Eun Young; Lee, Jun Beom; Jeong, Chang Jin; Park, Se Pill

    2015-06-01

    Bovine somatic cell nuclear transfer (SCNT) using vitrified-thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with feeder cells that had been preincubated for 15 h significantly improved the survival of VT oocytes and their in vitro developmental potential following SCNT in comparison to co-culture with feeder cells that had been preincubated for 2, 5, or 24 h (pEVT) group, 13.7%; VT group, 15.0%; p<0.05] and was comparable with that of the non-V group (25.9%). The reactive oxygen species level was significantly lower in the EAVT group than in the other vitrification groups (p<0.05). mRNA levels of maternal genes (ZAR1, BMP15, and NLRP5) and a stress gene (HSF1) were lower in the vitrification groups than in the non-V group (p<0.05), whereas the level of phospho-p44/42 mitogen-activated protein kinase did not differ among the groups. Among the vitrification groups, blastocysts in the EAVT group had the best developmental potential, as judged by their high mRNA expression of developmental potential-related genes (POU5f1, Interferon-tau, and SLC2A5) and their low expression of proapoptotic (CASP3) and stress (Hsp70) genes. This study demonstrates that SCNT using bovine frozen-thawed oocytes can be successfully achieved using optimized vitrification and co-culture techniques. PMID:25984830

  14. Comparison between the characteristics of follicular fluid and the developmental competence of bovine oocytes.

    Science.gov (United States)

    Iwata, H; Inoue, J; Kimura, K; Kuge, T; Kuwayama, T; Monji, Y

    2006-02-01

    There are great differences in the developmental competence of oocytes collected from individual cows. Oocytes grow and mature in the follicular fluid (FF). In the present study, characteristics of the FF of each ovary and the developmental competence of enclosed oocytes were investigated, and these data were then compared. A total of 37 pairs of ovaries were collected from beef heifers. The concentration of magnesium (Mg), aspirate aminotransferase (AST), nonesterified fatty acids (NEFA), and lactate dehydrogenase (LDH) in the FF were great compared with serum standard. Several significant correlations among these characteristics were detected. Forty-eight hours after fertilization, the stage of embryo development at an advanced developmental stage (>6 cell stage) is related to the rate of blastulation 8 days after fertilization. In addition, a significantly positive or negative correlation was observed between the developmental competence (the rate of cleavage in the embryo and blastulation) and the concentration of the icterus index (ICT) or blood urea nitrogen (BUN) in the FF. In conclusion, the quality of oocytes is affected by the environment in the follicle, and BUN or ICT is a predictable index of the developmental competence of oocytes. PMID:15961265

  15. The Effect of Lysophosphatidic Acid during In Vitro Maturation of Bovine Oocytes: Embryonic Development and mRNA Abundances of Genes Involved in Apoptosis and Oocyte Competence

    OpenAIRE

    Dorota Boruszewska; Ana Catarina Torres; Ilona Kowalczyk-Zieba; Patricia Diniz; Mariana Batista; Luis Lopes-da-Costa; Izabela Woclawek-Potocka

    2014-01-01

    In the present study we examined whether LPA can be synthesized and act during in vitro maturation of bovine cumulus oocyte complexes (COCs). We found transcription of genes coding for enzymes of LPA synthesis pathway (ATX and PLA2) and of LPA receptors (LPAR 1–4) in bovine oocytes and cumulus cells, following in vitro maturation. COCs were matured in vitro in presence or absence of LPA (10−5 M) for 24 h. Supplementation of maturation medium with LPA increased mRNA abundance of FST and GDF9 i...

  16. Grb10 characterization in bovine cumulus oocyte complexes from different follicle sizes

    Directory of Open Access Journals (Sweden)

    Paulo Roberto Antunes da Rosa

    2015-05-01

    Full Text Available The objective of this study was to investigate the mRNA expression and protein localization of Grb10 gene in bovine cumulus-oocyte complexes (COCs from different follicle sizes. Firstly, it was investigated the mRNA expression to correlate with maturation rates. COCs from follicles at 1-3, 4-6, 6-8 and >8mm were used to evaluate Grb10 gene expression by qRT-PCR assay and nuclear maturation rates. It was observed that more competent oocytes (from follicles at 6-8 and >8mm; P>0.05, had lower Grb10 mRNA expression levels when compared to the oocytes from follicles at 1-3 and 4-6mm (P>0.05. After it was performed an immunofluorescence analysis in COCs from different follicle sizes (1-3, 4-6, 6-8 and >8mm to investigate Grb10 protein localization. Samples were incubated with primary antibody: Polyclonal rabbit anti-Grb10 (1:100. Primary antibody was detected using goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:500. Positive fluorescence signal was detected in all analyzed samples but less evident in COCs from largest follicles. These results characterized Grb10 gene in bovine COC and provide evidences for its involvement during oocyte molecular maturation.

  17. A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa

    OpenAIRE

    Abdalla, H; Shimoda, M; M. Hirabayashi; Hochi, S

    2009-01-01

    Regardless of the presence of sperm-borne oocyte-activating factors, activation of bovine oocytes with exogenous activation stimuli is required for further development after intracytoplasmic sperm injection (ICSI). The current study was designed to develop a new activation regimen for improving the blastocyst yield after ICSI of bovine oocytes harvested from ovaries stored at 10 to 12 degrees C for 24 h. After ICSI, oocytes were treated with 5 mu M ionomycin for 5 min, 7% ethanol for 5 or 10 ...

  18. Effects of the reproductive status on morphological oocyte quality and developmental competence of oocytes after in vitro fertilization and somatic cell nuclear transfer in cat.

    Science.gov (United States)

    Naoi, H; Agung, B; Karja, N W K; Wongsrikeao, P; Shimizu, R; Taniguchi, M; Otoi, T

    2008-04-01

    This study was conducted to examine the effects of the reproductive cycle of donor cat on the quality of oocytes at recovery and developmental competence of oocytes after in vitro fertilization (IVF) and somatic cell nuclear transfer (NT). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular or luteal stages. After collection of oocytes, the oocytes were classified into four grades according to the morphological condition of oocyte cytoplasm and cumulus cells. A total of 16 558 oocytes were obtained from 198 ovarian pairs. The total mean numbers of oocytes and the mean numbers of oocytes with high quality (grade I) were significantly higher in ovarian pairs at the inactive stage (111.1 and 19.0 oocytes, respectively) than in ovarian pairs at the follicular stage (67.1 and 11.4 oocytes, respectively). A significant difference in the proportions of oocytes with grade I out of the total examined oocytes was observed between the follicular and luteal stages of ovaries (14.9% vs 20.2%, p quality of oocytes at recovery, irrespective of the reproductive status of ovaries. Moreover, there were no significant differences in the proportions of cleavage and development to the blastocyst stage of IVF and NT embryos among three oestrous stages of ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries has no apparent effects on the in vitro development of oocytes after IVF and NT, but the quality of oocytes at recovery influences the development of IVF embryos. PMID:18325005

  19. Fertilization of IVF/ICSI Using Sibling Oocytes from Couples with Subfertile Male or Unexplained Infertility

    Institute of Scientific and Technical Information of China (English)

    李志凌; 林虹; 肖婉芬; 王玉莲

    2004-01-01

    The significance of the performance of conventional in vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) using sibling oocytes from couples with subfertile male or unexplained infertility was evaluated. A total of 410 sibling oocyte cumulus-corona complexes (OCCC)from 21 couples with subfertile male (group A) and 11 unexplained infertile couples (group B) were randomly divided, in order of retrieval, into two groups inseminated either by conventional IVF or by ICSI. The treatment outcomes and the influence of infertility factors on fertilization in each group were compared. The results showed that although the two pronuclear (2PN) fertilization rate per injected sibling oocytes was significantly higher after ICSI (group A: 68.2 % ±28.8 %; group B: 66.2 %±24.9 %) than after conventional IVF (group A: 41.8 %±32.7 %; group B: 40. 1 %±22.1 %), the other variables studied included: the fertilization rates of per allocated sibling oocytes IVF/ICSI, the fertilization rates of sibling oocytes IVF/ICSI after excluding failed IVF fertilization cycles, as well as the cleavage rates of normal fertilization were not statistically significant (P>0.05). Similarly, though the total fertilization failure rate in the IVF group (group A: 42.9 %;group B: 36.4 %) was significantly higher than in the ICSI group (group A: 4.8 %; group B: 0),we did not cancel cycles due to the normal fertilization of sibling oocytes. Embryo transfer was possible in all 32 couples. There were 10 clinical pregnancies in the two groups. We also discovered a possible association between some semen parameters and sperm functions of group A, and women age and duration of infertility of group B and fertilization. It is suggested that adoption of the split IVF/ICSI technology in the above cases may help eliminate fertilization failures. This is also a useful method to investigate the effect of single factor on the employment of assisted reproductive tech nology.

  20. 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.

  1. Effects of fetal bovine serum and estrus buffalo serum on maturation of buffalo (Bubalus bubalis) oocytes in vitro

    OpenAIRE

    Gopal Puri; S. S. Chaudhary; Singh, V. K.; Sharma, A. K.

    2015-01-01

    Aim: The aim was to assess the effects of fetal bovine serum (FBS) and estrus buffalo serum (EBS) on in vitro maturation rate of oocytes in buffalo. Materials and Methods: Maturation rate of oocytes was assessed in two maturation media supplemented with 20% FBS and EBS. Oocytes maturation rate was evaluated on the basis of cumulus cell expansion and extrusion of polar body after 24 h of in vitro culture in CO2 incubator. Results: The average percentage of in vitro matured oocytes in FBS...

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

    LENUS (Irish Health Repository)

    Mamo, Solomon

    2011-03-16

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

  3. Oocyte vitrification in the 21st century and post-warming fertility outcomes: a systematic review and meta-analysis.

    Science.gov (United States)

    Potdar, Neelam; Gelbaya, Tarek A; Nardo, Luciano G

    2014-08-01

    Oocyte cryopreservation is a rapidly developing technology, which is increasingly being used for various medical, legal and social reasons. There are inconsistencies in information regarding survival rate and fertility outcomes. This systematic review and meta-analysis provides evidence-based information about oocyte survival and fertility outcomes post warming to help women to make informed choices. All randomized and non-randomized, controlled and prospective cohort studies using oocyte vitrification were included. The primary outcome measure was ongoing pregnancy rate/warmed oocyte. Sensitivity analysis for donor and non-donor oocyte studies was performed. Proportional meta-analysis of 17 studies, using a random-effects model, showed pooled ongoing pregnancy and clinical pregnancy rates per warmed oocyte of 7%. Oocyte survival, fertilization, cleavage, clinical pregnancy and ongoing pregnancy rates per warmed oocyte were higher in donor versus non-donor studies. Comparing vitrified with fresh oocytes, no statistically significant difference was observed in fertilization, cleavage and clinical pregnancy rates, but ongoing pregnancy rate was reduced in the vitrified group (odds ratio 0.74), with heterogeneity between studies. Considering the age of women and the reason for cryopreservation, reasonable information can be given to help women to make informed choices. Future studies with outcomes from oocytes cryopreserved for gonadotoxic treatment may provide more insight. PMID:24931362

  4. Effect of Equilibration Temperature on In vitro Viability and Subsequent Embryo Development of Vitrified-Warmed Immature Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    Hajarian Hadi

    2010-01-01

    Full Text Available Problem statement: Vitrification is replacing conventional slow freezing to cryopreserve gametes and embryos especially for in vitro production of embryo in domestic animal species. However, the results are still not satisfactory. The aim of this experiment was to study the effect of different equilibration temperatures on in vitro viability of immature bovine oocytes after vitrification. Approach: Oocytes were obtained from slaughterhouse ovaries. Only grade one oocytes were used. Oocytes were equilibrated in three different temperatures: 32, 37, or 41°C. Immature oocytes were equilibrated in VS1 (7.5 Ethylene Glycol (EG + 7.5% DMSO for 10-12 min and then exposed to VS2 (15% EG + 15%DMSO + 0.5M sucrose for one min. Thereafter oocytes were loaded on hand-made Cryotop and directly plunged into liquid nitrogen. After warming, oocytes were examined for viability, maturation, cleavage and blastocyst production. Results: Oocytes that were equilibrated at 37°C had significantly higher (pConclusion: In conclusion, these results indicated that immature bovine oocytes can be equilibrated successfully at 37°C while higher or lower temperature can significantly decrease their subsequent viability and development.

  5. Effects of Ooplasmic Transfer on Rabbit Oocyte Fertilization and Early Embryonic Development

    Institute of Scientific and Technical Information of China (English)

    LI Jun-feng; SONG Yan-hua; LI Hai-feng; WANG Xian-zhong; ZHANG Jia-hua

    2005-01-01

    In order to evaluate the effects of ooplasm on oocyte fertilization and early embryonic development and to study the mitochondrial DNA (mtDNA) heterogeneity of early embryos, microinjection was first performed to transfer a small amount (5 to 7%) of donor ooplasm into recipient oocytes, then the eggs were fertilized with rabbit sperm through intracytoplasmic sperm injection (ICSI). In group 1 (homogeneous ooplasmic transfer), both the donor and recipient rabbit oocytes were at metaphase Ⅱ (MⅡ). In group 2 (heterogeneous ooplasmic transfer), the donor was mouse MⅡ oocyte and the recipient was rabbit MⅡ oocyte. In the control group, only ICS! was done on rabbit oocyte without ooplasmic transfer.No significant difference (P>0.05) was observed in blastocyst development rates between group 1 (13.0%, 3/23) and the control group (16.7%, 4/24), but significant difference (P<0.05) was examined in blastocyst development rate between group 2 (0, 0/27) and the control group. Blastomeres cleaved unequally and embryonic fragments increased after ooplasmic transfer and ICSI. In early embryos, in group 2, donor mouse mtDNA was detected in 2-cell embryos (3/3), 4-cell embryos(3/4), 8-cell embryos (4/4), and morulae (2/2). The mtDNA fingerprinting analysis showed that mouse mtDNA detected in heterogeneous embryos of different developmental stages had exactly the same sequence as that of the donor mouse mtDNA, thus indicating that homogenous ooplasmic transfer had no significant influence on rabbit oocyte fertilization and early embryonic development, and that heterogeneous ooplasmic transfer did cause notable reduction in blastocyst development rate. Heterogeneous mtDNA sequence in early embryos did not mutate. Compared with the control group,the embryonic quality declined after ooplasmic transfer operation in the present experiment.

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

    Directory of Open Access Journals (Sweden)

    Mahdi Mahdipour

    Full Text Available Transforming acidic coiled-coil (TACC proteins are key players during mitosis via stabilization of the spindle. The roles of TACCs during meiosis are however less clear. We used bovine oocytes to study the expression and function of TACC3 during meiosis. TACC3 mRNA was detected in bovine oocytes during meiosis using qRT-PCR, and while it was also expressed in cleavage stage embryos, its expression was down-regulated at the morula and blastocyst stages. Immunofluorescence was used to demonstrate that TACC3 co-localized with tubulin in the metaphase I and II spindles. However, TACC3 was not detected at anaphase or telophase of the first meiotic division. Aurora A, which is known to phosphorylate and activate TACC3 in mitotic cells, showed a similar pattern of gene expression to that of TACC3 in meiotic oocytes and preimplantation embryos. Aurora A protein was however only very transiently associated to the meiotic spindle. Pharmaceutical inhibition of Aurora A activity inhibited TACC3 phosphorylation but did not prevent TACC3 appearance in the spindle. Inhibiting Aurora A activity did however lead to abnormal meiotic spindle formation and impaired maturation of bovine oocytes. Similar results were obtained by knock-down of TACC3 expression using siRNA injection. These results suggest that TACC3 is important for stabilizing the meiotic spindle, but phosphorylation of TACC3 by Aurora A is not required for its recruitment to the meiotic spindle although phosphorylation of TACC3 by other kinases cannot be excluded.

  7. Disruption of bovine oocytes and preimplantation embryos by urea and acidic pH.

    Science.gov (United States)

    Ocon, O M; Hansen, P J

    2003-04-01

    Feeding cattle diets high in degradable crude protein (CP) or in excess of requirements can reduce fertility and lower uterine pH. Objectives were to determine direct effects of urea and acidic pH during oocyte maturation and embryonic development. For experiment 1, oocytes were matured in medium containing 0, 5, 7.5, or 10 mM urea (0, 14, 21, or 28 mg/dl urea nitrogen, respectively). Cleavage rate was not reduced by any concentration of urea. However, the proportion of oocytes developing to the blastocyst stage at d 8 after insemination was reduced by 7.5 mM urea. In addition, the proportion of cleaved oocytes becoming blastocysts was decreased by 5 and 7.5 mM urea. For experiment 2, putative zygotes were collected -9 h after insemination and cultured in modified Potassium Simplex Optimized Medium (KSOM). Urea did not reduce the proportion of oocytes developing to the blastocyst stage, although 10 mM urea reduced cleavage rate slightly. For experiment 3, dimethadione (DMD), a weak nonmetabolizable acid, was used to decrease culture medium pH. Putative zygotes were cultured in modified KSOM containing 0, 10, 15, or 20 mM DMD for 8 d. DMD reduced cleavage rate at 15 and 20 mM and development to the blastocyst stage at all concentrations. Results support the idea that feeding diets rich in highly degradable CP compromises fertility through direct actions of urea on the oocyte and through diet-induced alterations in uterine pH.

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

    Science.gov (United States)

    Liu, X Johné

    2016-01-01

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

  9. Apoptotic effects on maturation of mouse oocytes, fertilization and fetal development by puerarin.

    Science.gov (United States)

    Huang, Fu-Jen; Chan, Wen-Hsiung

    2016-10-01

    Previously we identified puerarin, an isoflavone compound, as a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to retardation of embryonic development and cell viability. In the current study, we investigated whether puerarin exerts deleterious effects on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, puerarin caused significant impairment of these processes in vitro. Pre-incubation of oocytes with puerarin during in vitro maturation led to increased post-implantation embryo resorption and decreased mouse fetal weight. In an in vivo animal model, intravenous injection with or without puerarin (1, 3 and 5 mg/kg body weight/day) for 4 days caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked puerarin-triggered deleterious effects, clearly implying that embryonic injury induced by puerarin is mediated by a caspase-dependent apoptotic mechanism. These results clearly demonstrate that puerarin has deleterious effects on mouse oocyte maturation, fertilization and subsequent embryonic development in vitro and in vivo. PMID:26712108

  10. Oocyte Source and Hormonal Stimulation for In Vitro Fertilization Using Sexed Spermatozoa in Cattle

    Directory of Open Access Journals (Sweden)

    Giorgio A. Presicce

    2011-01-01

    Full Text Available The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.2% versus 12.8%, =.001. Ocytes derived from slaughterhouse (SH ovaries were also fertilized with sperm from the same bulls. Overall, non-sexed sperm used with oocytes derived from SH ovaries was significantly more efficient for blastocyst development than was sexed sperm with these same SH derived oocytes and sexed sperm with stimulated donor oocytes (39.8% versus 25.0% and 25.2%, =.001. In conclusion, the use of sexed sperm with OPU-derived oocytes resulted in a significantly higher blastocyst development when donors were hormonally stimulated; furthermore, the level of efficiency achieved was comparable to that attained when the same sexed sperm was tested on oocytes derived from SH ovaries.

  11. Transmembrane Signal Transduction in Oocyte Maturation and Fertilization: Focusing on Xenopus laevis as a Model Animal

    Directory of Open Access Journals (Sweden)

    Ken-ichi Sato

    2014-12-01

    Full Text Available Fertilization is a cell biological phenomenon of crucial importance for the birth of new life in a variety of multicellular and sexual reproduction species such as algae, animal and plants. Fertilization involves a sequence of events, in which the female gamete “egg” and the male gamete “spermatozoon (sperm” develop, acquire their functions, meet and fuse with each other, to initiate embryonic and zygotic development. Here, it will be briefly reviewed how oocyte cytoplasmic components are orchestrated to undergo hormone-induced oocyte maturation and sperm-induced activation of development. I then review how sperm-egg membrane interaction/fusion and activation of development in the fertilized egg are accomplished and regulated through egg coat- or egg plasma membrane-associated components, highlighting recent findings and future directions in the studies using Xenopus laevis as a model experimental animal.

  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

    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...... following in vitro maturation (IVM) of immature oocytes collected from medulla tissue, of which two survived vitrification and warming. This approach represents an add-on method to potentially augment the fertility opportunity for cancer patients, especially in young women with cancer where transplantation......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...

  13. 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.

  14. Effect of Follicle Size and Follicle Stimulating Hormone Concentration on Nuclear Maturation of Bovine Oocytes In Vitro

    Directory of Open Access Journals (Sweden)

    Uğur Şen

    2015-07-01

    Full Text Available The aim of the study was to investigate the effect of follicle size and follicle stimulating hormone (FSH concentration on nuclear maturation of bovine oocytes in vitro. Follicles on bovine ovary were classified into 3 groups according to the diameter; small (<3 mm, medium (3–8 mm and large (9–12 mm. Oocytes were aspirated from follicles with different size and matured in tissue culture medium (TCM–199 supplemented with 10% FCS and various concentrations of FSH (0.5, 1.0 or 10 and μg/ml for 22 hours filled with approximately 95% humidified and 5% CO2 in air at 38.5 °C. At the end of culture period, nuclear maturation (at metaphase II; MII of oocytes were determined by Bisbenzimide (Hoechst 33258 DNA staining under fluorescent microscope. In the present study, effect of follicle size on nuclear maturation of bovine oocytes were determined and the percentage of oocytes reached to M II stage was significantly lower in oocytes obtained small follicle than those of medium and large follicles. Supplementation of 10.0 μg/ml FSH into maturation media increased percentage of nuclear maturation compare to 0.5 and 1.0 μg/ml. Additionally, improving effect of high FSH concentration on nuclear maturation were more observed in oocytes obtained small follicles. The results of present study showed that oocytes from follicles with 3–8 mm diameters exhibited a more successful maturation, but oocytes obtained small follicles exhibited more maturation as a ratio under high FSH concentration.

  15. Effect of oviduct epithelial cells on the fertilization and development of sheep oocytes in vitro

    DEFF Research Database (Denmark)

    Holm, Peter; Irvine, Brendon J.; Armstrong, David T.;

    1994-01-01

    The study examined whether co-culture with oviductal epithelial cells was of benefit to ovine in vitro fertilization ( IVF) and embryo culture procedures utilizing ·a well charac- terized culture system based on a synthetic oviductal fluid medium (SOFM) supple- mented with serum in a 90% N2, 5% 0 2......, 5% C02, atmosphere at 38.6°C. Two experiments were carried out. In Experiment 1, comparison was made between the frequency of fertil- ization and development of in vitro matured ( IVM) oocytes cultured in the absence (Group 1) or presence of oviductal cells for a 24 h (Group 2), 48 h (Group 3) or 96...... h (Group 4) period post insemination. In Experiment 2, comparison was made between the develop- ment of IVM oocytes fertilized and cultured in vitro for 7. 5 days in the absence or presence of oviductal cells with IVM oocytes which had been fertilized in vitro for 20 h in the pres- ence of oviductal...

  16. 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.

  17. The Effect of Methamphetamine on Oocyte Quality, Fertilization Rate and Embryo Development in Mice

    Directory of Open Access Journals (Sweden)

    Maryam Nezhad Sistani

    2016-01-01

    Full Text Available Objective: Methamphetamine (METH is an illicit psychoactive drug. There are different reasons of abusing METH such as have afun and to develop sexual satisfaction. This study was designed to investigate the effects of short and long term use of METH on oocyte and embryo development in mice. Material and Method: In this study, 75 female NMRI mice were divided into five groups of 15. The groups consisted of a control group, experimental groups A and B, which received MA (10 mg/kg/day intraperitoneally for 2 and 14 consecutive days, respectively and two sham groups A and B, which only received saline. After the last injection, 10 IU PMSG and 10 IU HCG were administered intraperitoneally for induction ovulation. Then, all the mice were sacrificed to aspirate their oocytes for further evaluation. In-vitro fertilization was done by using mature oocytes and embryo developmentwas investigated to the blastocyst stage. Data analyzed by using SPSS and Non parametric Mann-Whitney test. Results: The number of fragmented oocytes in experimental group A was significantlyincreased in comparison with experimental group B and control group (P˂0.05. Also there were significant differences in the number of unfertilized oocytes and early cleavage stage embryos in experimental group B in comparison with other groups that Indicates the reduction offertilization ratein experimental group B (P˂0.05. Conclusion: Short-termadministrationof METH in mice can affect oocyte quality but had no effect on early embryo development,whilethelong-term administration may effect on oocyte and embryo development in early cleavage stage.

  18. 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.

  19. Preservation of fertility through cryopreservation and in vitro maturation of human ovarian follicles and oocytes

    OpenAIRE

    Hreinsson, Julius

    2003-01-01

    One of the most rapidly expanding fields in assisted reproduction is the preservation of fertility for young women at risk of premature ovarian failure. This may be caused by cytotoxic therapy or other reasons. Cryopreservation of follicles in ovarian tissue has been successful in animal models with live young being born. Furthermore, the survival of human ovarian follicles after cryopreservation and thawing has been shown. In vitro maturation of oocytes (IVM) is important f...

  20. A survey on oocyte donation: Turkish fertile and infertile women's opinions.

    OpenAIRE

    Akyüz, Aygül; Seven, Memnun; Karaşahin, Emre; Güvenç, Gülten; Cek, Suzan

    2014-01-01

    Background: There are various treatment options for infertility, and new techniques are also being developed as it is an important healthcare problem affecting approximately 15-20% of married couples. The purpose of this study was to evaluate the level of information of fertile and infertile Turkish women on oocyte donation in order to understand their awareness of the legal, ethical, social and religious issues regarding this technique and to compare these two groups in terms of these variab...

  1. Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals.

    Science.gov (United States)

    Karabinova, Pavla; Kubelka, Michal; Susor, Andrej

    2011-10-01

    Gametogenesis and fertilization are the key events in sexual reproduction. In the female, meiosis results in a large oocyte that is competent for fertilization and fundamental for the success of early embryonic development. Progression through meiosis is monitored by fine regulatory mechanisms. In this review, we focus on one of the most well-known regulatory elements, the E3 ligase APC/C, which mediates proteolytic degradation of a number of important substrates via the ubiquitin proteasome pathway (UPP). The UPP also indirectly regulates protein synthesis by affecting proteins involved in RNA metabolism, a process that is paramount for the transcriptionally silent oocyte. During the past few years, more evidence has accumulated to suggest that the UPP has an important role in zona pellucida penetration and gamete fusion in mammals. This review focuses on the function of the UPP in regulating oocyte meiotic maturation in mammals, with special attention to its role in chromosome segregation and polar body extrusion, its role in the acquisition of meiotic/developmental competence and recent advances in our understanding of the UPP role in fertilization.

  2. The Effects of Murine Cytomegalovirus on the Maturation,Fertilization, Cleavage and Blastula Formation of Mouse Oocytes In Vitro

    Institute of Scientific and Technical Information of China (English)

    WANG Xinrong; ZHANG Xinhong; CHEN Suhua; ZHU Guijin; AI Jihui

    2007-01-01

    To study the effects of mouse cytomegalovirus (MCMV) on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages (100 TCID50, 10 TCID50 and 1 TCID50). The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID50 of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.

  3. The influence of macrophage co-culture to the fertility rate and blastocyst formation rate of the mouse's oocytes

    Institute of Scientific and Technical Information of China (English)

    Chen Mei; Sun Zheng-yi; Yu Qi; He Fang-fang

    2007-01-01

    Objective: To investigate the effect of co-culture with peritoneal macrophage to the fertility rate and blastocyst formation rate of the mouse's oocytes.Methods: A total of 15 KunMing mice aged 10-14 weeks were undergone ovulation induction and oocytes collection.The oocytes were randomly divided into three groups.The first group was the control group and cultured without macrophage; the second group (experimental group 1) was co-cultured with macrophages right after oocytes collection; the third group (experimental group 2) was cultured alone after oocytes retrieval, and co-cultured with macrophages from 8 cell stage.Results: 1.The fertility rate in control group (91.0%) and experimental group 2 (93.7%) was significantly higher than that in experimental group 1 (72.0%, P<0.001).2.Blastocyst formation rate of the fertilized oocytes in the experimental group 2 (78.2%) was much higher than control group (60.3%, P<0.001).3.Blastocyst formation rate of the oocytes in experimental group 2 (73.2%) was much higher than control group (54.8%) and experimental group 1 (50.0%), which were statistically significant.Conclusions: 1.Co-culture with peritoneal macrophages had an adverse influence on the fertilization process.2.Peritoneal macrophages could enhance the mouse embryonic development and increase the blastocyst formation rate.

  4. Determining fertility in a bovine subject comprises detecting in a sample from the bovine subject the presence or absence of genetic marker alleles associated with a trait indicative of fertility of the bovine subject and/or off-spring

    DEFF Research Database (Denmark)

    2009-01-01

    for determining fertility in a bovine subject; and selecting bovine subjects for breeding purposes (all claimed). DETAILED DESCRIPTION - Determining fertility in a bovine subject comprises detecting in a sample from the bovine subject the presence or absence of two or more genetic marker alleles......NOVELTY - Determining fertility in a bovine subject comprises detecting in a sample from the bovine subject the presence or absence of two or more genetic marker alleles that are associated with a trait indicative of fertility of the bovine subject and/or off-spring. USE - The methods are useful...... that are associated with a trait indicative of fertility of the bovine subject and/or off-spring, where the two or more genetic marker alleles are single nucleotide polymorphisms selected from Hapmap60827-rs29019866, ARS-BFGL-NGS-40979, Hapmap47854-BTA-119090, ARS-BFGL-NGS-114679, Hapmap43841-BTA-34601, Hapmap43407...

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Laser-assisted in vitro fertilization facilitates fertilization of vitrified-warmed C57BL/6 mouse oocytes with fresh and frozen-thawed spermatozoa, producing live pups.

    Science.gov (United States)

    Woods, Stephanie E; Qi, Peimin; Rosalia, Elizabeth; Chavarria, Tony; Discua, Allan; Mkandawire, John; Fox, James G; García, Alexis

    2014-01-01

    The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen

  7. Laser-assisted in vitro fertilization facilitates fertilization of vitrified-warmed C57BL/6 mouse oocytes with fresh and frozen-thawed spermatozoa, producing live pups.

    Directory of Open Access Journals (Sweden)

    Stephanie E Woods

    Full Text Available The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W oocytes. Laser-assisted in vitro fertilization (LAIVF facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762 of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1 LAIVF using V-W oocytes, 2 LAIVF using fresh oocytes, 3 conventional IVF using V-W oocytes and 4 conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml. LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298 and fresh oocytes (69%, 135/197, compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively. Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343 using V-W oocytes (P<0.05, compared to fresh spermatozoa, and 73% (195/266 using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168 and fresh (5%, 15/323 oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784, advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908. Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen

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

    Directory of Open Access Journals (Sweden)

    Wei Shi

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

  9. Compromised fertility disrupts Peg1 but not Snrpn and Peg3 imprinted methylation acquisition in mouse oocytes

    Directory of Open Access Journals (Sweden)

    Michelle M Denomme

    2012-07-01

    Full Text Available Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERβ and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20-80 μm control oocytes at three imprinted genes, Snrpn, Peg3 and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition, individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3 or Peg1 in oocytes from Ers2-deficient females, and no perturbation in Snrpn or Peg3 de novo methylation in oocytes from Gja4-null females. However, Gja4-deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development.

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

    Science.gov (United States)

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

    2016-10-01

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

  11. DNA methylation and mRNA expression profiles in bovine oocytes derived from prepubertal and adult donors.

    Science.gov (United States)

    Diederich, Mike; Hansmann, Tamara; Heinzmann, Julia; Barg-Kues, Brigitte; Herrmann, Doris; Aldag, Patrick; Baulain, Ulrich; Reinhard, Richard; Kues, Wilfried; Weissgerber, Christian; Haaf, Thomas; Niemann, Heiner

    2012-09-01

    The developmental capacity of oocytes from prepubertal cattle is reduced compared with their adult counterparts, and epigenetic mechanisms are thought to be involved herein. Here, we analyzed DNA methylation in three developmentally important, nonimprinted genes (SLC2A1, PRDX1, ZAR1) and two satellite sequences, i.e. 'bovine testis satellite I' (BTS) and 'Bos taurus alpha satellite I' (BTαS). In parallel, mRNA expression of the genes was determined by quantitative real-time PCR. Oocytes were retrieved from prepubertal calves and adult cows twice per week over a 3-week period by ultrasound-guided follicular aspiration after treatment with FSH and/or IGF1. Both immature and in vitro matured prepubertal and adult oocytes showed a distinct hypomethylation profile of the three genes without differences between the two types of donors. The methylation status of the BTS sequence changed according to the age and treatment while the methylation status of BTαS sequence remained largely unchanged across the different age and treatment groups. Relative transcript abundance of the selected genes was significantly different in immature and in vitro matured oocytes; only minor changes related to origin and treatment were observed. In conclusion, methylation levels of the investigated satellite sequences were high (>50%) in all groups and showed significant variation depending on the age, treatment, or in vitro maturation. To what extent this is involved in the acquisition of developmental competence of bovine oocytes needs further study. PMID:22733804

  12. Analysis of mRNA associated factors during bovine oocyte maturation and early embryonic development.

    Science.gov (United States)

    Siemer, Corinna; Smiljakovic, Tatjana; Bhojwani, Monika; Leiding, Claus; Kanitz, Wilhelm; Kubelka, Michal; Tomek, Wolfgang

    2009-12-01

    Regulation of gene expression at the translational level is particularly essential during developmental periods, when transcription is impaired. According to the closed-loop model of translational initiation, we have analyzed components of the 5 -mRNA cap-binding complex eIF4F (eIF4E, eIF4G, eIF4A), the eIF4E repressor 4E-BP1, and 3 -mRNA poly-(A) tail-associated proteins (PABP1 and 3, PAIP1 and 2, CPEB1, Maskin) during in vitro maturation of bovine oocytes and early embryonic development up to the 16-cell stage. Furthermore, we have elucidated the activity of distinct kinases which are potentially involved in their phosphorylation. Major phosphorylation of specific target sequences of PKA, PKB, PKC, CDKs, ATM/ATR, and MAPK were observed in M II stage oocytes. Furthermore, main changes in the abundance and/or phosphorylation of distinct mRNA-binding factors occur at the transition from M II stage oocytes to 2-cell embryos. In conclusion, the results indicate that, at the transition from oocyte to embryonic development, translational initiation is regulated by striking differences in the abundance and/or phosphorylation of 5 -end and 3 -end mRNA associated factors, mainly the poly-(A) bindings proteins PABP1 and 3, their repressor PAIP2 and a Maskin-like protein with distinct eIF4E-binding properties which prevents eIF4E/cap binding and eIF4F formation in vitro. Nevertheless, from the M II stage to 16-cell embryos a substantial amount of eIF4E and, to a lesser extent, of eIF4G was precipitated by (7)m-GTP-Separose indicating eIF4F complex formation. Therefore, it is likely that in general the reduction in PABP1 and 3 abundance represses overall translation during early embryonic development.

  13. 牛卵母细胞体外成熟减数分裂进程及核型变化的研究%Research on the Bovine Oocyte Meiosis in-vitro Maturation and Karyotype Changes

    Institute of Scientific and Technical Information of China (English)

    吐逊·吾守尔

    2016-01-01

    To further understand the bovine oocyte meiosis in-vitro maturation and karyotype changes along with the process, this paper studies the recovery status of meiosis in the process of maturation and the corresponding karyotype by the regular method of bovine oocyte in-vitro maturation and karyotype analysis. It is showed that the recovery rate of meiosis reaches 45.26% when bovine oocyte matures in vitro for 8 hours, while the rate reaches to 81.63%and 91.67%for 12 hours and 24 hours respectively, which demonstates that oocytes can grow and reach to the status prior to fertilization with normal meiosis process during the period of in-vitro maturation of bovine oocytes, and that the form of cellula karyotype and its rules in all period of meiosis can be identified clearly when the oocytes are dyed, which provides reference for the study of molecular mechanisms of bovine oocyte meiosis maturation and the improvement of efficiency of in-vitro maturation.%为了更加深入了解牛卵母细胞体外成熟减数分裂及其伴随该进程中核型的变化,实验采用常规的牛卵母细胞体外成熟方法及核型染色方法对成熟过程中减数分裂恢复情况及其对应的核型进行了研究.结果表明:牛卵母细胞体外成熟8h减数分裂抑制恢复率达45.26%,成熟12h时绝大部分卵母细胞(81.63%)已经恢复了减数分裂抑制状态,正常成熟24h后GVBD率达91.67%.说明在对牛卵母细胞进行体外成熟过程中卵母细胞可以按照正常的减数分裂进程发育至受精前阶段,经染色后可以较为清楚识别减数分裂各时期细胞核核型形态及其变化规律,为研究牛卵母细胞体外成熟减数分裂抑制恢复的分子机制探讨及体外成熟效率的提高提供了参考价值.

  14. Complete in vitro generation of fertile oocytes from mouse primordial germ cells.

    Science.gov (United States)

    Morohaku, Kanako; Tanimoto, Ren; Sasaki, Keisuke; Kawahara-Miki, Ryouka; Kono, Tomohiro; Hayashi, Katsuhiko; Hirao, Yuji; Obata, Yayoi

    2016-08-01

    Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells. PMID:27457928

  15. Influence of L-arginine during bovine in vitro fertilization.

    Science.gov (United States)

    Silva, Thiago Velasco Guimarães; da Silva, Bruno Baraúna; de Sá, André Luiz Alves; da Costa, Nathalia Nogueira; Sampaio, Rafael Vilar; Cordeiro, Marcela da Silva; Santana, Priscila Di Paula Bessa; Adona, Paulo Roberto; Santos, Simone do Socorro Damasceno; Miranda, Moysés dos Santos; Ohashi, Otávio Mitio

    2014-12-01

    The objective of this work was to evaluate the effect of using L-arginine during in vitro fertilization (IVF) on in vitro embryonic development using Bos taurus and Bos indicus semen. Effect of different concentrations (0, 1, 10 and 50 mM) of L-arginine, added to the IVF medium, was evaluated on the fertilization rate at 18 h post-fertilization (hpf), NO3(-)/NO2(-) production during IVF by the Griess colorimetric method (30 hpf), cleavage and blastocyst rates (on Day 2 and Day 7 of culture, respectively) and total blastocyst cell number (Day 7 of culture). The results reveal that the addition of 50 mM L-arginine to IVF medium, with either Bos taurus or Bos indicus spermatozoa, decreased the cleavage rate and blastocyst rate compared to the control group. Other concentrations did not affect embryo production. However, 1 mM L-arginine with Bos indicus semen increased the proportion of hatched blastocysts. These results indicate that high L-arginine concentrations may exhibit toxic effects on bovine gametes during in vitro fertilization. PMID:25651608

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

    Directory of Open Access Journals (Sweden)

    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

  17. Effect of phosphorus 32 on mouse oocytes: fertility, histological changes and genetic injury

    International Nuclear Information System (INIS)

    Sexually mature female mice received single intraperitoneal treatment with 50 microcurie/mouse radioactive phosphorus. The fertility in cross-breeds with intact mice was followed up over a period of one month. The number of oocytes in the primordial follicles decreased at the end of the first and of the second month after treatment, the prenatal losses occurred in the first progeny from irradiated female animals. Underthe ndicated conditions of internal phosphorus 32 irradiation the fertility of females dropped to half that of the controls. The histologic examinations, carried out at the end of the first month, showed a marked depletion of the oocyte stock of primordial follicles (9,3 per cent of the controls) and complete disappearance at the end of the second month. The prenatal loss of the first generation - expression of radiation induced genetic injury - was twice as high as the spontaneous one. The relation between the results of the three parameters of radiation injury in this study is discussed. (author)

  18. Effects of green tea polyphenols, insulin-like growth factor I and glucose on developmental competence of bovine oocytes

    Directory of Open Access Journals (Sweden)

    Zhengguang Wang

    2012-12-01

    Full Text Available The present study examined the effects of green tea polyphenols (GTP, insulin-like growth factor-I (IGF-I and glucose on oocyte in vitro maturation, subsequent embryo development and blastocyst quality in bovine. Cumulus-oocyte complexes (COC were aspirated from the ovaries and cultured in synthetic oviduct fluid supplemented with MEM amino acids (SOFaa media supplemented with one of the following supplements: GTP (0, 10, 15 and 20 µM, IGF-I (0, 50, 100 and 150 ng/mL or glucose (0, 1.5, 5.6 and 20 mM for 24 h. The results showed that oocytes cultured in media supplemented with 15 µM GTP, 100 ng/mL IGF-I and 5.6 mM glucose, in separate experiments, have higher cleavage and blastocyst rates compared with oocytes cultured in media without or with other concentration of GTP, IGF-I and glucose. Then these three substances with the concentration above were added together into SOFaa media and constituted a modified medium (Modified SOFaa. The COC were cultured in control SOFaa media and modified SOFaa media, respectively. The results showed that modified SOFaa media increased the intracellular glutathione concentration of matured oocytes, blastocyst rates and total cell numbers and cell numbers of inner cell mass per blastocyst compared with the control. Supplementing of GTP, IGF-I and glucose synchronously to maturation media can increase the intracellular GSH concentration of oocytes after in vitro maturation, and improve the embryo development and blastocyst quality in bovine.

  19. Effect of growth factors on oocyte maturation and allocations of inner cell mass and trophectoderm cells of cloned bovine embryos.

    Science.gov (United States)

    Arat, Sezen; Caputcu, Arzu Tas; Cevik, Mesut; Akkoc, Tolga; Cetinkaya, Gaye; Bagis, Haydar

    2016-08-01

    This study was conducted to determine the additive effects of exogenous growth factors during in vitro oocyte maturation (IVM) and the sequential culture of nuclear transfer (NT) embryos. Oocyte maturation and culture of reconstructed embryos derived from bovine granulosa cells were performed in culture medium supplemented with either epidermal growth factor (EGF) alone or a combination of EGF with insulin-like growth factor-I (IGF-I). The maturation rates of oocytes matured in the presence of EGF or the EGF + IGF-I combination were significantly higher than those of oocytes matured in the presence of only fetal calf serum (FCS) (P 0.05). IGF-I alone or in combination with EGF in sequential embryo culture medium significantly increased the ratio of inner cell mass (ICM) to total blastocyst cells (P media of cloned bovine embryos increased the ICM without changing the total cell number. These unknown and uncontrolled effects of growth factors can alter the allocation of ICM and trophectoderm cells (TE) in NT embryos. A decrease in TE cell numbers could be a reason for developmental abnormalities in embryos in the cloning system. PMID:26444069

  20. Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming.

    Directory of Open Access Journals (Sweden)

    José F W Sprícigo

    Full Text Available The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM. Four different maturation systems were tested: 1 in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136; 2 in vitro maturation using immature oocytes obtained by ovum pick-up (OPU from unstimulated heifers (IMA; n = 433; 3 in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444; and 4 in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658. A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT, while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P0.05 for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%. MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1 + H]+, which was more highly expressed in MII compared to FSH (P<0.05. The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.

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

    Directory of Open Access Journals (Sweden)

    Jolanta Opiela

    2014-01-01

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

  2. Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess.

    Directory of Open Access Journals (Sweden)

    Adam F Summers

    Full Text Available Aspiration of bovine follicles 12-36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; mean = 102 and another with Low A4 (<20 ng/ml; mean = 9 in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold, A4 (158-fold and testosterone (31-fold in the dominant follicle of High A4 cows. However, estrone (3-fold and estradiol (2-fold concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07. There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07 to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.

  3. 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

  4. Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection (ICSI) using ejaculated and testicular spermatozoa

    Institute of Scientific and Technical Information of China (English)

    Yun Qian; Ting Feng; Juan Chen; Ling-Bo Cai; Jia-Yin Liu; Yun-Dong Mao; Wei Ding; Jia-Hao Sha

    2005-01-01

    Aim: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). Methods: Fifty-six completed cycles in 46women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners.Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. Results: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours,75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %,P < 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4% (216/247) vs. 88.9% (32/36); 21.3% (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. Conclusion: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.

  5. 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.

  6. In Vitro Developmental Potential of Cloned Embryos Derived from Bovine Somatic Cells and Rabbits Oocyte

    Institute of Scientific and Technical Information of China (English)

    LIU Ya; LI Bin; ZHAO Huan; CHENG Li-zi; ZHANG Xiao-rong; CHEN Da-yuan; ZHANG Yun-hai; ZHANG Zhi-guo; JING Ren-tao; WANG Cun-li; ZHANG Mei-lin; LI Dong-wei

    2003-01-01

    180 reconstituted embryos were produced by nuclear transplantation using bovine ear fibroblasts at G0 or non-G0 stage as donor nuclei and oocytes collected from superovulated multiparous or young rabbits as recipients. After cultivation in two kinds of medium M199+ 10%FBS or RD+ 10%FBS, 112 of them developed to 2-cell stage (62.2%) and 26 to morula stage (14.4%) and 20 of them eventually developed to blastocyst stage (11. 1% ). There is no significant difference for the cleavage rates in two groups of reconstituted embryos derived from G0-stage and non-G0 stage donor cells respectively. However, G0-stage donor cells could result in higher rate of 8-cell - 16-cell stage embryos significantly (P<0.05), as well as higher rate of blastocysts (P<0.01). It seems that using two different culture systems had no significant effects on the cleavage rate, morula rate or blastocyst rate (P>0.05).

  7. Effect of Somatic Cell Types and Culture Medium on in vitro Maturation, Fertilization and Early Development Capability of Buffalo Oocytes

    Directory of Open Access Journals (Sweden)

    H. Jamil*, H. A. Samad, N. Rehman, Z. I. Qureshi and L. A. Lodhi

    2011-04-01

    Full Text Available This study was designed to evaluate the efficacy of different somatic cell types and media in supporting in vitro maturation (IVM, in vitro fertilization (IVF and early embryonic development competence of buffalo follicular oocytes. Cumulus oocyte complexes were collected for maturation from follicles (>6mm of buffalo ovaries collected at the local abattoir. Oocytes were co-cultured in tissue culture medium (TCM-199 with either granulosa cells, cumulus cells, or buffalo oviductal epithelial cells (BOEC @ 3x106 cells/ml or in TCM-199 without helper cells (control at 39°C and 5%CO2 in humidified air. Fresh semen was prepared in modified Ca++ free Tyrode medium. Fertilization was carried out in four types of media: i Tyrode lactate albumin pyruvate (TALP, ii TALP+BOEC, iii modified Ca++ free Tyrode and iv modified Ca++ free Tyrode+BOEC. Fertilized oocytes were cultured for early embryonic development in TCM-199 with and without BOEC. Higher maturation rates were observed in the granulosa (84.24% and cumulus cells (83.44% than BOEC co culture system (73.37%. Highest fertilization rate was obtained in modified Ca++ free Tyrode with BOEC co culture (70.42%, followed by modified Ca++ free Tyrode alone (63.77%, TALP with BOEC (36.92% and TALP alone (10.94%. Development of early embryos (8-cell stage improved in TCM-199 with BOEC co culture than TCM-199 alone. From the results of this study, it can be concluded that addition of somatic cells (granulosa cells, cumulus cells results in higher maturation rates of buffalo follicular oocytes than BOEC co culture system, while fertilization rate improved in modified Ca++ free Tyrode with and without BOEC. Addition of BOEC to TCM-199 improved the developmental capacity of early embryo.

  8. Effect of Rat Medicated Serum Containing You Gui Wan on Mouse Oocyte In Vitro Maturation and Subsequent Fertilization Competence

    Directory of Open Access Journals (Sweden)

    Xiao-Hui Jiang

    2014-01-01

    Full Text Available You Gui Wan (YGW is a classic herbal formula in traditional Chinese medicine (TCM used for the clinical treatment of infertility. This study was to explore whether YGW has an impact on mouse oocyte maturation in vitro and subsequent fertilization competence. Rat medicated serum containing YGW was prepared by orally administrating YGW. Mouse immature oocytes were cultured with YGW medicated serum and compared to those cultured with or without normal rat serum or follicle-stimulating hormone (FSH. YGW medicated serum significantly increased the percentages of matured oocytes when compared to the groups with or without normal rat serum (P < 0.01. Furthermore, YGW medicated serum increased the rate of in vitro fertilization (IVF when compared to the groups treated with FSH and with or without normal rat serum (P < 0.001. YGW medicated serum also had significant effects on the mRNA expressions of PKA, CREB, MAPK, PKC, PKG, and MPF and the concentrations of cAMP, cGMP, and NO in matured oocytes. These results indicate that YGW can promote mouse oocyte maturation and IVF in vitro. Signaling pathways, such as the cAMP/PKA/MAPK, the PKC-MAPK, and the NO-cGMP-PKG pathway, which are similar to those induced by FSH, may be responsible for this action.

  9. Developmental competence of oocytes isolated from surplus medulla tissue in connection with cryopreservation of ovarian tissue for fertility preservation

    DEFF Research Database (Denmark)

    Wilken-Jensen, Helle N; Kristensen, Stine G; Jeppesen, Janni V;

    2014-01-01

    OBJECTIVE: Evaluating the developmental competence of immature oocytes collected from surplus medulla tissue in connection with ovarian tissue cryopreservation for fertility preservation. DESIGN: Cohort comparative study. SETTING: University laboratory in Denmark from 2011-2012. POPULATION: 69...... on the ovaries, and collected from the saline solution, containing surplus medulla tissue, following dissection of the ovarian cortical tissue for cryopreservation. The immature oocytes were cultured for 48 h in an Embryoscope™ Time-lapse System or in culture dishes overlaid with liquid paraffin using commercial...

  10. 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.

  11. Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos Efeito do fator de crescimento semelhante à insulina-1 durante a maturação in vitro dos oócitos e cultivo in vitro de embriões bovinos

    OpenAIRE

    M.D. Quetglas; L.A Coelho; Garcia, J M; E.B. Oliveira Filho; C.R. Esper

    2001-01-01

    The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) and on in vitro embryo development (experiment II) of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR), blastocyst (BR) and hatching (HR) rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium) and gonadotropins (14 U/ml PMSG and 7 U/ml hCG)....

  12. Prospective Randomized Study on the Influence of Myoinositol in PCOS Women Undergoing IVF in the Improvement of Oocyte Quality, Fertilization Rate, and Embryo Quality

    Science.gov (United States)

    Lesoine, Bernd

    2016-01-01

    Polycystic ovarian syndrome (PCOS) is one of the pathological factors involved in the failure of in vitro fertilization (IvF). The aim of the present study was to investigate if the combination of myoinositol + folic acid was able to improve the oocyte quality, the ratio between follicles and retrieved oocytes, the fertilization rate, and the embryo quality in PCOS patients undergoing IvF treatments. 29 patients with PCOS underwent IvF protocols for infertility treatment and were randomized prospectively into two groups. Group A (placebo) with 15 patients and group B (4000 mg myoinositol + 400 μg folic acid per day) with 14 patients. The patients of group B used for two months myoinositol + folic acid before starting the IvF protocol and data were obtained concerning number of follicles, number of oocytes, quality of oocytes, fertilization rates, and embryo quality in both groups. The ratio follicle/retrieved oocyte was better in the myoinositol group (= group B). Out of the 233 oocytes collected in the myoinositol group 136 were fertilized, whereas only 128 out of 300 oocytes in the placebo group were fertilized. More metaphase II and I oocytes were retrieved in relation to the total amount of oocytes in the myoinositol. More embryos of grade I quality were obtained in the myoinositol. The duration of stimulation was 9,7 days (±3,3) in the myoinositol group and 11,2 (±1,8) days in the placebo group and the number of used FSH units was lower in the myoinositol group: 1750 FSH units (mean) versus 1850 units (mean). Our evidence suggests that myoinositol therapy in women with PCOS results in better fertilization rates and a clear trend to a better embryo quality. As the number of retrieved oocytes was smaller in the myoinositol group, the risk of hyper stimulation syndrome can be reduced in these patients. PMID:27635136

  13. Prospective Randomized Study on the Influence of Myoinositol in PCOS Women Undergoing IVF in the Improvement of Oocyte Quality, Fertilization Rate, and Embryo Quality

    Directory of Open Access Journals (Sweden)

    Bernd Lesoine

    2016-01-01

    Full Text Available Polycystic ovarian syndrome (PCOS is one of the pathological factors involved in the failure of in vitro fertilization (IvF. The aim of the present study was to investigate if the combination of myoinositol + folic acid was able to improve the oocyte quality, the ratio between follicles and retrieved oocytes, the fertilization rate, and the embryo quality in PCOS patients undergoing IvF treatments. 29 patients with PCOS underwent IvF protocols for infertility treatment and were randomized prospectively into two groups. Group A (placebo with 15 patients and group B (4000 mg myoinositol + 400 μg folic acid per day with 14 patients. The patients of group B used for two months myoinositol + folic acid before starting the IvF protocol and data were obtained concerning number of follicles, number of oocytes, quality of oocytes, fertilization rates, and embryo quality in both groups. The ratio follicle/retrieved oocyte was better in the myoinositol group (= group B. Out of the 233 oocytes collected in the myoinositol group 136 were fertilized, whereas only 128 out of 300 oocytes in the placebo group were fertilized. More metaphase II and I oocytes were retrieved in relation to the total amount of oocytes in the myoinositol. More embryos of grade I quality were obtained in the myoinositol. The duration of stimulation was 9,7 days (±3,3 in the myoinositol group and 11,2 (±1,8 days in the placebo group and the number of used FSH units was lower in the myoinositol group: 1750 FSH units (mean versus 1850 units (mean. Our evidence suggests that myoinositol therapy in women with PCOS results in better fertilization rates and a clear trend to a better embryo quality. As the number of retrieved oocytes was smaller in the myoinositol group, the risk of hyper stimulation syndrome can be reduced in these patients.

  14. Prospective Randomized Study on the Influence of Myoinositol in PCOS Women Undergoing IVF in the Improvement of Oocyte Quality, Fertilization Rate, and Embryo Quality

    Science.gov (United States)

    Lesoine, Bernd

    2016-01-01

    Polycystic ovarian syndrome (PCOS) is one of the pathological factors involved in the failure of in vitro fertilization (IvF). The aim of the present study was to investigate if the combination of myoinositol + folic acid was able to improve the oocyte quality, the ratio between follicles and retrieved oocytes, the fertilization rate, and the embryo quality in PCOS patients undergoing IvF treatments. 29 patients with PCOS underwent IvF protocols for infertility treatment and were randomized prospectively into two groups. Group A (placebo) with 15 patients and group B (4000 mg myoinositol + 400 μg folic acid per day) with 14 patients. The patients of group B used for two months myoinositol + folic acid before starting the IvF protocol and data were obtained concerning number of follicles, number of oocytes, quality of oocytes, fertilization rates, and embryo quality in both groups. The ratio follicle/retrieved oocyte was better in the myoinositol group (= group B). Out of the 233 oocytes collected in the myoinositol group 136 were fertilized, whereas only 128 out of 300 oocytes in the placebo group were fertilized. More metaphase II and I oocytes were retrieved in relation to the total amount of oocytes in the myoinositol. More embryos of grade I quality were obtained in the myoinositol. The duration of stimulation was 9,7 days (±3,3) in the myoinositol group and 11,2 (±1,8) days in the placebo group and the number of used FSH units was lower in the myoinositol group: 1750 FSH units (mean) versus 1850 units (mean). Our evidence suggests that myoinositol therapy in women with PCOS results in better fertilization rates and a clear trend to a better embryo quality. As the number of retrieved oocytes was smaller in the myoinositol group, the risk of hyper stimulation syndrome can be reduced in these patients.

  15. Prospective Randomized Study on the Influence of Myoinositol in PCOS Women Undergoing IVF in the Improvement of Oocyte Quality, Fertilization Rate, and Embryo Quality.

    Science.gov (United States)

    Lesoine, Bernd; Regidor, Pedro-Antonio

    2016-01-01

    Polycystic ovarian syndrome (PCOS) is one of the pathological factors involved in the failure of in vitro fertilization (IvF). The aim of the present study was to investigate if the combination of myoinositol + folic acid was able to improve the oocyte quality, the ratio between follicles and retrieved oocytes, the fertilization rate, and the embryo quality in PCOS patients undergoing IvF treatments. 29 patients with PCOS underwent IvF protocols for infertility treatment and were randomized prospectively into two groups. Group A (placebo) with 15 patients and group B (4000 mg myoinositol + 400 μg folic acid per day) with 14 patients. The patients of group B used for two months myoinositol + folic acid before starting the IvF protocol and data were obtained concerning number of follicles, number of oocytes, quality of oocytes, fertilization rates, and embryo quality in both groups. The ratio follicle/retrieved oocyte was better in the myoinositol group (= group B). Out of the 233 oocytes collected in the myoinositol group 136 were fertilized, whereas only 128 out of 300 oocytes in the placebo group were fertilized. More metaphase II and I oocytes were retrieved in relation to the total amount of oocytes in the myoinositol. More embryos of grade I quality were obtained in the myoinositol. The duration of stimulation was 9,7 days (±3,3) in the myoinositol group and 11,2 (±1,8) days in the placebo group and the number of used FSH units was lower in the myoinositol group: 1750 FSH units (mean) versus 1850 units (mean). Our evidence suggests that myoinositol therapy in women with PCOS results in better fertilization rates and a clear trend to a better embryo quality. As the number of retrieved oocytes was smaller in the myoinositol group, the risk of hyper stimulation syndrome can be reduced in these patients. PMID:27635136

  16. MicroRNA expression profile in bovine cumulus-oocyte complexes during late oogenesis

    Science.gov (United States)

    During late oogenesis, the mammalian oocyte synthesizes and stores mRNA necessary to guide the early stages of embryo development prior to the activation of embryonic transcription. The oocyte also contains many post-transcriptional regulatory mechanisms that coordinate mRNA stability and translati...

  17. Cumulus Cell Transcripts Transit to the Bovine Oocyte in Preparation for Maturation

    DEFF Research Database (Denmark)

    Macaulay, Angus D; Gilbert, Isabelle; Scantland, Sara;

    2016-01-01

    the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocyte's polyribosomes...

  18. Effects of Saffron (Crocus sativus L. Aqueous Extract on In vitro Maturation, Fertilization and Embryo Development of Mouse Oocytes

    Directory of Open Access Journals (Sweden)

    Poopak Eftekhari-Yazdi

    2012-01-01

    Full Text Available Objective: Lower pregnancy rates of in vitro matured oocytes compared to those of invivo stimulated cycles indicate that optimization of in vitro maturation (IVM remains achallenge. Reduced developmental competence of in vitro matured oocytes shows thatcurrent culture systems for oocyte maturation do not adequately support nuclear and/orcytoplasmic maturation. Therefore this study evaluates the effects of different concentrationsof saffron (Crocus sativus L. aqueous extract (SAE, as an antioxidant agent on IVMof immature mouse oocytes.Materials and Methods: In this experimental study ,cumulus-oocyte complexes (COCswere collected from 6-8 weeks old novel medical research institute (NMRI female miceovaries. COCs were cultured in IVM medium supplemented with 0 (control, 5, 10, 20 and40 μg/ml of SAE in 5% CO2 at 37°C. The rates of maturation, fertilization and developmentwere recorded. ANOVA and Duncan’s protected least significant test, using the SASprogram was applied for all statistical analysis.Results: The maturation rate was significantly higher in all groups treated with differentconcentrations of SAE compared with the control group (p<0.05. However, the lowerconcentrations of SAE (10 and 5 μg/ml in maturation medium respectively increased thefertilization rate of oocytes and in vitro developmental competence when compared withthe control group (p<0.05.Conclusion: The results of this study indicate that lower concentrations of SAE are moreappropriate to be added to maturation medium when compared with other experimental andcontrol groups. Generally, we conclude that addition of appropriate amounts of natural extractssuch as SAE to maturation medium improves oocyte maturation and embryo development.

  19. 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...

  20. TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

    Science.gov (United States)

    Carvacho, Ingrid; Ardestani, Goli; Lee, Hoi Chang; McGarvey, Kaitlyn; Fissore, Rafael A.; Lykke-Hartmann, Karin

    2016-01-01

    The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development. PMID:27681336

  1. Follicular progesterone concentrations and messenger RNA expression of MATER and OCT-4 in immature bovine oocytes as predictors of developmental competence.

    Science.gov (United States)

    Urrego, R; Herrera-Puerta, E; Chavarria, N A; Camargo, O; Wrenzycki, C; Rodriguez-Osorio, N

    2015-04-15

    The ability of bovine embryos to develop to the blastocyst stage and to implant and generate healthy offspring depends greatly on the competence of the oocyte. Oocyte competence is attributed to its close communication with the follicular environment and to its capacity to synthesize and store substantial amounts of messenger RNA. Higher developmental competence of bovine oocytes has been associated with both the expression of a cohort of developmental genes and the concentration of sex steroids in the follicular fluid. The aim of this study was to identify differences in the expression of FST in cumulus cells and OCT-4 and MATER in oocytes and the influence of the follicular progesterone and follicular estrogen concentration on the competence of bovine oocytes retrieved 30 minutes or 4 hours after slaughter. Cumulus-oocyte complexes (COCs) were left in postmortem ovaries for 30 minutes (group I) or 4 hours (group II) at 30 °C. Aspirated oocytes were then subjected to IVM, IVF, and IVC or were evaluated for MATER and OCT-4 messenger RNA abundance by quantitative real-time polymerase chain reaction. Total RNA was isolated from pools of 100 oocytes for each experimental replicate. Progesterone and estradiol concentration in follicular fluid was evaluated by immunoassay using an IMMULITE 2000 analyzer. Three repeats of in vitro embryo production were performed with a total of 455 (group I) and 470 (group II) COCs. There were no significant differences between the cleavage rates (72 hours postinsemination [hpi]) between both groups (63.5% vs. 69.1%). However, blastocyst (168 hpi) and hatching (216 hpi) rates were higher (P < 0.05) in group II compared with those of group I (21.3% vs. 30.7% and 27.6% vs. 51.5%, respectively). Group II oocytes exhibited the highest MATER and OCT-4 abundance (P < 0.05). Follicular estradiol concentration was not different between both the groups, whereas the progesterone concentration was lower (P ≤ 0.05) in group II follicles. These

  2. Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

    Science.gov (United States)

    Bernstein, Lori R; Mackenzie, Amelia C L; Lee, Se-Jin; Chaffin, Charles L; Merchenthaler, István

    2016-03-01

    Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women. PMID:26713784

  3. In Vitro Maturation, Fertilization and Embryo Culture of Oocytes Obtained from Vitrified Auto-Transplanted Mouse Ovary

    Directory of Open Access Journals (Sweden)

    Arash Behbahanian

    2013-01-01

    Full Text Available Background: The purpose of this study was to investigate the in vitro survival and developmentalpotential of oocytes obtained from vitrified mouse ovaries transplanted to a heterotopic site.Materials and Methods: In this experimental study, two-week-old mice were unilaterallyovariectomized after anesthesia. The ovaries were vitrified by cryotop. After two weeks, the ovarieswere thawed and autotransplanted to the gluteus muscle tissue. Three weeks later the mice werekilled, after which we removed and dissected the transplanted and opposite right ovaries. Cumulusoocyte complexes (COCs and denuded oocytes were evaluated for in vitro maturation (IVM, invitro fertilization (IVF and in vitro development (IVD. The control group consisted of sevenweek-old age-matched mice ovaries.Results: All vitrified-transplanted (Vit-trans ovaries contained some oocytes that survived.Following IVM, IVF and IVD, there were 41.7% out of 12 cultured zygotes that reached the 8-cellstage.Conclusion: Our experiment supports the progressive role of long-term graft survival after wholeovariancryopreservation by vitrification and subsequent heterotopic transplantation. It is possible torecover viable follicles and oocytes that have the ability to develop in vitro.

  4. Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

    Science.gov (United States)

    Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

    2008-01-01

    The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had ???8 cells at 84 hpa, while 32% of the bovine NT embryos had ???8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had ???8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

  5. Follicular-fluid anti-Mullerian hormone (FF AMH is a plausible biochemical indicator of functional viability of oocyte in conventional in vitro fertilization (IVF cycles

    Directory of Open Access Journals (Sweden)

    Bindu N Mehta

    2013-01-01

    Full Text Available Context: Oocyte quality may be a governing factor in influencing in vitro fertilization (IVF outcomes. However, morphological evaluation of oocyte quality is difficult in conventional IVF cycles. Follicular-fluid (FF, the site for oocyte growth and development, has not yet been sufficiently explored to obtain a marker indicative of oocyte quality. Anti-Mullerian hormone (AMH is produced by granulosa cells of preantral and early-antral follicles and is released in FF. Aim: To investigate AMH as a biochemical indicator of functional viability/quality of oocyte produced in the FF micro-environmental milieu. Settings and Design: Prospective study involving 132 cycles of conventional IVF-embryo transfer (ET in infertile women. Subjects and Methods: AMH concentration was estimated in pooled FF on day of oocyte pickup. Cycles were sorted into low and high groups according to median (50 th centile values of measurement. Main outcome measure was oocyte viability, which included morphological assessment of oocyte quality, fertilization rate, clinical pregnancy, and implantation rates. Statistical Analysis: Graph-pad Prism 5 statistical package. Results: Low FF AMH group shows significantly higher percentage of top-quality oocytes (65.08 ± 24.88 vs. 50.18 ± 25.01%, P =0.0126, fertilization (83.65 ± 18.38 vs. 75.78 ± 21.02%, P =0.0171, clinical pregnancy (57.57 vs. 16.67%, P <0.0001, and embryo implantation rates (29.79 vs. 7.69%, P <0.0001 compared to high FF AMH group. FF AMH shares an inverse correlation with FF E2 (Pearson r = −0.43, r 2 = 0.18 and clinical pregnancy (Pearson r = −0.46, r 2 = 0.21. Threshold value of FF AMH for pregnancy is <1.750 ng/mg protein. Conclusion: FF AMH is a plausible biochemical indicator of functional viability of oocyte in conventional IVF cycles.

  6. Soluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway.

    Science.gov (United States)

    Nakano, Takeshi; Kyozuka, Keiichiro

    2015-12-01

    Matured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation. PMID:25318389

  7. Effects of oocyte quality, incubation time and maturation environment on the number of chromosomal abnormalities in IVF-derived early bovine embryos.

    Science.gov (United States)

    Demyda-Peyrás, Sebastian; Dorado, Jesus; Hidalgo, Manuel; Anter, Jaouad; De Luca, Leonardo; Genero, Enrique; Moreno-Millán, Miguel

    2013-01-01

    Chromosomal aberrations are one of the major causes of embryo developmental failures in mammals. The occurrence of these types of abnormalities is higher in in vitro-produced (IVP) embryos. The aim of the present study was to investigate the effect of oocyte morphology and maturation conditions on the rate of chromosomal abnormalities in bovine preimplantational embryos. To this end, 790 early cattle embryos derived from oocytes with different morphologies and matured under different conditions, including maturation period (24 v. 36h) and maturation media (five different serum supplements in TCM-199), were evaluated cytogenetically in three sequential experiments. The rates of normal diploidy and abnormal haploidy, polyploidy and aneuploidy were determined in each embryo. Throughout all the experiments, the rate of chromosomal abnormalities was significantly (P<0.05) affected by oocyte morphology and maturation conditions (maturation time and culture medium). Lower morphological quality was associated with a high rate of chromosome abnormalities (P<0.05). Moreover, polyploidy was associated with increased maturation time (P<0.01), whereas the maturation medium significantly (P<0.05) affected the rates of haploidy and polyploidy. In general, supplementing the maturation medium with oestrous cow serum or fetal calf serum resulted in higher rates of chromosomal aberrations (P<0.05) compared with the other serum supplements tested (bovine steer serum, anoestroues cow serum, bovine amniotic fluid and bovine serum albumin). On the basis of the results of the present study, we conclude that the morphological quality of oocytes and the maturation conditions affect the rate of chromosomal abnormalities in IVP bovine embryos.

  8. On-Chip Enucleation of Bovine Oocytes using Microrobot-Assisted Flow-Speed Control

    Directory of Open Access Journals (Sweden)

    Akihiko Ichikawa

    2013-06-01

    Full Text Available In this study, we developed a microfluidic chip with a magnetically driven microrobot for oocyte enucleation. A microfluidic system was specially designed for enucleation, and the microrobot actively controls the local flow-speed distribution in the microfluidic chip. The microrobot can adjust fluid resistances in a channel and can open or close the channel to control the flow distribution. Analytical modeling was conducted to control the fluid speed distribution using the microrobot, and the model was experimentally validated. The novelties of the developed microfluidic system are as follows: (1 the cutting speed improved significantly owing to the local fluid flow control; (2 the cutting volume of the oocyte can be adjusted so that the oocyte undergoes less damage; and (3 the nucleus can be removed properly using the combination of a microrobot and hydrodynamic forces. Using this device, we achieved a minimally invasive enucleation process. The average enucleation time was 2.5 s and the average removal volume ratio was 20%. The proposed new system has the advantages of better operation speed, greater cutting precision, and potential for repeatable enucleation.

  9. Bovine females with thecal cell androgen excess result in altered oocyte maternal effect gene abundance

    Science.gov (United States)

    In Nebraska, 1.9 million cows are calved/yr; however, 550,000 more (30%) are bred but fail to calve. If markers to identify females with reduced fertility identified even 5% of sub-fertile females this would reduce costs associated with their development and breeding. Within the UNL physiology herd,...

  10. Acrylamide toxic effects on mouse oocyte quality and fertility in vivo.

    Science.gov (United States)

    Duan, Xing; Wang, Qiao-Chu; Chen, Kun-Lin; Zhu, Cheng-Cheng; Liu, Jun; Sun, Shao-Chen

    2015-06-25

    Acrylamide is an industrial chemical that has attracted considerable attention due to its presumed carcinogenic, neurotoxic, and cytotoxic effects. In this study we investigated possible acrylamide reproductive toxic effects in female mice. Mice were fed an acrylamide-containing diet for 6 weeks. Our results showed the following effects of an acrylamide-containing diet. (1) Ovary weights were reduced in acrylamide-treated mice and oocyte developmental competence was also reduced, as shown by reduced GVBD and polar body extrusion rates. (2) Acrylamide feeding resulted in aberrant oocyte cytoskeletons, as shown by an increased abnormal spindle rate and confirmed by disrupted γ-tubulin and p-MAPK localization. (3) Acrylamide feeding resulted in oxidative stress and oocyte early stage apoptosis, as shown by increased ROS levels and p-MAPK expression. (4) Fluorescence intensity analysis showed that DNA methylation levels were reduced in acrylamide-treated oocytes and histone methylation levels were also altered, as H3K9me2, H3K9me3, H3K4me2, and H3K27me3 levels were reduced after acrylamide treatment. (5) After acrylamide feeding, the litter sizes of acrylamide-treated mice were significantly smaller compared to thus of control mice. Thus, our results indicated that acrylamide might affect oocyte quality through its effects on cytoskeletal integrity, ROS generation, apoptosis induction, and epigenetic modifications.

  11. Effect of exogenous glutathione in medium fertilization to improve blastocyst rates of bovine embryos

    Directory of Open Access Journals (Sweden)

    E Triwulaninngsih

    2002-06-01

    Full Text Available Glutathione (C10H17N3O6S is a tripeptide (γ-Glu-Cys-Gly widespread in living organism. Glutathione (GSH at the 5 mM concentration increased the motility and fertility of frozen-thawed sperm. Intracellulair glutathione improved the cleavage rate and embryo development to the blastocyst rate. Research on in vitro embryos production through the implementation of GSH during IVF (in vitro fertilization on embryo development has been conducted at the Laboratorium Reproductive of Physiology, Research Institute for Animal Production. Ovaries of beef cattle as source of oocytes were collected from the slaughterhouse in a thermo flask with 350C PBS as medium and transported to the laboratory. The oocytes were fertilized in vitro with selected motile sperm using Percoll gradient (90 and 45%. Ten COCs (cumulus oocytes complexes were transfered to 44 μl of fertilization medium (mTALP was performed with either 0; 0.25; 0.50; 0.75 and 1.00 mM of glutathione as treatment A, B, C, D and E respectively, and inseminated with 2 μl of capacitated sperm and added 2 μl of heparin and 2 μl of PHE (consisting of 20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine. Incubation between sperm and oocytes in the 5% CO2 incubator and RH 90% in fertilization media (TALP for 20 hours. All zygotes were cultured in modification of CR1aa medium up to blastocyst and were fed serum 5 μl/50μl medium on day 6. Results of this experiment showed that the effect of concentration of glutathione (0, 0.25; 0.50; 0.75 and 1.00 mM on fertilization medium to the percentage of cleavage rates were 69.35 + 29.40; 79.07 + 13.17; 67.88 + 10.65; 98.10 + 3.30 and 82.62 + 24.19% not significant different (P>0.05 among treatments A, B, C, D dan E respectively.The precentages of morula and blastocyst for treatment D were 50.45 + 42.64% and 31.28 + 24.28%; while 36.55 + 24.13% and 17.74 + 17.86% for treatment E respectively.

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

    Directory of Open Access Journals (Sweden)

    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

  13. Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 microm diode laser beam.

    Science.gov (United States)

    Germond, M; Nocera, D; Senn, A; Rink, K; Delacretaz, G; Pedrazzini, T; Hornung, J P

    1996-05-01

    The safety of microdrilling the zona pellucida of mouse oocytes with a 1.48 microm diode laser has been investigated by determining the ability of mouse oocytes to fertilize in vitro and develop in vivo. Mice born after transfer of control and zona pellucida-microdrilled embryos into foster mothers were submitted to anatomical and immunohistochemical investigations, and their aptitude to breed was assessed in two subsequent generations. Decoronization of the oocytes with hyaluronidase induced a reduction of the fertilization and implantation rates, which was attributed to a zona hardening phenomenon. After laser zona pellucida microdrilling, these rates were restored to those obtained with embryos derived from untreated oocyte-cumulus complexes. Pups derived from zona pellucida microdrilled embryos were comparable with those obtained from control embryos, confirming the lack of deleterious effects of the laser treatment. In conclusion, the 1.48 microm diode laser allows safe microdrilling of the zona pellucida of mouse oocytes after decoronization with hyaluronidase. Based on the health of the F2 generation and the lack of neuroanatomical and neurochemical differences, we concluded that this technology may be investigated in the human, particularly when the zona pellucida represents the main impediment for fertilization or embryo hatching.

  14. Strategies to support human oocyte development in vitro.

    Science.gov (United States)

    Telfer, Evelyn E; McLaughlin, Marie

    2012-01-01

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

  15. Lowering storage temperature during ovary transport is beneficial to the developmental competence of bovine oocytes used for somatic cell nuclear transfer.

    Science.gov (United States)

    Wang, Y S; Zhao, X; Su, J M; An, Z X; Xiong, X R; Wang, L J; Liu, J; Quan, F S; Hua, S; Zhang, Y

    2011-03-01

    The objective of this study was to determine the effect of storage temperature during ovary transport on the developmental competence of bovine oocytes for use in somatic cell nuclear transfer (SCNT). Ovaries obtained from a slaughterhouse were stored in physiological saline for 3-4h at one of the three temperatures: 15 °C, 25 °C, or 35 °C. The developmental competence of oocytes used for SCNT was ascertained by cleavage and blastocyst formation rate, total cell number, apoptosis index, and the relative abundance of Bax and Hsp70.1 in day 7 blastocysts. Ovaries stored at 35 °C for 3-4h reduced the recovery rate of grade I and II oocytes compared with those stored at 25 °C or 15 °C (45.1±0.7% vs. 76.7±1.2% or 74.8±2.0%, Povaries stored at 15 °C, however, produced blastocysts with higher cell numbers (97.3±8.6 vs. 80.2±10.8 or 77.4±11.7; Povaries stored at 15 °C was lower than those stored at 25 °C or 35 °C (Pquality and developmental competence of oocytes used for SCNT due to the alleviation of stresses on the oocytes compared with those subjected to storage temperatures of 25 °C or 35 °C. PMID:21333472

  16. Identification of Polymorphisms in the Enhancer Region of the Bovine Prolactin Gene and Association with Fertility in Beef Cows

    Science.gov (United States)

    Objectives were to investigate the polymorphic nature of the enhancer region of the bovine prolactin (PRL) gene and determine the association of these polymorphisms with fertility in beef cows. Primers were designed to amplify a 500 base pair fragment 892 to 1392 bases upstream of the bovine PRL gen...

  17. CARBENDAZIM (MBC) DISRUPTS OOCYTE SPINDLE FUNCTION AND INDUCES ANEUPLOIDY IN HAMSTERS EXPOSED DURING FERTILIZATION (MEIOSIS II)

    Science.gov (United States)

    Peri-fertilization exposure to Carbendazim (MBC; a microtubule poison) induces infertility and early pregnancy loss (EPL) in hamsters. resently, both in vivo and in vitro techniques were employed to characterize the effects of MBC on cellular aspects of fertilization in hamsters....

  18. The influence of follicle numbers per ovary on the quality of oocytes and the day length of forming blastocyte s by in vitro fertilization

    Directory of Open Access Journals (Sweden)

    Cece Sumantri

    1999-12-01

    Full Text Available The aim of this research is to know the influence of the number of follicles produced per ovary on the quality of oocytes and the day length of developing blastocytes by in vitro fertilization (IVF at 7-,8-, and 9-days. Material of the research is ovary of Fries Holland dairy cattle gathered from slaughterhouse. A number of 138 ovaries are grouped into four based on the number of follicles per ovary, including group I (≤5 follicles, group II (6-10 follicles, group III (11-20 follicles, and group IV (≥20 follicles. The result showed that the number of follicles per ovary are not significantly (P>0.05 influencing both on oocyte quality and the days length of forming blastocytes at 7-, 8-, and 9-days. Number of oocytes for grade A-B (percentage of the number of oocytes IVF per the number of oocytes obtained per ovary for group I - IV consecutively is 83.5%, 91.3%, 97.1%, and 91.2%. The number of developing blastocytes (percentage the number of blastocytes per the number of oocytes for group I - IV at 7-days consecutively is 5.19%, 5.56%, 5.45%, and 6.03%; at 8-days is 15.58%, 12.94 %, 13.64%, and 11.64%; at 9-days is 1818%, 8.96%, 8.18%, and 10.35%.

  19. Cryopreservation of oocytes

    International Nuclear Information System (INIS)

    Various approaches have been utilized in attempting to cryopreserve oocytes, beginning with slow cooling and more recently the advent of technique of vitrification. Now it seems that oocyte cryopreservation is no longer an experimental technique and it is being increasingly utilized in clinics around the world. As successful outcome in oocyte cryopreservation can be assessed by survival through the freeze-thaw process, potential for fertilization, embryo development and dynamics of meiotic spindles. This study aimed to analyse these features in context of vitrification and slow freezing. Methods: In this laboratory based study, mature MII mouse oocytes from F1(C57BL6/J X CBA) mice (n=43) were divided randomly into two groups of equal numbers and were cryopreserved by slow freezing and by vitrification. Upon re-warming these oocytes were assessed for survival and for fertilization potential. Oocytes were fixed and stained to compare the effect of both protocols on spindle reassembly and chromosome configuration 10min, 1h and 3h after warming. Unfrozen oocytes were used as controls. Results: A greater number of vitrified oocytes survived cryopreservation than slow frozen oocytes (70.3% vs. 12.5%, p=0.024). After insemination, fertilization rates were higher for vitrified oocytes as compared to slow frozen oocytes (15.86% vs. 4.6%, p=0.046). Morphology of the meiotic spindle was found to be in a disorganized configuration in slow frozen oocytes at all-time points 10 mins, 1 h and 3h), whereas in vitrified oocytes the spindles were found to be aligned at all-time points. Chromosomes were seen to be displaced from equatorial region in both groups. Conclusion: Cryopreservation of mouse oocytes was conducted with greater success using vitrification, compared to slow freezing, with survival, fertilization, and spindle assembly more favourable to a successful outcome in this model. (author)

  20. Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

    Science.gov (United States)

    van Tol, H T; van Eijk, M J; Mummery, C L; van den Hurk, R; Bevers, M M

    1996-10-01

    Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage. Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 microns vs. 240 microns in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group) nor on cumulus expansion (246 microns vs. 240 microns in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles. PMID:8914080

  1. Effects of pig follicular fluid on maturation of pig oocytes in vitro and on their subsequent fertilizing and developmental capacity in vitro.

    Science.gov (United States)

    Yoshida, M; Ishizaki, Y; Kawagishi, H; Bamba, K; Kojima, Y

    1992-07-01

    This study examines the effects of pig follicular fluid on the maturation of pig oocytes and on their subsequent fertilizing and developmental capacity in vitro. The addition of pig follicular fluid or its fractions obtained by ultrafiltration, gel filtration and ion-exchange chromatography to maturation medium significantly increased the rates of nuclear maturation, normal fertilization and normal cleavage of pig oocytes after fertilization in vitro: the rates of normal fertilization and cleavage were 2-4 times higher than those in the control medium. The efficacy of pig follicular fluid was lost after heating at 56 degrees C for 30 min, whereas no significant decrease in activity was observed after defatting. In addition, the effective component(s) was partially purified by ultrafiltration, gel filtration and ion-exchange chromatography: the activity was observed in the fraction (UF2; M(r) 10,000-20,000) obtained by ultrafiltration. Activity was found in the first fraction (G1) obtained by gel filtration of UF2. Among three fractions obtained by ion-exchange chromatography of G1, only the third fraction had the activity. The results indicate that pig follicular fluid contains an acidic substance(s) (M(r) 10,000-200,000) that promotes oocyte maturation.

  2. A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro fertilization procedure

    Institute of Scientific and Technical Information of China (English)

    Ge Lin; Qi OuYang; Xiaoying Zhou; Yifan Gu; Ding Yuan; Wen Li; Gang Liu; Tiancheng Liu; Guanexiu Lu

    2007-01-01

    Homozygous human embryonic stem cells (hESCs) are thought to be better cell sources for hESC banking because their human leukocyte antigen (HLA) haplotype would strongly increase the degree of matching for certain populations with relatively smaller cohorts of cell lines. Homozygous hESCs can be generated from parthenogenetic embryos, but only heterozygous hESCs have been established using the current strategy to artificially activate the oocyte without second polar body extrusion. Here we report the first successful derivation of a human homozygous ESC line (chHES-32) from a one-pronuclear oocyte following routine in vitro fertilization treatment. cAHES-32 cells express common markers and genes with normal hESCs. They have been propagated in an undifferentiated state for more than a year (>P50) and have maintained a stable karyotype of 46, XX. When differentiated in vivo and in vitro, c/zHES-32 cells can form derivatives from all three embryonic germ layers. The almost undetectable expression of five paternally expressed imprinted genes and their HLA genotype identical to the oocyte donor indicated their parthenogenetic origin. Using genome-wide single-nucleotide polymorphism analysis and DNA fingerprinting, the homozygosity of c/zHES-32 cells was further confirmed. The results indicated that 'unwanted' one-pronuclear oocytes might be a potential source for human homozygous and parthenogenetic ESCs, and suggested an alternative strategy for obtaining homozygous hESC lines from parthenogenetic haploid oocytes.

  3. 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].

  4. Kinetics of fertilization and development, and sex ratio of bovine embryos produced using the semen of different bulls.

    Science.gov (United States)

    Alomar, M; Tasiaux, H; Remacle, S; George, F; Paul, D; Donnay, I

    2008-08-01

    The between bulls variation in in vitro fertility and the shift of sex ratio towards male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the kinetics of fertilization, embryo development and the sex ratio of the resulting embryos using the frozen/thawed semen of four different bulls. In a first experiment, the kinetics of pronucleus (PN) formation was evaluated at 8, 12 and 18 h post-insemination (hpi). Based upon the pronuclei sizes and the distance between the two pronuclei, inseminated oocytes were classified in three PN stages. Differences between bulls were observed at each time point, but were more important at 12 hpi. At 8 and 12 hpi bull III showed a significantly faster PN evolution by comparison with the three other bulls (Pcinematography. The analysis of embryos reaching the blastocyst stage revealed significant differences in the mean time of first cleavage (range of 22.7-25.6h, P<0.05), while the lengths of the subsequent three cell cycles did not differ between bulls. The early mean time of first cleavage with bull III was associated with an early blastulation and a high blastocyst rate at Day 7, in opposition to what was observed with bull II showing a later timing of first cleavage (first cleavage 22.1 hpi versus 25.5 hpi; blastulation 140.4 hpi versus 152.5 hpi; D7 blastocyst rates: 31.3% versus 21.9%; P<0.05). In a third experiment, 65-76 Day 8 blastocysts per bull were sexed by PCR. Only blastocysts obtained with bull III showed a shift in sex ratio towards male embryos (76% male embryos; P<0.05). Such shift was already observed at the 2-cell and morula stages. In conclusion, the bull influences the kinetics of PN formation, of embryo development and the sex ratio of the embryos. Moreover, those parameters might be related. PMID:17629423

  5. PENICILLIN-STREPTOMYCIN IN THE CULTURE MEDIUM DURING IN VITRO MATURATION (IVM OF BOVINE OOCYTES AFFECTS NUCLEAR MATURATION AND SUBSEQUENT EMBRYO DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    SHIRAZI A

    2001-01-01

    Full Text Available Introduction: Standard concentrations of antibiotics in culture media are thought to have no detectable toxic effects on the cultured cells. However, since antibiotics are biologically active substances, the possibility that they interfere to some extent with cellular processes occurring in the cultured cells can not always be totally excluded. This study, therefore, was conducted to assess whether the presence of penicllin-streptomycin (pen-strep during in vitro maturation (IVM of bovine cumulus oocyte complexes (COCs affect nuclear and cytoplasmic maturation and subsequent embryo development. Materials and Methods: Bovine COCs were matured at 39oC in a humidified atmosphere with 5 % CO2 in air for 24 h in: 1- culture medium M 199 supplemented with 10 % FCS (Fetal calf serum, 0.05 IU/ml rhFSH (recombinant human FSH and 100 units penicillin and 100 ?g streptomycin/ ml. 2- culture medium M 199 without FCS and rhFSH in the presence of pen-strep. Cultures without antibiotics served as control. Six series of experiments, each consisted of at least 3 replicates, were performed. Results: In vitro maturation in the presence of pen-strep in culture medium supplemented with FCS and rhFSH significantly (P<0.05 increased the percentage of MII oocytes, however, when the COCs were divided, on the basis of appearance of the cumulus investment, into bright and dark groups, this effect was less obvious in both types of COCs, 76% vs 72% in bright COCs (P= 0.149 or 83% vs 80% in dark COCs (P=0.296 in treated and control groups respectively. The percentage of oocytes with type III of cortical granules (CGs distribution was not affected in the presence of pen-strep. The COCs expansion after IVM was not affected by the presence of antibiotics in culture medium. The subsequent embryo development of IVM/IVF produced ova, which were exposed to pen-strep during IVM, was significantly (P<0.05 decreased with respect to blastocyst formation by day 9. In vitro maturation in

  6. Cryopreservation of mammalian oocytes by using sugars: Intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates.

    Science.gov (United States)

    Eroglu, Ali

    2010-07-01

    Accumulation of intra- and extracellular sugars such as trehalose, glucose, and raffinose is central to survival strategies of a variety of organisms coping with extreme conditions including freezing and almost complete drying. The objective of the present study was to investigate the potential application of intra- and extracellular raffinose in combination with low concentrations of dimethylsulfoxide (Me(2)SO) to mammalian oocyte cryopreservation. To this end, the fertilization and embryonic development of cryopreserved metaphase II (M II) mouse oocytes were studied in comparison to unfrozen controls. For cryopreservation, M II oocytes were microinjected with 0.1M raffinose, and then cooled to -196 degrees C in the presence of either 0.3M raffinose and 0.5M Me(2)SO (cryopreservation group 1) or 0.3M raffinose and 1.0M Me(2)SO (cryopreservation group 2). The control groups included untreated oocytes (untreated control) and oocytes microinjected with raffinose, but not frozen (injection control). The post-thaw survival rates were 83.9% and 80.6% for the cryopreservation group 1 and 2, respectively. The fertilization and blastocyst rates in the cryopreservation group 1 (90.0% and 77.8%, respectively) and 2 (94.6% and 72.5%, respectively) were also high and similar to the ones of the injection controls (97.8% and 78.5%, respectively) and untreated controls (98.8% and 83.6%, respectively). These results are consistent with the findings of our earlier studies and support the use of sugars as intra- and extracellular cryoprotectants. Furthermore, the results of the present study indicate that the presence of intra- and extracellular sugars alleviates high concentrations of conventional penetrating cryoprotectants, and thus minimizes their toxicity.

  7. Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development.

    Science.gov (United States)

    Ashry, Mohamed; Lee, KyungBon; Mondal, Mohan; Datta, Tirtha K; Folger, Joseph K; Rajput, Sandeep K; Zhang, Kun; Hemeida, Nabil A; Smith, George W

    2015-03-01

    Brilliant cresyl blue (BCB) is a super-vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocyte's developmental potential, and the transcript abundance for select TGFβ-superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus-cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low-quality oocytes, based on BCB staining, was also determined. Cumulus-oocyte complexes (COCs) from abattoir-derived ovaries were subjected to BCB staining, and germinal-vesicle-stage oocytes and cumulus cells were harvested from control, BCB+, and BCB- (low-quality oocyte) groups for real-time PCR or Western-blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB- oocytes. Western-blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB- oocytes. Embryo-culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY-1 treatment, improve the developmental competence of BCB- oocytes. These results contribute to a better understanding of molecular indices of oocyte competence. PMID:25704641

  8. Evaluation of Embryos Derived from in vitro Fertilized Oocytes Reconstructed by Meiosis-II Chromosome Transplantation from Aged Mice to Ooplasms of Young Mice

    Directory of Open Access Journals (Sweden)

    Abdolhossein Shahverdi

    2010-01-01

    Full Text Available Background: To assess embryos derived by the transfer of meiosis-II chromosomes (M-II-t fromaged mice oocytes into ooplasms from younger mice to overcome the problem of age-relateddecline in female fertility.Materials and Methods: The developmental capacity, karyotype, and ultrastructure of reconstructedoocytes derived from meiosis-II chromosome transplantation from aged mice into the ooplasms ofyoung mice by piezo-micromanipulation were assessed.Results: The survival rate of enucleated young oocytes was 54% and the percent of fertilizedreconstructed oocytes was 23%. The rate of embryo development to the two-cell stage aftercultivation was 40%. Since 82.4% of the analyzed embryos derived from reconstructed oocyteshad condensed nuclei, it was not possible to analyze their chromosomal integrity. However, 17.6%of analyzable reconstructed old oocyte derived embryos (old-ODEs, had normal diploid sets ofchromosomes. Major structural differences were not observed between young, old, and M-II-tderived two-cell embryos.Conclusion: Our findings suggested that ooplasms from younger mice may overcome ageassociatedproblems in older mice.

  9. Birth after human chorionic gonadotropin-primed oocyte in vitro maturation and fertilization with testicular sperm in a normo-ovulatory patient

    Science.gov (United States)

    González-Ortega, Claudia; Piña-Aguilar, Raul Eduardo; Cancino-Villareal, Patricia; Gutiérrez-Gutiérrez, Antonio Martin

    2016-01-01

    In this report, we present a case of in vitro maturation (IVM) with surgical retrieved testicular sperm in a normo-ovulatory female. Human chorionic gonadotropin-primed IVM, testicular biopsy for sperm retrieval and intracytoplasmic sperm injection with fresh sperm were performed. Fourteen cumulus-oocyte complexes were obtained in germinal vesicle or metaphase I stage, eight oocytes reached metaphase II, seven presumptive zygotes were obtained, and three cleavage stages embryos in day 2 were transferred producing a singleton pregnancy. A single healthy newborn was obtained. Our results suggest that IVM may be an alternative for in vitro fertilization in normo-ovulatory women even if surgical retrieval of sperm is needed. Further research is required to depict contributing factors to the success of IVM in indications different from polycystic ovaries syndrome and the role of male gamete. PMID:27803591

  10. The effect of intrauterine inoculation with Ureaplasma diversum on bovine fertility.

    Science.gov (United States)

    Kreplin, C M; Ruhnke, H L; Miller, R B; Doig, P A

    1987-01-01

    To determine the influence of Ureaplasma diversum on bovine fertility 11 uninfected virgin heifers with normal ovarian cyclic activity were randomly allocated to test or control groups. At a synchronized estrus, five test heifers were given an intrauterine broth inoculum containing 1.09 x 10(8) to 1.4 x 10(9) colony forming units of U. diversum and six control animals were infused with sterile ureaplasma broth medium. All animals were artificially inseminated within one hour of infusion. Pregnancy was diagnosed in one of five test heifers and all of six controls by serum progesterone concentrations measured to 25 days postinsemination. The difference in pregnancy rates between the two groups was statistically significant (p = 0.0152). It was concluded that under the conditions of this experiment U. diversum is capable of causing infertility in cattle. PMID:3453263

  11. 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.

  12. The influence of follicle numbers per ovary on the quality of oocytes and the day length of forming blastocyte s by in vitro fertilization

    OpenAIRE

    Cece Sumantri; Anneke Anggraeni

    1999-01-01

    The aim of this research is to know the influence of the number of follicles produced per ovary on the quality of oocytes and the day length of developing blastocytes by in vitro fertilization (IVF) at 7-,8-, and 9-days. Material of the research is ovary of Fries Holland dairy cattle gathered from slaughterhouse. A number of 138 ovaries are grouped into four based on the number of follicles per ovary, including group I (≤5 follicles), group II (6-10 follicles), group III (11-20 follicles), an...

  13. Body composition, dietary carbohydrates and fatty acids determine post-fertilisation development of bovine oocytes in vitro.

    Science.gov (United States)

    Adamiak, S J; Powell, K; Rooke, J A; Webb, R; Sinclair, K D

    2006-02-01

    This study assessed the interactive effects of carbohydrate type (fibre vs starch) and fatty acid (FA) supplementation (0% vs 6% calcium soaps of palm oil FA) on the post-fertilisation development of oocytes recovered from low and moderate body condition score (BCS) heifers. A secondary objective was to compare the FA composition of plasma to that of granulosa cells (GCs) and cumulus-oocyte complexes (COCs) from these animals, and to relate these findings to the developmental potential of oocytes. Plasma, GCs and COCs were recovered from 32 heifers on day 5 of a synchronised oestrous cycle for FA analyses. Oocytes were also recovered on days 10 and 15 of the same cycle after short-term ovarian stimulation (FSH + GnRH), and matured, fertilised and cultured to the blastocyst stage in vitro. High levels of dietary starch increased (P < 0.01) plasma insulin but, together with dietary FA, reduced (P < 0.05) blastocyst yields in low, but not in moderate, BCS heifers. Diet-induced alterations to the FA content of plasma were less apparent in GCs and COCs. In summary, although dietary lipids increased the FA content of COCs, the selective uptake of saturated FAs at the expense of mainly polyunsaturated FAs within the follicular compartment ensured that the FA composition of COCs was largely unaffected by diet. However, the concentration of saturated FAs within COCs was inherently high, and so further increases in FA content may have impaired post-fertilisation development. The data establish a robust nutritional framework for more detailed studies into the mechanistic effects of dietary composition on the post-fertilisation developmental potential of oocytes. PMID:16452718

  14. Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes

    OpenAIRE

    Nakagawa, Yoshiko; Sakuma, Tetsushi; Sakamoto, Takuya; Ohmuraya, Masaki; NAKAGATA, Naomi; Yamamoto, Takashi

    2015-01-01

    Background Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing permits the rapid production of genetically engineered mice. To make the most of this innovative technology, a streamlined procedure is needed for the robust construction of CRISPR/Cas9 vectors, the efficient preparation of mouse oocytes, and refined genotyping strategies. Although we previously demonstrated the applicability of oocyte cryopreservation tech...

  15. Three-Day-Old Human Unfertilized Oocytes after In Vitro Fertilization/Intracytoplasmic Sperm Injection Can Be Activated by Calcium Ionophore A23187 or Strontium Chloride and Develop to Blastocysts

    OpenAIRE

    Liu, Ying; Han, Xiao-jie; Liu, Ming-hui; Wang, Shu-Yu; Jia, Chan-Wei; Yu, Lan; Ren, Guoqing; Wang, Li; Li, Wei

    2014-01-01

    Our objective was to observe the effectiveness of the calcium ionophore A23187 or strontium chloride on the activation and subsequent embryonic development of 3-day-old human unfertilized oocytes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). A total of 279 3-day-old unfertilized oocytes after IVF or ICSI were randomized to be activated by the calcium ionophore A23187 (n=138) or strontium chloride (n=141). The activated oocytes were cultured in vitro for 3–5 da...

  16. Transporte de oócitos bovinos em meio de maturação sem controle de atmosfera gasosa Transport of bovine oocytes in maturation medium without a controlled gaseous atmosphere

    Directory of Open Access Journals (Sweden)

    Fábio Gallas Leivas

    2004-02-01

    aspirated from 2 to 8mm follicles obtained from bovine slaughterhouse ovaries (11 replications were randomly distributed in four treatments. Oocytes were matured for 24h with modified TCM-199 Earle salts, plus 25mM bicarbonate, 25 mM HEPES, rFSH-h, Estrus Cow Serum (ECS, and piruvate at 39ºC, in incubator with 5% CO2 and saturated humidity (Control Group, n=296 or exposed to a simulated transport for 6 (T6, n=286, 12 (T12, n=294 or 18h (T18, n=301 in maturation medium containing TCM + HEPES, in a 39ºC water bath, with the same components used in the Control Group, but with 1mM bicarbonate. At the conclusion of each transport period, oocytes were transferred to dishes with maturation medium to reach 24h in incubator, under the same conditions described for the Control group. Fertilization was accomplished during 18h, with the same temperature and gaseous atmosphere, in FERT-TALP plus heparin. The insemination dose was 1x106 spermatozoa/mL, sorted by swim-up. Presumptive zygotes were cultured in SOF medium + 5% ECS for 8 days, in incubator at 39ºC using gasified bags with 5% CO2, 5% O2 and 90% N2. Cleavage rates did not differ between treatments. Embryonic development rates at D7 were similar for Control (20.9%, T6 (19.2% and T12 (21.4% groups, with a reduction (P0.05 in hatched blastocyst rate. The average number of cells of hatched blastocysts was similar (P>0.05 in Control (136, T6 (125.5 and T12 (126.8 groups. These results indicate the possibility of transporting bovine oocytes in maturation medium containing TCM + HEPES, without controlled gaseous atmosphere environment, at 39ºC, for up to 12 hours. This technique offers a practical and efficient alternative for the transport of bovine oocytes for in vitro production of bovine embryos (IVP.

  17. Impact of GnRH analogues on oocyte/embryo quality and embryo development in in vitro fertilization/intracytoplasmic sperm injection cycles: a case control study

    Directory of Open Access Journals (Sweden)

    Rigó János

    2009-09-01

    Full Text Available Abstract Background Despite the clinical outcomes of ovarian stimulation with either GnRH-agonist or GnRH-antagonist analogues for in vitro fertilization (IVF being well analysed, the effect of analogues on oocyte/embryo quality and embryo development is still not known in detail. The aim of this case-control study was to compare the efficacy of a multiple-dose GnRH antagonist protocol with that of the GnRH agonist long protocol with a view to oocyte and embryo quality, embryo development and IVF treatment outcome. Methods Between October 2001 and December 2008, 100 patients were stimulated with human menopausal gonadotrophin (HMG and GnRH antagonist in their first treatment cycle for IVF or intracytoplasmic sperm injection (ICSI. One hundred combined GnRH agonist + HMG (long protocol cycles were matched to the GnRH antagonist + HMG cycles by age, BMI, baseline FSH levels and by cause of infertility. We determined the number and quality of retrieved oocytes, the rate of early-cleavage embryos, the morphology and development of embryos, as well as clinical pregnancy rates. Statistical analysis was performed using Wilcoxon's matched pairs rank sum test and McNemar's chi-square test. P Results The rate of cytoplasmic abnormalities in retrieved oocytes was significantly higher with the use of GnRH antagonist than in GnRH agonist cycles (62.1% vs. 49.9%; P Conclusion Antagonist seemed to influence favourably some parameters of early embryo development dynamics, while other morphological parameters seemed not to be altered according to GnRH analogue used for ovarian stimulation in IVF cycles.

  18. Effect of male hepatitis B virus infection on outcomes of in vitro fertilization and embryo transfer treatment: insights from couples undergoing oocyte donation.

    Science.gov (United States)

    Bu, Zhiqin; Kong, Huijuan; Li, Jing; Wang, Fang; Guo, Yihong; Su, Yingchun; Zhai, Jun; Sun, Yingpu

    2014-01-01

    It is common to see HBV infected couple seeking fertility treatment in reproductive medical centers. However, it is still unclear whether HBV infection has any relationship with IVF outcome. To assess the impact of male HVB infection on the outcomes of IVF, we retrospectively analyzed data from two hundred and seventy-seven subfertile couples undergoing oocyte donation cycles in our center. Twenty men (7.2%) were HBV seropositive in 277 couples. 20 couples with seropositive husbands had similar semen parameters and fertilization rate when compared with their controls. Among the 215 couples undergoing their first oocyte donation cycles, 19 couples with seropositive husbands/seronegative wives had lower implantation rate (26.7% vs. 40.6%; P > 0.05), and lower clinical pregnancy rate (42.1% vs. 63.8%; P > 0.05), but the difference was not statistically significant. In binary regression model, male HBV infection had no association with clinical pregnancy. Our study shows that male HBV infection has little impact on IVF outcomes. PMID:25126191

  19. Improvement of Preimplantation Development of In Vitro-Fertilized Bovine Zygotes by Glucose Supplementation to a Chemically Defined Medium

    OpenAIRE

    SAKAGAMI, Nobutada; Nishino, Osamu; Adachi, Satoshi; UMEKI, Hidenobu; UCHIYAMA, Hiroko; ICHIKAWA, Kyoko; TAKESHITA, Kazuhisa; KANEKO, Etsushi; AKIYAMA, Kiyoshi; Kobayashi, Shuji; Tamada, Hiromichi

    2014-01-01

    ABSTRACT The influences of glucose supplementation on early development of bovine embryos in BSA-free synthetic oviduct fluid were examined. Among the groups supplemented with 1.5, 2.0, 4.0 or 5.6 mM glucose either at 0, 72 or 144 hr after fertilization, blastocysts yield significantly increased in the group supplemented with 4.0 mM glucose 144 hr after fertilization compared to the controls without glucose supplementation. The results suggest that appropriate amounts of glucose supplemented ...

  20. RELATIONSHIP BETWEEN OOCYTE MATURITY FOR FERTILIZATION AND PRE-OVULATORY FOLLICULAR FLUID HORMONE LEVELS IN INDUCED OVULATORYCY CLE

    Institute of Scientific and Technical Information of China (English)

    LIUYong

    1989-01-01

    This paper describes the relationship between haman oocyte matarity for fcrtilization andpre-ovulalory follicular fluid hormone levels in induced ova]story cycle by trealmcm withclomiphenz+HMG or clomiphene+HMC+HCG. 32 hours after urine LH--surge or 34

  1. Effects of bovine viral diarrhoea virus on the fertility of cows.

    Science.gov (United States)

    Yavru, Sibel; Kale, Mehmet; Gulay, Mehmet Sukru; Yapici, Orhan; Bulut, Oya; Ata, Ayhan

    2013-06-01

    The aim of the present study was to determine the possible relationship between bovine viral diarrhoea (BVD) virus infection and the appearance of cervical mucous discharge (CMD) and the reproductive performance of cows in oestrus. For this purpose, CMD from 97 Holstein cows in oestrus was evaluated visually before artificial insemination (AI). Cows in oestrus were inseminated with frozen semen free from BVD virus (BVDV). Blood samples were tested by enzyme-linked immunoassay (ELISA) for antigen (Ag) and antibodies (Ab) of BVDV. The presence of the BVDV genome in cervical mucus samples was tested by reverse transcriptase-polymerase chain reaction (RT-PCR). The presence of BVDV Ab, Ag or genome was not associated with abnormal cervical mucous discharge (A-CMD). However, the presence of BVDV Ag (but not of the BVDV Ab) in blood samples was associated with a lower first service conception rate (FSCR; 27.8 vs. 70.9%; P < 0.01), indicating that BVDV viraemia at the time of AI has a negative effect on the fertility of cows. PMID:23661395

  2. Monitoring in-vitro bovine embryo development during the first days after fertilization (Conference Presentation)

    Science.gov (United States)

    Kandel, Mikhail E.; Rubessa, Marcello; Fernandes, Daniel; Nguyen, Tan H.; Wheeler, Matthew B.; Popescu, Gabriel

    2016-03-01

    Conventional label-based contrast enhancement techniques (e.g., fluorescence) frequently modify the genetic makeup of tagged cells, making them poor candidates for use in in-vitro fertilization applications. Instead, we choose a label-free form of contrast, based on interferometric imaging, sensitive to optical path length differences. Compared to, single HeLa cells, typical mammalian ova and embryos are more than an order of magnitude thicker. As a result, regions of large phase variation lead to phase wrapping and an overall reduction in signal intensity occurs due to multiple scattering. These effects manifest themselves in low-spatial frequencies (blurs), with the desired details buried in the background. We present a phase shifting interferometer that yields the derivative of the phase, a quantity whose value is particularly sensitive to local variations and fine details. We demonstrate that our new real-time imaging platform is valuable in measuring the multiday development of bovine embryos. Reconstructing the derivative of the image phase and amplitude, we characterize the motion of previously low-contrast structures, which are relevant for embryo viability tests.

  3. Vitrificación de ovocitos bovinos y su uso en el desarrollo partenogenético de embriones Vitrification of bovine oocytes and its use in the parthenogenetic development of embryos

    Directory of Open Access Journals (Sweden)

    J Ruiz

    2010-01-01

    Full Text Available El objetivo de este trabajo fue evaluar el efecto de la vitrificación en la viabilidad de ovocitos activados químicamente para la producción de embriones partenogenéticos bovinos. Ovocitos bovinos aspirados de ovarios obtenidos en el matadero fueron madurados in vitro por 20-22 horas y se distribuyeron en los siguientes grupos. I (n=76: ovocitos vitrificados/descongelados, II (n=119: ovocitos expuestos a crioprotectantes sin vitrificación y III (n=142: ovocitos control. Los ovocitos fueron vitrificados en microgotas sobre un papel de aluminio preenfriado flotando en nitrógeno líquido, utilizando una solución de equilibrio con 4% de etilenglicol y una solución de vitrificación con 35% de etilenglicol 5% de polivinilpirrolidona y 0,4 M de trehalosa. Las microgotas vitrificadas fueron almacenadas en nitrógeno líquido y fueron descongeladas 1-3 días después del almacenamiento. Los tres grupos de ovocitos se activaron partenogenéticamente por exposición de 4 minutos a 5 µM de ionomicina de Ca a temperatura ambiente seguido de una incubación por 5 horas en 6-dimetilaminopurina a 38,5 ºC en una atmósfera húmeda con 5% CO2. Los embriones se cultivaron en medio mSOF durante 8-9 días. Las tasas de ovocitos sobrevivientes fueron 55,1% y 93,7% para ovocitos vitrificados/descongelados (I y expuestos (II respectivamente. Las tasas de segmentación de 55,3%, 72,3% y 74,6%, y de desarrollo hasta blastocistos fueron 7,1%, 17,4% y 21,7% en los grupos I, II y III respectivamente. Estos resultados demuestran que la técnica de vitrificación ha quedado establecida y permite la producción de embriones partenogenéticos bovinos.The aim of this study was to evaluate vitrification effects on the viability of chemically activated oocytes in order to produce parthenogenetic bovine embryos. Bovine oocytes retrieved from ovaries obtained in a slaughterhouse were matured in vitro for 20-22 hours and then assigned to the following groups: I (n=76

  4. Effect of recombinant-LH and hCG in the absence of FSH on in vitro maturation (IVM) fertilization and early embryonic development of mouse germinal vesicle (GV)-stage oocytes.

    Science.gov (United States)

    Dinopoulou, Vasiliki; Drakakis, Peter; Kefala, Stella; Kiapekou, Erasmia; Bletsa, Ritsa; Anagnostou, Elli; Kallianidis, Konstantinos; Loutradis, Dimitrios

    2016-06-01

    During in vitro maturation (IVM), intrinsic and extrinsic factors must co-operate properly in order to ensure cytoplasmic and nuclear maturation. We examined the possible effect of LH/hCG in the process of oocyte maturation in mice with the addition of recombinant LH (r-LH) and hCG in our IVM cultures of mouse germinal vesicle (GV)-stage oocytes. Moreover, the effects of these hormones on fertilization, early embryonic development and the expression of LH/hCG receptor were examined. Nuclear maturation of GV-stage oocytes was evaluated after culture in the presence of r-LH or hCG. Fertilization rates and embryonic development were assessed after 24h. Total RNA was isolated from oocytes of different stages of maturation and from zygotes and embryos of different stages of development in order to examine the expression of LH/hCG receptor, using RT-PCR. The in vitro nuclear maturation rate of GV-stage oocytes that received hCG was significantly higher compared to the control group. Early embryonic development was increased in the hCG and LH cultures of GV oocytes when LH was further added. The LH/hCG receptor was expressed in all stages of in vitro matured mouse oocytes and in every stage of early embryonic development. Addition of hCG in IVM cultures of mouse GV oocytes increased maturation rates significantly. LH, however, was more beneficial to early embryonic development than hCG. This suggests a promising new technique in basic science research or in clinical reproductive medicine. PMID:27288338

  5. Dose inseminante para fertilização artificial de ovócitos de dourado Insemination dose for artificial fertilization of dourado oocytes

    Directory of Open Access Journals (Sweden)

    Eduardo Antônio Sanches

    2009-11-01

    Full Text Available Objetivou-se determinar a dose inseminante adequada para uso na fertilização artificial de ovócitos de dourado (Salminus brasiliensis. Os ovócitos foram distribuídos em delineamento inteiramente casualizado, e fertilizados com uma das relações espermatozoides/ovócito 6,0×10³; 6,0×10(4; 6,0×10(5; 6,0×10(6 ou 3,0×10(7, cada uma com quatro repetições. Considerou-se unidade experimental uma incubadora de volume útil de 2,5 L, contendo 2,0 mL de ovócitos não-hidratados. As taxas de fertilização foram mensuradas 8 horas após o início da fertilização. Com intuito de verificar possíveis efeitos da diluição seminal na movimentação dos espermatozoides, realizou-se a mensuração do tempo de duração da motilidade espermática dos espermatozoides de dourado, ativados por meio de diferentes relações de diluição: 6,8×10-5; 6,8×10-4; 6,8×10-3; 6,8×10-2; 3,4×10-1 e 1,0 mL de sêmen por mL de água. O tempo de duração da motilidade foi avaliado em delineamento inteiramente casualizado composto de seis tratamentos e três repetições. As taxas de fertilização apresentaram relação quadrática com o número de espermatozoides por ovócito. As relações de diluição do sêmen tiveram efeito inversamente proporcional sobre a duração da motilidade espermática. A relação que proporcionou melhores taxas de fertilização artificial de ovócitos de dourado (Salminus brasiliensis foi de 30.722 espermatozoides por ovócio.The objective of the present study was to determine the proper insemination dose of dourado (Salminus brasiliensis oocytes. The oocytes were placed in a randomized complete design and fertilized with one of the spermatozoa.oocytes-1 ratio, 6.0×10³, 6.0×10(4, 6.0×10(5, 6.0×10(6, 3.0×10(7 SPZ:OOC, each one with four replications. An experimental unit was considered to be an incubator with a 2.5L useful volume containing 2.0 mL non-hydrated oocytes. The fertilization rates were measured eight hours

  6. Different gonadotropin releasing hormone agonist doses for the final oocyte maturation in high-responder patients undergoing in vitro fertilization/intra-cytoplasmic sperm injection

    Directory of Open Access Journals (Sweden)

    Emre Goksan Pabuccu

    2015-01-01

    Full Text Available Context: Efficacy of gonadotropin releasing hormone agonists (GnRH-a for ovulation in high-responders. Aims: The aim of the current study is to compare the impact of different GnRH-a doses for the final oocyte maturation on cycle outcomes and ovarian hyperstimulation syndrome (OHSS rates in high-responder patients undergoing ovarian stimulation. Settings And Designs: Electronic medical records of a private in vitro fertilization center, a retrospective analysis. Subjects and Methods: A total of 77 high-responder cases were detected receiving GnRH-a. Group I consisted of 38 patients who received 1 mg of agonist and Group II consisted of 39 patients who received 2 mg of agonist. Statistical Analysis: In order to compare groups, Student′s t-test, Mann-Whitney U-test, Pearson′s Chi-square test or Fisher′s exact test were used where appropriate. A P < 0.05 was considered as statistically significant. Result: Number of retrieved oocytes (17.5 vs. 15.0, P = 0.510, implantation rates (46% vs. 55.1%, P = 0.419 and clinical pregnancy rates (42.1% vs. 38.5%, P = 0.744 were similar among groups. There were no mild or severe OHSS cases detected in Group I. Only 1 mild OHSS case was detected in Group II. Conclusion: A volume of 1 or 2 mg leuprolide acetate yields similar outcomes when used for the final oocyte maturation in high-responder patients.

  7. Ovarian Grafts 10 Days after Xenotransplantation: Folliculogenesis and Recovery of Viable Oocytes.

    Science.gov (United States)

    Campos-Junior, Paulo Henrique Almeida; Alves, Thalys Jair Melo; Dias, Marco Tulio; Assunçao, Carolina Marinho; Munk, Michele; Mattos, Matheus Silvério; Kraemer, Lucas Rocha; Almeida, Brígida Gomes; Russo, Remo Castro; Barcelos, Lucíola; Camargo, Luiz Sérgio Almeida; Viana, Joao Henrique Moreira

    2016-01-01

    Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62). Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (Panimals than in control and non-grafted (P<0.01). BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01). Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01). Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes. PMID:27362486

  8. Special nutrition in mouse developmental oocytes

    OpenAIRE

    Yu, Ling; Wang, Shu-Fang; Yao, Yuan-qing

    2012-01-01

    Investigation of nutrition-related proteins in mouse oocytes and zygotes is crucial for the development of an effective therapy for patients with infertility. Currently, we are concerned with the role of nutrition in the process of oocyte development in order to better reveal the relationship between nutrition and infertility. We collected mouse oocytes at three different developmental stages: germinal vesicle (GV) stage, metaphase II-arrested (MII) stage and fertilized oocytes (zygotes). Sem...

  9. Vitrificação de ovócitos imaturos de bovinos utilizando etilenoglicol associado à trehalose e polivinilpirrolidona Vitrification of immature bovine oocytes, by the ethylene glycol associated with trehalose and polivinylpyrrolidone

    Directory of Open Access Journals (Sweden)

    M.R. Souza

    2003-10-01

    Full Text Available Avaliaram-se os efeitos da vitrificação de ovócitos imaturos de bovinos utilizando o etilenoglicol (EG associado à trehalose e à polivinilpirrolidona (PVP. Utilizaram-se ovócitos provenientes de ovários de vacas abatidas em matadouro, distribuídos aleatoriamente em três tratamentos (T. TI - ovócitos não desnudados e não congelados, TII - ovócitos vitrificados com cumulus oophorus e TIII - ovócitos desnudados vitrificados. A percentagem de ovócitos recuperados e ovócitos com morfologia normal após a vitrificação foi diferente entre TII e TIII (92,2 e 72,6%; 79,0 e 63,6%, respectivamente. Os ovócitos normais foram cultivados à 38,5ºC em atmosfera de 5% de CO2 por 24 horas. Após o cultivo, os ovócitos foram fecundados e os embriões cultivados in vitro por sete dias. Foram encontradas diferenças entre tratamentos quanto às taxas de maturação nuclear, fecundação e clivagem (83,9, 70,0 e 44,0%; 17,5, 23,7 e 5,1%; 0,0, 0,0 e 0,0% para os tratamentos I, II e III, respectivamente. Apenas no TI foram obtidas mórulas e blastocistos (21,4%. Os procedimentos de vitrificação, segundo os protocolos utilizados, não são indicados para a criopreservação de ovócitos imaturos de bovinos.This study aimed to evaluate the effects of vitrification procedure of immature bovine oocytes using ethylene glycol (EG associated with trehalose and polivinylpyrrolidone on the percentage of recovered oocytes with normal morphology and nuclear maturation, fecundation and cleavage rates for in vitro cultivated embryos. Ovary oocytes of slaughtered cows were randomly allotted to three treatments (T: TI - oocytes neither undenuded nor vitrified, TII - vitrified oocytes with cumulus oophorus, TIII - undenuded vitrified oocytes. The percentage of recovered oocytes and oocytes with normal morphology after vitrification was different for TII and TIII (92.2 and 72.6%, 79.0 and 63.3% for TII and TIII, respectively. All normal oocytes were cultivated

  10. 猪卵母细胞体外受精影响因素的研究%Study on Factors Affecting in-vitro Fertilization of Porcine Oocytes

    Institute of Scientific and Technical Information of China (English)

    张立苹

    2015-01-01

    The effects of caffeine concentration in fertilization medium mTBM , sperm floating capacitation time, and sperm-oo-cyte co-incubation time on the in-vitro fertilization and subsequent embryonic development of porcine oocytes were explored .The re-sults showed that adding different concentrations of caffeine into mTBM had no significant effect on the blastocyst rate of pig oocytes after in-vitro fertilization, but the fertilized cleavage rate in 3 mmol/L caffeine supplement group was significantly (P<0.05) higher than that in 5 mmol/L and 0 mmol /L caffeine supplement groups .When the time of sperm floating capacitation was lengthened , both the cleavage rate and the blastocyst rate of fertilized ovum increased first and then decreased , and the cleavage rate and blastocyst rate in 1.0 h floating group were significantly (P<0.05 ) higher than those in 1.5 h and2 .0 h floating groups.The time of sperm-oo -cyte co -incubation had no significant effect on the fertilized cleavage rate , while the blastocyst rate in 9 h co-incubation group was significantly lower than that in 3 h and 6 h co-incubation groups.%探讨了 mTBM 中咖啡因浓度、精子上浮时间和精卵孵育时间对猪卵母细胞体外受精胚胎早期发育的影响。结果表明:咖啡因的添加浓度对猪卵母细胞体外受精胚胎的囊胚率无显著影响,但添加3 mmol/L 咖啡因组的精子入卵率显著高于添加5 mmol /L 咖啡因组和未添加咖啡因组的(P<0.05);随着精子上浮时间的延长,受精卵卵裂率和囊胚率均呈先上升后下降的趋势,上浮1.0 h 组的卵裂率和囊胚率显著高于上浮1.5 h 组和2.0 h 组的(P<0.05);精卵共孵育时间对精子入卵率无显著影响,但受精9 h 处理组的囊胚率显著低于受精3 h 组和6 h 组的。

  11. Effect of temporary meiosis block during prematuration of bovine cumulus-oocyte complexes on pregnancy rates in a commercial setting for in vitro embryo production.

    Science.gov (United States)

    Guemra, Samuel; da Silva Santo, Eriko; Zanin, Renato; Monzani, Paulo Sergio; Sovernigo, Tobias Canan; Ohashi, Otávio Mitio; Verde Leal, Cláudia Lima; Adona, Paulo Roberto

    2014-04-15

    Ovum pick up (OPU) associated with in vitro production (IVP) of embryos has been shown as an important tool in cattle breeding to increase the number of descendants from animals of high genetic value. In herds maintained distant from the laboratory, collecting cumulus-oocyte complexes (COCs) and transporting them to the laboratory may take several hours and decrease COCs viability, representing a challenge for commercial settings. In this study, a prematuration culture to induce temporary meiosis block was evaluated in a commercial scale IVP setting as a strategy to transport bovine OPU-derived COCs from Nelore and Brangus donors. Effects on embryo yield and pregnancy rates were assessed. Viable COCs from each donor were destined to one of the experimental groups (control, blocks 1 and 2). Control group COCs were placed in cryotubes with 1 mL TCM199-HEPES. In block groups (1 and 2), COCs were placed in cryotubes with 300 μL TCM 199 + 12 μM butyrolactone I (block medium). All groups were gassed and kept in a thermos bottle for 4 hours at 36 °C. Next, COCs in the control group were transferred to IVM medium and block 1 group to block medium, and cultured for 22 hours and 15 hours, respectively, at 38.5 °C and 5% CO2 in air. Block 2 COCs were kept in the cryotubes and in the thermos bottle for another 15 hours at 36 °C to simulate long-term transport conditions. After meiosis block in prematuration culture, blocks 1 and 2 COCs were matured in vitro for 22 hours as for the control group. After IVM, COCs in all groups were submitted to IVF and IVC, and blastocyst rates were evaluated on day 7. Embryos were transferred and pregnancy rates evaluated at 60 days of gestation. The mean total number of COCs retrieved by OPU did not differ between Nelore and Brangus donors (16.8 and 17.2, respectively, P > 0.05), but Nelore donors produced more viable COCs than Brangus (10.1 and 7.6, respectively, P Brangus cattle, respectively (P > 0.05). Pregnancy rates did

  12. Follicle stimulating hormone and anti-Mullerian hormone per oocyte in predicting in vitro fertilization pregnancy in high responders: a cohort study.

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    Andrea Weghofer

    Full Text Available BACKGROUND: Follicle stimulating hormone (FSH and Anti-Müllerian hormone (AMH are utilized to differentiate between good and poor response to controlled ovarian hyperstimulation. Their respective roles in defining functional ovarian reserve remain, however, to be elucidated. To better understand those we investigated AMH and FSH per oocyte retrieved (AMHo and FSHo. METHODOLOGY/PRINCIPAL FINDINGS: Three-hundred and ninety-six women, undergoing first in vitro fertilization cycles, were retrospectively evaluated. Women with oocyte yields >75(th percentile for their age group were identified as high responders. In a series of logistic regression analyses, AMHo and FSHo levels were then evaluated as predictive factors for pregnancy potential in high responders. Patients presented with a mean age of 38.0±5.0 years, mean baseline FSH of 11.8±8.7 mIU/mL and mean AMH of 1.6±2.1 ng/mL. Those 88 women, who qualified as high responders, showed mean FSH of 9.7±6.5 mIU/mL, AMH of 3.1±3.1 ng/mL and oocyte yields of 15.8±7.1. Baseline FSH and AMH did not predict pregnancy in high responders. However, a statistically significant association between FSHo and pregnancy was observed in high responders, both after univariate regression (p = 0.02 and when adjusted for age, percentage of usable embryos, and number of embryos transferred (p = 0.03. Rate of useable embryos also significantly affected pregnancy outcome independently of FSHo (p = 0.01. AMHo was also associated with clinical pregnancy chances in high responders (p = 0.03 and remained significant when adjusted for usable embryos and number of embryos transferred (p = 0.04. CONCLUSIONS: AMHo and FSHo are predictive of pregnancy potential in high responders, but likely reflect different responsibilities in recruitment and maturation of growing follicle cohorts.

  13. Vitrificação de ovócitos desnudados ou não e previamente maturados in vitro Cryopreservation of bovines oocytes desnuded or not and previously in vitro matured

    Directory of Open Access Journals (Sweden)

    Letícia Martins Fagundes

    2004-10-01

    Full Text Available Objetivou-se avaliar os efeitos da vitrificação de ovócitos maturados in vitro de bovinos, utilizando o etilenoglicol (EG associado a trehalose e polivinilpirrolidona (PVP. Utilizaram-se ovócitos provenientes de ovários de vacas abatidas em matadouro, distribuídos aleatoriamente em três tratamentos. Tratamento 0 (T0 - testemunha: ovócitos não desnudados e não congelados. Tratamento 1 (T1: vitrificação de ovócitos com cumulus oophorus e maturados in vitro. Tratamento 2 (T2: vitrificação de ovócitos desnudados e maturados in vitro. A porcentagem de ovócitos recuperados e com morfologia normal após a desvitrificação foi diferente entre T1 e T2 (94,7 e 76,8%; 69,5 e 49,85%, para T1 e T2, respectivamente. Após a reidratação, os ovócitos vitrificados foram fecundados e cultivados in vitro por sete dias. Foi verificada, em nível ultra-estrutural, liberação prematura dos grânulos corticais em ovócitos vitrificados. As taxas de fecundação e de clivagem foram diferentes entre os tratamentos (56,2; 41,7 e 12,5%; 36,3; 0,0 e 0,0% para T0, T1 e T2, respectivamente. Apenas no T0 foram obtidos mórulas e blastocistos (34,5%. Estes resultados indicam que o procedimento de vitrificação, segundo os protocolos utilizados, não é indicado para a criopreservação de ovócitos maturados de bovinos.This study aimed at the evaluation of the effects from cryopreservation of bovine oocytes in vitro matured, by using ethylene glycol (EG associated to trehalose and polyvinylpyrrolidone (PVP, of ovary oocytes of slaughtered cows, randomly assigned to three treatments. Treatment 0 (T0 - control: oocytes that were desnuded and not vitrified. Treatment 1 (T1: cryopreservation of in vitro matured oocytes with cumulus oophorus. Tratamento 2 (T2: cryopreservation of in vitro matured desnuded oocytes. The percentage of recovered oocytes after cryopreservation and with normal morphology was different for vitrified oocytes (94.7 and 76.8%; 69.5 and

  14. Effect of calcium ionophore on unfertilized oocytes after ICSI cycles

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    Maryam Eftekhar

    2012-01-01

    Full Text Available Background: Fertilization failure is one of the most problems in assisted reproduction technology (ART. Objective: The aim of this study was the evaluation of oocytes activation by addition of calcium ionophore in unfertilized oocytes in ICSI cycles. Materials and Methods: This study was done on 15 ICSI cycles (stimulated with standard long protocol. Mature retrieved oocytes with normal morphology that had no evidence of fertilization 24 hours after ICSI were included in the study. The oocytes with fertilization and unfertilized oocytes with degeneration were excluded from the study. The unfertilized oocytes were washed with GIVF medium and were transferred to GIVF medium that contained 5 μmol of calcium ionophore and were incubated for 10 minutes. Then again oocytes were washed with GIVF medium and consequently were transferred to GIVF medium and were incubated at 37°C in 6% CO2. After 18 hours, the oocytes were examined and activated oocytes were defined with observation of at least one pronucleus or cleaved oocytes. Results: After ovarian stimulation and oocytes retrieval, 175 mature oocytes were obtained and injection of sperm was done for all of them. 114 of 175 oocytes (66% showed evidence of fertilization after 24 hours. A total of 61 oocytes (34% showed no evidence of fertilization and 10 oocytes were degenerated and were excluded from the study. Only 51 unfertilized oocytes with normal morphology were selected and were exposed to calcium ionophore. 37 (72.5% of treated oocytes were fertilized (2PN and 32 (62.7% of them showed evidence of cleavage. 6 (11.8% embryos had good quality. Conclusion: According to our results, oocytes activation with calcium ionophore had an acceptable fertilization rate, however high quality embryos remained low. We propose future studies to evaluate embryo quality.

  15. 亮甲酚蓝染色对牛卵母细胞体外成熟的影响%Effect of Brilliant Cresyl Blue on in Vitro Maturity of Bovine Oocytes

    Institute of Scientific and Technical Information of China (English)

    王士勇; 杨月春; 郑军军; 刘宗岳; 于淼; 杨福合

    2014-01-01

    研究旨在观察亮甲酚蓝(brilliant cresyl blue,BCB)对牛卵母细胞的染色效果及体外成熟的影响,从而筛选出更高质量的卵母细胞用于胚胎工程研究。从本地屠宰场收集牛卵巢后采用抽吸法获取卵泡液,在体视显微镜下选择卵丘完整、胞质均匀的卵丘-卵母细胞复合体( COCs )随机分组,分别进行BCB染色时间和不同浓度BCB染色的试验。结果表明,染色90 min后卵母细胞着色率为68.1%,显著高于30、60 min组( P<0.05),辨识难度为“中”;39μmol/L 组着色率显著高于13μmol/L 组( P<0.05),与26、52μmol/L 组差异不显著(P>0.05),成熟数最高(62),成熟率显著高于52μmol/L组,与其它组差异不显著(P>0.05);BCB+组和BCB-组的成熟率与对照组比较差异均不显著( P>0.05)。结果提示,39μmol/L BCB染色90 min能有效筛选牛COCs用于IVM,但是并不能显著提高牛卵母细胞的成熟率。%This experiment aimed at the observation of the staining effect of brilliant cresyl blue ( BCB ) and its influence on bovine oocyte in vitro maturation so as to select the higher quality oocytes for the embryo engineering research .Bovine ovaries were collected from a local slaughterhouse , and with the suction method the follicular fluid was obtained ,cumulus oocyte complexes ( COCs) with intact cumulus,homogeneous cyto-plasm were selected by using a stereoscopic microscope ,and then were randomly divided into two groups ,and the staining time and BCB concentration were determined , respectively .The results showed that , the staining rate of oocyte 90 min after staining was 68.1%,it was significantly higher than that of the groups of 30 and 60 min,and the distinguishable difficulty was “common”.The rate of stained oocytes in the 39μmol/L group was significantly higher than that in the 13μmol/L group ( P0.05),the No.of mature oocytes was the highest (62

  16. Effect of germinal vesicle translation process on in vitro fertilization of mouse oocytes%生发泡移植过程对小鼠卵母细胞体外受精的影响

    Institute of Scientific and Technical Information of China (English)

    崔龙波; 周雪莹; 崔志峰; 黄秀英; 孙方臻

    2009-01-01

    To study oocytes reconstructed by germinal vesicle (GV) translation can mature and develop fully in vitro, effects of GV translation process and in vitro maturation on in vitro fertilization (IVM) of mouse oocytes were compared. The maturation rate of the reconstructed oocytes obtained by zona cutting and GV translation was 87.0% (147/169), and the rates of pronuclear and 2-cell embryo developed from these oocytes after IVF were 76.2%(112/147)and 42.2%(62/147), respectively, which were comparable (P>0.05) with the control oocytes obtained by zona cutting but not GV translation. The results showed the GV translation process had no harmful effect on early embryo developmental capacity of the oocytes after IVM. The rates of fertilization and cleavage of the oocytes matured in vitro, however, were significantly lower than that matured in vivo, showing the further developping capacity of the oocytes matured in vitro was affected.%为研究生发泡(GV)移植后的重组卵母细胞能充分在体外成熟和发育,本试验比较了GV移植过程及体外成熟对小鼠卵母细胞体外受精的影响.通过透明带切口及GV移植获得的重组GV期卵母细胞的成熟率为87.0%(147/169),体外受精后发育到原核期胚和2-细胞期胚的比率分别为76.2%(112/147)和42.2%(62/147),与仅做透明带切口但不进行GV移植的对照组相似(P>0.05),表明GV移植过程未对卵母细胞体外受精后的早期胚胎发育能力产生不利影响.但与体内成熟的卵母细胞相比,体外成熟卵母细胞的受精率和卵裂率都明显降低,表明其进一步发育的能力受到影响.

  17. 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

    OpenAIRE

    F. Perecin; S.C. Méo; C.L.V. Leal; Garcia, J M

    2007-01-01

    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 rosco...

  18. Anesthetic management for oocyte retrieval: An exploratory analysis comparing outcome in in vitro fertilization cycles with and without pre-implantation genetic diagnosis

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    Alexander Ioscovich

    2013-01-01

    Full Text Available Purpose: To date, there has been no comparison of outcomes in women undergoing anesthesia for in vitro fertilization (IVF oocyte retrieval for the purpose of pre-implantation genetic diagnosis (PGD because of their or their partner′s genetic disease relative to the outcome in women requiring IVF because of fertility issues. Materials and Methods: A prospective observational study, wherein all demographic and anesthetic management data were collected from IVF and PGD units′ records for a 6-month period. Descriptive analyses and parametric tests were employed. Results: There were 307 cases IVF and 76 cases PGD: most (97.4% and 99.7%, respectively received general anesthesia with propofol and fentanyl ± dipyrone (90.5% and 93.3%, respectively with no adverse effects. The only statistically significant difference between IVF and PGD groups that was potentially clinically significant was post-procedure recovery time (23.0 ± 20.4 vs. 29.4 ± 35.8 min, respectively; P < 0.0001, but is explainable as greater caution by Anesthesiologists for higher-risk PGD cases having autosomal dominant diseases that may impact anesthesia management (myotonic dystrophy, neurofibromatosis, Marfan′s; two of these cases also recovered in the general post-anesthesia care unit, as a precaution for early diagnosis and treatment of potential post-procedural complication. Conclusions: Results of this first-ever survey of anesthesia for PGD compared with IVF cases imply that propofol-and-fentanyl-based anesthesia is safe and can be recommended, bearing in mind that with patients who have autosomal dominant diseases impacting anesthetic management it is prudent to be more cautious post-recovery.

  19. Effect of myo-inositol and alpha-lipoic acid on oocyte quality in polycystic ovary syndrome non-obese women undergoing in vitro fertilization: a pilot study.

    Science.gov (United States)

    Rago, R; Marcucci, I; Leto, G; Caponecchia, L; Salacone, P; Bonanni, P; Fiori, C; Sorrenti, G; Sebastianelli, A

    2015-01-01

    The aim of the present study was to evaluate the effectiveness of the combined administration of myo-inositol and α-lipoic acid in polycystic ovary syndrome (PCOS) patients with normal body mass index (BMI), who had previously undergone intracytoplasmic sperm injection (ICSI) and received myo-inositol alone. Thirty-six of 65 normal-weight patients affected by PCOS who did not achieve pregnancy and one patient who had a spontaneous abortion were re-enrolled and given a cycle of treatment with myo-inositol and α-lipoic acid. For all female partners of the treated couples, the endocrine-metabolic and ultrasound parameters, ovarian volume, oocyte and embryo quality, and pregnancy rates were assessed before and after three months of treatment and compared with those of previous in vitro fertilization (IVF) cycle(s). After supplementation of myo-inositol with α-lipoic acid, insulin levels, BMI and ovarian volume were significantly reduced compared with myo-inositol alone. No differences were found in the fertilization and cleavage rate or in the mean number of transferred embryos between the two different treatments, whereas the number of grade 1 embryos was significantly increased, with a significant reduction in the number of grade 2 embryos treated with myo-inositol plus α-lipoic acid. Clinical pregnancy was not significantly different with a trend for a higher percentage for of myo-inositol and α-lipoic acid compared to the myo-inositol alone group. Our preliminary data suggest that the supplementation of myo-inositol and α-lipoic acid in PCOS patients undergoing an IVF cycle can help to improve their reproductive outcome and also their metabolic profiles, opening potential for their use in long-term prevention of PCOS. PMID:26753656

  20. Effect of myo-inositol and alpha-lipoic acid on oocyte quality in polycystic ovary syndrome non-obese women undergoing in vitro fertilization: a pilot study.

    Science.gov (United States)

    Rago, R; Marcucci, I; Leto, G; Caponecchia, L; Salacone, P; Bonanni, P; Fiori, C; Sorrenti, G; Sebastianelli, A

    2015-01-01

    The aim of the present study was to evaluate the effectiveness of the combined administration of myo-inositol and α-lipoic acid in polycystic ovary syndrome (PCOS) patients with normal body mass index (BMI), who had previously undergone intracytoplasmic sperm injection (ICSI) and received myo-inositol alone. Thirty-six of 65 normal-weight patients affected by PCOS who did not achieve pregnancy and one patient who had a spontaneous abortion were re-enrolled and given a cycle of treatment with myo-inositol and α-lipoic acid. For all female partners of the treated couples, the endocrine-metabolic and ultrasound parameters, ovarian volume, oocyte and embryo quality, and pregnancy rates were assessed before and after three months of treatment and compared with those of previous in vitro fertilization (IVF) cycle(s). After supplementation of myo-inositol with α-lipoic acid, insulin levels, BMI and ovarian volume were significantly reduced compared with myo-inositol alone. No differences were found in the fertilization and cleavage rate or in the mean number of transferred embryos between the two different treatments, whereas the number of grade 1 embryos was significantly increased, with a significant reduction in the number of grade 2 embryos treated with myo-inositol plus α-lipoic acid. Clinical pregnancy was not significantly different with a trend for a higher percentage for of myo-inositol and α-lipoic acid compared to the myo-inositol alone group. Our preliminary data suggest that the supplementation of myo-inositol and α-lipoic acid in PCOS patients undergoing an IVF cycle can help to improve their reproductive outcome and also their metabolic profiles, opening potential for their use in long-term prevention of PCOS.

  1. The role of sex chromosomes in mammalian germ cell differentiation: can the germ cells carrying X and Y chromosomes differentiate into fertile oocytes?

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    Teruko Taketo

    2015-06-01

    Full Text Available The sexual differentiation of germ cells into spermatozoa or oocytes is strictly regulated by their gonadal environment, testis or ovary, which is determined by the presence or absence of the Y chromosome, respectively. Hence, in normal mammalian development, male germ cells differentiate in the presence of X and Y chromosomes, and female germ cells do so in the presence of two X chromosomes. However, gonadal sex reversal occurs in humans as well as in other mammalian species, and the resultant XX males and XY females can lead healthy lives, except for a complete or partial loss of fertility. Germ cells carrying an abnormal set of sex chromosomes are efficiently eliminated by multilayered surveillance mechanisms in the testis, and also, though more variably, in the ovary. Studying the molecular basis for sex-specific responses to a set of sex chromosomes during gametogenesis will promote our understanding of meiotic processes contributing to the evolution of sex determining mechanisms. This review discusses the fate of germ cells carrying various sex chromosomal compositions in mouse models, the limitation of which may be overcome by recent successes in the differentiation of functional germ cells from embryonic stem cells under experimental conditions.

  2. 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

  3. 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.

  4. Criopreservação de ovócitos de bovinos imaturos desnudados ou não, utilizando o etilenoglicol pelo método da vitrificação Cryopreservation of bovines immature oocytes desnudes or not, by the ethylene glycol vitrification method

    Directory of Open Access Journals (Sweden)

    Eduardo Paulino da Costa

    2002-06-01

    . Treatment 0 (control: frozen-thawed undesnude oocytes; treatment 1, immature vitrificated undesnude oocytes dehydrated for 5 minutes in each of the following solutions of 20, 20 and 40% of ethylene glycol, respectively, associated to 0.3 Mol l-1 of trehalose and 20% of PVP, in media Talp Hepes, and, treatment 2, the same as treatment 1, but desnudes oocytes. After frozen-thawed of the oocytes (imersion in water bath at 30ºC for 20 seconds, the oocytes were gradually rehydrated, in the following sequence of solutions: media Talp Hepes with 20% of ethylene glycol + 0.3 Mol l-1 of trehalose + 10% of PVP and media Talp Hepes without ethylene glycol, trehalose and PVP, were washed three times. Ultimately, the oocytes were cultured at 38.5ºC, with 95% umidity and atmosphere of 5% of CO2 for 24 hours. After culture, the oocytes were fertilized and the embryos cultured in vitro for seven days. The nuclear maturation were 81 (68/84, 19 (7/36 and 0% (0/31, for treatments 0, 1 and 2, respectively. The cleavage and development rates were: 56.4(102/181 and 54,9% (56/102, 1,7. (1/60 and 0,0% (1/60, 0,0 (0/71 and 0,0% (0/71, for the treatments 1, 2 e 3, respectively. These results show that the vitrification procedures, by the used protocols, are not indicated for bovine oocytes cryopreservation.

  5. Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development.

    Science.gov (United States)

    Wang, S; Panter, K E; Gaffield, W; Evans, R C; Bunch, T D

    2005-02-01

    alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.

  6. Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes.

    Science.gov (United States)

    Warzych, E; Peippo, J; Szydlowski, M; Lechniak, D

    2007-02-01

    The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.

  7. Desarrollo de embriones de bovino obtenidos por fecundación in vitro cultivados con células oviductales o medio condicionado y transferidos a hembras receptoras Bovine embryo development produced by in vitro fertilization cultured with oviductal cell or conditioned medium and transfer to recipients

    Directory of Open Access Journals (Sweden)

    M RATTO

    1999-01-01

    Full Text Available Se comparó el desarrollo in vitro de ovocitos obtenidos de ovarios de vaca de matadero, madurados, fecundados y cultivados in vitro bajo dos sistemas. Los ovocitos fueron cultivados en un medio de maduración a 39 °C, 5 % de CO2 y humedad relativa de 95 % durante 22 horas. Posteriormente, fueron incubados con espermatozoides seleccionados a través de una gradiente discontinua de Percoll. La tasa de maduración nuclear y fecundación fueron de 93,7 % (74/79 y 76,9 % (50/65 respectivamente. Un total de 252 ovocitos fecundados fueron cultivados in vitro. El porcentaje de desarrollo in vitro a las 2 días post-inseminación (embriones de 4-8 células fue de 62,7 % (64/102 para los cigotos cultivados con células oviductales y de 67 % (100/150 para los cultivados en medio condicionado (P0,05. El porcentaje de desarrollo de mórulas fue de 17,6 % (18/102 para los cigotos cultivados con células oviductales y de 13,3 % (20/150 para los cultivados con medio condicionado (P0,05. Se obtuvo una tasa de desarrollo del 15,7 % (16/102 de blastocistos para aquellos cigotos cultivados con células oviductales. No se obtuvo blastocistos a partir de cigotos cultivados en medio condicionado. Cuatro blastocistos fueron transferidos a dos hembras receptoras. A los 42 y 57 días se encontró la presencia de un feto en cada hembraThe in vitro development of matured and fertilized bovine oocytes was compared between two culture systems. Oocytes were collected by aspiration of follicles of 3-8 mm in diameter using an 18g needle. After morphological selection the oocytes were incubated at 39 0C, 5 % C02 y 95 % relative humidity, during 22 hours. Afterwards, oocytes were incubated with spermatozoa selected by Percoll gradient system. The rate of nuclear maturation and fertilization was 93,7 % (74/79 and 76,9 % (50/65, respectively. A total of 252 zygotes were cultured, 102 with oviductal cells and 150 in conditioned medium. The in vitro development on day 2 of culture

  8. Gonadotropin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes.

    Science.gov (United States)

    Armstrong, D T; Irvine, B J; Earl, C R; McLean, D; Seamark, R F

    1994-01-01

    Crossbred beef x dairy calves were randomly allocated at 3 wk of age to different gonadotropin treatment regimens for stimulation of follicle development and induction of oocyte maturation in vivo. Follicular responses were assessed laparoscopically, and oocytes were aspirated for assessment of maturational state or for in vitro fertilization (IVF) and culture to determine developmental capacity. Follicle-stimulating Hormone (FSH), administered in a single subcutaneous injection together with a low dosage of PMSG, was as effective as the same total dosage of FSH administered in 6 injections over a 3-d period. Without accompanying PMSG, this dose of FSH was ineffective in stimulating follicle development. The mean number of preovulatory follicles (> 5mm, with hyperemic appearance) doubled with each successive stimulation at 3-wk intervals, reaching 35 follicles per calf at 9 wk of age. Oocyte yields ranged from 55 to 81% of follicles aspirated, and did not differ significantly among age, FSH regimen and oocyte maturation stimulus. A combination of LH + FSH was more effective in stimulating cumulus cell expansion than LH by itself (73 vs 22% of recovered oocyte-cumulus cell complex (OCC) respectively; Poocytes (cumulus unexpanded) subjected to in vitro maturation (IVM) and IVF, 30% developed to blastocysts during co-culture with bovine oviduct epithelial cells, which was not significantly different from 25% of 36 oocytes from adult ovaries which reached the blastocyst stage under similar conditions. The results indicate that follicle responses of calf ovaries to FSH stimulation increase progressively from 3 to 9 wk of age, and that oocytes recovered laparoscopically from these follicles produce blastocysts in culture at rates similar to oocytes from adult cattle ovaries collected at slaughter. The approach offers promise for embryo production from donor calves of superior genetic merit for embryo transfer, thereby enhancing the rate of genetic gain above that

  9. Efeito de diferentes meios de cultivo no desenvolvimento e proporção do sexo de embriões bovinos produzidos in vitro Effect of different culture media on development and sex ratio of bovine embryos fertilized in vitro

    Directory of Open Access Journals (Sweden)

    S.G.T. Gilardi

    2004-10-01

    Full Text Available Avaliou-se o efeito da suplementação de meios de cultivo sobre o desenvolvimento e proporção do sexo de embriões bovinos fertilizados in vitro. Complexos cumulus-oócitos obtidos de ovários de matadouro foram maturados e fertilizados in vitro. Os zigotos (n= 484 foram distribuídos aleatoriamente em meio CR2aa, contendo soro fetal bovino (SFB (T1, albumina sérica bovina (BSA (T2 ou BSA mais insulina:transferrina:selênio e vitaminas (BSA+ (T3, no cultivo embrionário in vitro, a uma atmosfera de 5% CO2 a 38,8ºC em ar. A taxa de clivagem foi observada 72-76 horas pós-fertilização (PF e a taxa de blastocistos com sete e oito dias PF. Os blastocistos (n= 63 foram sexados pela técnica de reação em cadeia de polimerase. A taxa de clivagem em T2 foi maior (P0,05 entre T2 e T3, porém menor (P0,05 entre os tratamentos. O T1 influenciou o desenvolvimento de blastocistos, mas não teve efeito sobre a proporção do sexo.The effect of culture media on the development and on the sex ratio of bovine embryos fertilized in vitro was studied. Cumulus oocyte-complexes from slaughterhouse ovaries were matured and fertilized in vitro. Zygotes (n= 484 were randomly allotted to different culture media and cultured with their cumulus cells in CR2aa medium and an atmosphere of 5% CO2 in air at 38.8ºC. The fetal calf serum (FCS, bovine seric albumin (BSA or BSA plus insulin:transferrin:selenium and vitamins (BSA+ supplementation effect on embryo culture was evaluated. Cleavage rate was assessed at 72-76h post-fertilization (PF and blastocyst rate on days 7 and 8 PF. The blastocysts (n= 63 were also sexed using polymerase chain reaction. Cleavage rate for BSA medium supplemented was higher (P0.05, but lower (P<0.01 than FCS. Culture medium FCS supplemented affected blastocyst development but not the sex ratio.

  10. High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows.

    Science.gov (United States)

    Rocha, A; Randel, R D; Broussard, J R; Lim, J M; Blair, R M; Roussel, J D; Godke, R A; Hansel, W

    1998-02-01

    Two experiments were conducted to assess the effects of environmental temperature and humidity on the quality and developmental capabilities of bovine oocytes. In Experiment 1, Bos taurus (Holstein and crossbred Angus) cows were subjected to 5 weekly sessions of ultrasound-guided follicle aspiration from February 16 through March 23 (cool season) and 5 sessions from May 22 through June 20 (hot season). In Experiment 2, Bos taurus (Holstein) and Bos indicus (Brahman) cows were superstimulated (Super-Ov) during the months of August (hot season) or January (cool season), and each cow was subjected to a single oocyte aspiration session. In each experiment, oocytes were classified as normal or abnormal based on ooplasm morphology and cumulus cell layers. In Experiment 1, oocytes classified as normal were in vitro matured and fertilized (IVM/IVF), and the resulting embryos cultured for 8 d. All oocytes recovered from superstimulated cows in Experiment 2 were matured and fertilized in vitro and the subsequent embryos cultured for 8 d, regardless of their morphological appearance. In Experiment 1, Bos taurus cows produced a higher (P = 0.02) percentage of normal oocytes during the cool season (75.9 +/- 8.0) than during the hot season (41.0 +/- 9.5). The percentage of fertilized oocytes developing to the 2-cell (82.4), 8-cell (65.4) and morula (46.6) stages were also greater (P Holstein) had a lower (P = 0.01) percentage of normal oocytes in the hot season (24.5 vs 80.0) and a lower (P or = 0.57) in the percentage of normal oocytes or in embryo development was detected between seasons in Bos indicus (Brahman) cows. In conclusion, high environmental temperature and humidity resulted in a marked decline in the quality of oocytes retrieved from Bos taurus cows and markedly decreased their in vitro developmental capabilities. In contrast, a high percentage of oocytes retrieved from Bos indicus cows exhibited normal morphology and yielded a high proportion of blastocysts

  11. Ovarian Grafts 10 Days after Xenotransplantation: Folliculogenesis and Recovery of Viable Oocytes

    Science.gov (United States)

    Campos-Junior, Paulo Henrique Almeida; Alves, Thalys Jair Melo; Dias, Marco Tulio; Assunçao, Carolina Marinho; Munk, Michele; Mattos, Matheus Silvério; Kraemer, Lucas Rocha; Almeida, Brígida Gomes; Russo, Remo Castro; Barcelos, Lucíola; Camargo, Luiz Sérgio Almeida; Viana, Joao Henrique Moreira

    2016-01-01

    Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62). Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (P<0.05), when compared to non-grafted fragments. There was a higher blood perfusion under the back skin in the transplantation sites of host animals than in control and non-grafted (P<0.01). BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01). Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01). Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes. PMID:27362486

  12. Ovarian Grafts 10 Days after Xenotransplantation: Folliculogenesis and Recovery of Viable Oocytes.

    Directory of Open Access Journals (Sweden)

    Paulo Henrique Almeida Campos-Junior

    Full Text Available Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62. Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (P<0.05, when compared to non-grafted fragments. There was a higher blood perfusion under the back skin in the transplantation sites of host animals than in control and non-grafted (P<0.01. BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01. Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01. Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes.

  13. 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.

  14. Elective oocyte cryopreservation: who should pay?

    OpenAIRE

    Mertes, Heidi; Pennings, Guido

    2012-01-01

    Despite the initial reactions of disapproval, more and more fertility clinics are now offering oocyte cryopreservation to healthy women in order to extend their reproductive options. However, so-called social freezing is not placed on an equal footing with 'regular' IVF treatments where public funding is concerned. In those countries or states where IVF patients receive a number of free cycles, we argue that fertilization and transfer cycles of women who proactively cryopreserved their oocyte...

  15. Association of recombinant bovine somatotropin (rBST with equine chorionic gonadotropin (eCG on antral follicle count and oocyte production in Holstein and Tabapuã heifers

    Directory of Open Access Journals (Sweden)

    Hévea de Morais

    2012-12-01

    Full Text Available The objective of this study was to investigate whether the use of rbST and eCG prior to ultrasound-guided follicular aspiration (OPU improves oocyte yield and quality in Tabapuã and Holstein heifers. The study was conducted in two phases, 20 days apart, in a change-over design. The dominant follicle was ablated two days (D-2 before two treatments: stimulation (6 Holstein and 8 Tabapuã, 500 mg of rbST (Boostin® on D0 and 500 IU of eCG (Folligon on D2; and control (6 Holstein and 8 Tabapuã, in which heifers received injections of the excipient. Heifers were aspirated on D4. Oocytes were subjected to a well established commercial in vitro embryo production protocol (Vitrogen® and embryos were evaluated seven days after fertilization. There was an effect from the interaction of treatment and breed, so that hormonal stimulation increased antral follicle count (2-8 mm in Tabapuã (29.9±2.6 to 41.4±2.6, but not in Holstein heifers (14.4±2.6 to 15.5±2.6. Tabapuã heifers had higher mean antral follicle count than Holsteins (35.6±1.8 vs. 15.0±2.1. The number of viable oocytes was not increased by stimulation in Tabapuã (from 4.7±1.0 to 5.2±1.1 in control and simulation, respectively or in Holstein heifers (1.3±1.9 to 2.0±1.6 in control and simulation, respectively. There was no difference in the percentage of heifers with more than five viable oocytes in the group treated (33 vs 27%. The number of blastocysts was not affected by treatment (1.75 vs. 1.00 in hormonal stimulation and control, respectively. The increase in antral follicle count in the stimulated Tabapuã heifers did not reflect upon oocyte yield. The differential breed response to the hormonal treatment underscores the need for additional tests, especially for the Holstein breed, in order to enhance OPU efficiency.

  16. EVALUATION OF FROZEN SEMEN BY A CROSOMAL INTEGRITY AND SPERM CONCENTRATION - TWO VITAL QUALITY PARAMETERS OF MALE FERTILITY IN BOVINES

    Directory of Open Access Journals (Sweden)

    Sumit Chowdhury

    2014-06-01

    Full Text Available Acrosomal integrity and sperm concentration are two important parameters to assess the quality of frozen semen doses which in terms validates the fertilizing capacity and conception rate. The present study was undertaken to evaluate acrosomal integrity by Giemsa’s stain and sperm concentration of FSS using improved neubauer chamber in Exotic pure Jersey, Crossbred Jersey, Indigenous Gir cattle and Indigenous Murrah buffalo prior to the field use. The overall values of Giemsa’s stain were observed as 73.74±0.31, 18.65±0.33 and 7.79±0.25 percent for Intact Acrosome, Partially Damaged Acrosome and Fully Damaged Acrosome, respectively. Overall values of sperm concentration were 21.98±0.28 million per straw. The study indicated that there was no significant difference (P<0.05 among the breeds and the values mostly correlates with the guideline of Minimum Standard Protocol for Production of bovine semen, 2012 of Govt. of India.

  17. Perinatal outcomes in 375 children born after oocyte donation

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  18. Effect of calcium ionophore on unfertilized oocytes after ICSI cycles

    OpenAIRE

    Maryam Eftekhar; Farnaz Mohammadian; Fariba Yousefnejad; Parisa Khani; Abbas Aflatoonian

    2012-01-01

    Background: Fertilization failure is one of the most problems in assisted reproduction technology (ART). Objective: The aim of this study was the evaluation of oocytes activation by addition of calcium ionophore in unfertilized oocytes in ICSI cycles. Materials and Methods: This study was done on 15 ICSI cycles (stimulated with standard long protocol). Mature retrieved oocytes with normal morphology that had no evidence of fertilization 24 hours after ICSI were included in the study. The oocy...

  19. Meat and Livestock Association Plenary Lecture 2005. Oocyte signalling molecules and their effects on reproduction in ruminants.

    Science.gov (United States)

    McNatty, Kenneth P; Lawrence, Stephen; Groome, Nigel P; Meerasahib, Mohammed F; Hudson, Norma L; Whiting, Lynda; Heath, Derek A; Juengel, Jennifer L

    2006-01-01

    Sheep (Ovis aries) are a highly diverse species, with more than 900 different breeds that vary significantly in their physiological characteristics, including ovulation rate and fecundity. From examination of inherited patterns of ovulation rate, several breeds have been identified with point mutations in two growth factor genes that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9b) gene and one in GDF9. Animals heterozygous for the GDF9 and/or the BMP15 mutations have higher ovulation rates than their wild-type counterparts. In contrast, those homozygous for any of the aforementioned BMP15 or GDF9 mutations are sterile owing to arrested follicular development. In bovine and ovine ovaries, GDF9 was expressed exclusively in oocytes throughout follicular growth from the primordial stage of development, whereas in sheep BMP15 was expressed exclusively in oocytes from the primary stage: no data for the ontogeny of BMP15 expression are currently available for cattle. In vitro, ovine growth differentiation factor 9 (oGDF9) has no effect on (3)H-thymidine incorporation by either bovine or ovine granulosa cells, whereas ovine bone morphogenetic protein 15 (oBMP15) has modest (1.2- to 1.6-fold; P reproduction in mammals, including rodents, humans and ruminants. Moreover, in vivo manipulation of these oocyte signalling molecules provides new opportunities for the management of the fertility of ruminants.

  20. 兔卵母细胞体外成熟、体外受精及受精卵体外培养条件的研究%IVM,IVF of Follicular Oocytes and IVC of in Vitro Fertilized Oocytes in Rabbit

    Institute of Scientific and Technical Information of China (English)

    王辉田; 罗光彬; 王忠彬; 孙超; 陈思; 吴宏平; 王娇娇

    2013-01-01

    为了建立一个完善的兔胚胎体外生产体系,探讨了TCM-199、M16和KSOM基础培养液对兔卵母细胞体外成熟,上游法和直接洗涤法洗涤处理精液的方法对卵母细胞体外受精及不同浓度的L-谷氨酰胺对受精卵发育的影响.结果表明:以TCM-199为基础培养液体外成熟率(72.29%)显著高于M16成熟率(59.11%)和KSOM成熟率(49.57%),三者之间彼此差异显著(P<0.05);上游法处理精子后,体外受精卵母细胞的卵裂率(64.89%)和囊胚发育率(18.56%)均高于直接洗涤法(51.9%,11.97%),且差异显著(P <0.05);0.5mmol·L-1L-谷氨酰胺受精卵培养液组的2~4细胞发育率(46.55%)、4~8细胞发育率(39.20%) 、8~16细胞发育率(30.40%)、桑葚胚发育率(26.65%),均显著高于含1mmol·L-1 L-谷氨酰胺受精卵培养液组(分别为38.83% 、32.02% 、25.14%和22.09%)和含2mmol·L-1 L-谷氨酰胺受精卵培养液组(分为33.96%、26.70%、21.43%和17.67%),且差异显著(P<0.05),而含0.5mmol· L-1 L-谷氨酰胺受精卵培养液组囊胚发育率(15.52%)与含1mmol·L-1、2 mmol·L-1 L-谷氨酰胺受精卵培养液组囊胚发育率(分别为10.59%和7.17%)差异显著(P<0.05).%In order to establish a perfect in vitro production system of rabbit embryo,We explored the effects of the TCM-199,M16 and KSOM three different basic nutrient solutions on the in vitro maturation of rabbit oocytes,and the upstream method and direct washing method of sperm washing processing methods treating semen of rabbit oocyte in vitro fertilization and different concentrations of L-glutamine on the influence of the rabbit zygote.The results showed that TCM-199 as the foundation nutrient solution in vitro maturation rate (72.29%) was statistically higher than that in the M16 mature rate (59.11%) and KSOM mature rate (49.57%)(P < 0.05).Upstream method processing sperm in vitro fertilized oocytes mother cell of the cleavage rate (64

  1. Mitochondrial functions on oocytes and preimplantation embryos

    Institute of Scientific and Technical Information of China (English)

    Li-ya WANG; Da-hui WANG; Xiang-yang ZOU; Chen-ming XU

    2009-01-01

    Oocyte quality has long been considered as a main limiting factor for in vitro fertilization (IVF). In the past decade,extensive observations demonstrated that the mitochondrion plays a vital role in the oocyte cytoplasm, for it can provide adenosine triphosphate (ATP) for fertilization and preimplantation embryo development and also act as stores of intracellular calcium and proapoptotic factors. During the oocyte maturation, mitochondria are characterized by distinct changes of their distribution pattern from being homogeneous to heterogeneous, which is correlated with the cumulus apoptosis. Oocyte quality decreases with the increasing maternal age. Recent studies have shown that low quality oocytes have some age-related dysfunctions, which include the decrease in mitochondrial membrane potential, increase of mitochondrial DNA (mtDNA) damages, chromosomal aneuploidies,the incidence of apoptosis, and changes in mitochondrial gene expression. All these dysfunctions may cause a high level of developmental retardation and arrest of preimplantation embryos. It has been suggested that these mitochondrial changes may arise from excessive reactive oxygen species (ROS) that is closely associated with the oxidative energy production or calcium overload,which may trigger permeability transition pore opening and subsequent apoptosis. Therefore, mitochondria can be seen as signs for oocyte quality evaluation, and it is possible that the oocyte quality can be improved by enhancing the physical function of mitochondria. Here we reviewed recent advances in mitochondrial functions on oocytes.

  2. 超表达Cdc20基因不影响牛卵母细胞第一极体排出%Over-expression of Cdc20 Gene Has No Effect on Bovine Oocytes First Polar Body Extrusion

    Institute of Scientific and Technical Information of China (English)

    杨文琳; 安鹏; 李伟; 赵贵民; 史芸安; 雷安民

    2012-01-01

    As one of the co-activator of anaphase-promoting complex ( APC) , cell division cycle 20 (CDC20) protein also functions as the target of the spindle assembly checkpoint ( SAC), which is essential for the cell cycle regulation. To investigate the function of Cdc20 during the first polar body extrusion ( PBE I) , Cdc20 CDS was cloned and eukaryotic expression vector pCdc20-Venus was constructed. Using the linear pCdc20-Venus as template, the capped Cdc20-Venus mRNA was synthesized via T7 Mmessage Mmachine Kit ( Ambion). Cdc20 over-expression was performed by microinjection of Cdc20-Venus mRNA into the cytoplasm of bovine oocytes. The results showed that Venus tagged Cdc20 dispersed around the nucleus in HeLa cells. In bovine oocytes, the fluorescence appeared in the whole cytoplasm. However, the PBE I rate in over-expressed group (48. 9% ) is not significant, compared to Venus mRNA injection group (50.9%) and non-injection group (51.1%). Our study demonstrated that the over-expression of Cdc20 in bovine oocytes does not affect the PBE I rate ( P > 0.05).%CDC20(cell division cycle 20)是后期促进复合物(anaphase-promoting complex,APC)的共激活剂之一,也是纺锤体组装检查点(spindle assembly checkpoint,SAC)的靶点,在细胞周期调控中扮演重要角色.为探讨Cdc20在第一极体排出(first polar body extrusion,PBE I)中的作用,Cdc20基因被成功克隆并构建了真核表达载体pCdc20-Venus,随后用T7 Mmessage Mmachine Kit(Ambion)以线性化pCdc20-Venus为模板体外转录(in vitro transcription)获得带帽的Cdc20-Venus mRNA,将Ccdc20-Venus mRNA显微注射到体外培养的牛卵母细胞胞质中进行超量表达.结果表明,真核表达载体pCdc20-Venus转染HeLa细胞后能够正常表达,绿色荧光在细胞核周围呈弥散状分布;将Cdc20-Venus mRNA注射到牛卵母细胞胞质后,胞质内有绿色荧光出现.Cdc20-Venus mRNA注射组卵母细胞的PBE I率(48.9%)与Venus mRNA注射组卵母细胞的PBE I率(50

  3. 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.

  4. Relationship between the number of retrieved oocytes and the outcome of in vitro fertilization - embryo transfer%获卵数目与体外受精-胚胎移植结局的关系

    Institute of Scientific and Technical Information of China (English)

    樊桂玲; 彭鹏; 韩宝生; 李宜学; 郭剑

    2011-01-01

    目的:分析和探讨体外受精-胚胎移植( IVF - ET)周期最合适的获卵数目.方法:对唐山市妇幼保健院生殖遗传科2009年8月~2010年12月实施的237个1VF - ET新鲜周期进行回顾性总结和分析,根据不同获卵数分为4组:A组≤3、B组4~9、C组10 ~ 15及D组>16个卵子,分析各组的周期数、年龄、基础FSH、平均促性腺激素(Gn)启动剂量、用量及天数、HCG注射日的E2值、优胚率和取卵周期临床妊娠率.结果:①获卵数随年龄增加呈下降趋势;②FSH值各组差异无统计学意义;③Gn启动剂量随获卵数增加呈下降趋势,A组与C组、A组与D组、B组与C组差异显著(P<0.01),其余各组差异无统计学意义(P>0.05);④Gn用药天数各组差异无统计学意义;⑤各组Gn总剂量随获卵数增加呈下降趋势但差异无统计学意义(P>0.05);⑥注射HCG日的血清E2值随获卵数增加而明显上升,与获卵数呈正相关,各组之间差异有统计学意义(P<0.01);⑦C组的优胚率及妊娠率高于其他组,差异有统计学意义(P<0.01);⑧D组周期取消数最高,且均因卵巢过度刺激综合征(OHSS).结论:IVF - ET周期中获卵数在10~15个之间既可以降低OHSS发生,又可获得较理想的妊娠结局.%Objective; To analyze and explore the optimal number of retrieved oocytes during in vitro fertilization - embryo transfer (IVF - ET) cycle. Methods; 237 fresh IVF - ET cycles carried out in the department of reproductive genetics of the hospital from August 2009 to December 2010 were summarized and analyzed retrospectively, then they were divided into four groups according to different numbers of retrieved oocytes; group A ( ^3 oocytes) , group B (4 -9 oocytes) , group C (10-15 oocytes) and group D ( > 16 oocytes) . The numbers of cycles, ages, basic follicle stimulating hormone (FSH) levels, average start doses, dosages and days of gonadotropin, estradiol values on the day of human chorionic

  5. Oocyte Pickup from Live Cows Through Laparoscopic Guided Aspiration

    Institute of Scientific and Technical Information of China (English)

    Ni Hemin; Guo Yong; Zhu Yuding; Liu Yunhai; Dou Zhongying

    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.

  6. The effect of IVM and IVC media on in vitro development of bovine embryos

    Directory of Open Access Journals (Sweden)

    E.T Mergawati

    2000-12-01

    Full Text Available The purpose of this study was to examine the effect of medium combination of IVM and IVC on the in vitro development of bovine embryos. The study involved 4 groups in a 2 (IVM medium x 2 (IVC medium factorial in a randomized block design. Each group was replicated for 5 times. The treatments were as follows: TCM-199/CR1aa (T1; TCM-199/SOF (T2; B- 199/CR1aa (T3 and B-199/SOF (T4. Oocytes were aspirated from ovaries collected at local abattoirs using aspiration medium of PBS supplemented with 3% FCS and 0.1% Penicillin and Streptomycin. The oocytes were matured in medium of TCM-199 or B-199 supplemented with 10% FCS, hormones: 10μg/ml FSH+ 10μg/ml hCG+ 1μg/ml Estradiol. Maturation was maintained at 37oC for 22 hours in 5% CO2 incubator with high humidity. A method of BRACKETT & Oliphant (BO was used to fertilize the matured oocytes. The fertilization was incubated for 7 hours in the 5% CO2 incubator. Two culture media of CR1aa or SOF/AA/BSA were used to develop the fertilized oocytes undergo to morula and blastocyst embryos. The findings showed that the proportion of oocytes cleaved and formation of blastocysts were affected significantly by a combination of IVM and IVC media (P<0.05. A combination of B-199/SOF (T4 resulted in a higher blastocyst rate (32% than others (T3= 29%; T2=T1= 23%. This study suggests that either SOF/AA/BSA or CR1aa has similar competence in development of bovine embryos in vitro.

  7. 水牛和黄牛同种及种间显微授精(ICSI)的初步研究%A Preliminary Study on the Intraspecific and Interspecific Fertilization between Buffalo and Cattle by Intracytoplasmic Sperm Injection

    Institute of Scientific and Technical Information of China (English)

    牛向丽; 陆凤花; 石德顺; 张艳玲; 卢晟盛

    2011-01-01

    试验探讨了水牛和黄牛同种及种间显微授精的可行性.体外成熟22~24 h的水牛和黄牛卵母细胞分别注入冷冻/解冻的尼里-拉菲水牛和利木赞黄牛精子,进行同种或种间的显微授精操作,构建的ICSI胚胎一部分培养到16~18 h固定染色检查原核形成情况,另外一部分胚胎培养2~9 d分别记录胚胎的分裂情况及囊胚发育情况.结果发现,水牛同种(水牛+水牛)和水牛异种(水牛+黄牛)显微授精胚胎的双原核率(47.73%和36.67%)、分裂率(68.00%和72.64%)和囊胚发育率(16.44%和22.39%)均无显著差异(P>0.05);黄牛同种(黄牛+黄牛)和黄牛异种(黄牛+水牛)显微授精胚胎的双原核率(60.00%和45.28%)、分裂率(73.33%和71.31%)和囊胚发育率(28.57%和33.70%)亦无显著差异(P>0.05).以上结果表明:①水牛精子注射到黄牛卵子和黄牛精子注射到水牛卵子的种间授精胚胎均可以体外发育到囊胚阶段;②黄牛卵子无论同种和种间显微授精的效果均优于水牛卵子.%The possibility of intraspecific and interspecific fertilization between buffalo and cattle was explored in this study. Buffalo and bovine oocytes matured in vitro lor 22 to 24 h were fertilized by injection of buffalo or bovine frozen-thawed spermatozoa. At 16 to 18 h after injection, a portion of ICSI oocytes were fixed in acetic ethanol to examine the pronuclear formation,others were cultured in vitro to evaluate their cleavage rate and embryonic developmental ability. There was no difference in the two pronuclear formation rate (47. 73% and 36. 67%),cleavage rate (68. 00% and 72. 64%) and blastocyst development 16. 44% and 22. 39%) of buffalo oocytes fertilized with buffalo (intraspecies) and bovine sperm (interspecies) (P>0. 05). The two pronuclear formation rate (60. 00%),cleavage rate (73. 33%) and blastocyst rate (28. 57%) of bovine oocytes fertilized with bovine sperm (intraspecies) were also not

  8. [Successful pregnancies after oocyte and embryo vitrification].

    Science.gov (United States)

    Salazar, Francisco Hernández; Loza, Erik Omar Okhuysen; Lucas, Maria Teresa Huerta J; Gutiérrez, Gustavo Romero

    2008-02-01

    Cryopreservation of human oocytes represents a solution for ethic conflict about frozen embryo storage for patients with risk to develop ovarian hyperstimulation syndrome; also is an available technique to preserve fertility in women with cancer under treatment, in poor response patients, in case of premature ovarian failure or aging and for other medical or social conditions that require to delay pregnancies, as well as to make easier oocyte donation programs. This paper reports two cases of successful pregnancies after embryo and oocyte vitrification, as well as their results. The technique of vitrification with the cryotop method is an excellent alternative, efficient, fast and cheap for oocyte and embryo cryopreservation with high ranges of fertilization, cleavage and pregnancies with a normal evolution. PMID:18798404

  9. 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.

  10. 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.

  11. Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos Efeito do fator de crescimento semelhante à insulina-1 durante a maturação in vitro dos oócitos e cultivo in vitro de embriões bovinos

    Directory of Open Access Journals (Sweden)

    M.D. Quetglas

    2001-04-01

    Full Text Available The effects of insulin-like growth factor-I (IGF-I on in vitro maturation (IVM (experiment I and on in vitro embryo development (experiment II of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR, blastocyst (BR and hatching (HR rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium and gonadotropins (14 U/ml PMSG and 7 U/ml hCG. For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5ºC in 5% CO2 in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P>0.05 among treatments for CR, BR or HR. In experiment II, the addition of IGF-I to the embryo culture medium (ECM resulted in a significant increase in CR while for BR and HR this effect was not observed. The addition of 200 ng/ml IGF-I to ECM increased CR (71.1% when compared to 100 ng/ml IGF-I (57.6% or control (56.7% groups, however, there were no differences when compared to 50 (69.4% or 10 ng/ml (73.1% groups. There was no beneficial effect of the addition of IGF-I in the IVM or ECM media on the in vitro development of embryos produced from oocytes matured and fertilized in vitro.Avaliaram-se o efeito do IGF-I na maturação in vitro (MIV (experimento I e no desenvolvimento embrionário (DE (experimento II de oócitos bovinos fecundados in vitro, quanto às taxas de clivagem (TC, de blastocistos (TB e de eclosão (TE. Para MIV, complexos cumulus-oócitos imaturos foram cultivados em meio TCM-199 suplementado com HEPES, bicarbonato e piruvato de sódio, aditivos, soro

  12. Fertility Preservation for Female

    Institute of Scientific and Technical Information of China (English)

    Jack Huang; Seang Lin Tan; Ri-Cheng Chian

    2006-01-01

    Preservation of female fertility is an important issue today. However, there are few effective clinical options for preserving female fertility. Firstly, conventional in vitro fertilization (IVF) followed by embryo cryopreservation is an accepted procedure but is not applicable to all women. Embryo freezing is suitable only for women with a male partner and may not be acceptable to some patients due to moral and religious reasons. Ovarian tissue freezing is another option of female fertility preservation but is an invasive procedure and the efficacy of this technique remains to be determined.Oocyte cryopreservation is also method for fertility preservation. Egg freezing is minimally invasive and can avoid the ethical and moral concerns related to cryopreservation of embryos. However, conventional slow freezing/rapid thawing methods are associated with low survival of oocytes. Recent development in vitrification of oocytes appears promising. Therefore, vitrification of unfertilized eggs may be a novel method to preserve female fertility.

  13. 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.

  14. 未成熟卵体外成熟技术在卵巢高反应患者IVF-ET中的应用%In vitro maturation of human immature oocyte in in vitro fertilization and embryo transfer

    Institute of Scientific and Technical Information of China (English)

    黄绘; 洪焱; 冯玉蓉; 刘敏利; 胡皓睿; 潘世春

    2012-01-01

    目的:探讨未成熟卵体外成熟(IVM)技术在卵巢高反应患者体外受精-胚胎移植(IVF - ET)中的应用价值.方法:在IVF - ET促排治疗中,对双卵巢卵泡数过多,有可能发生卵巢过度刺激综合征(OHSS)或继续治疗可能发生重度OHSS的患者,根据其意愿即刻停药,全部取卵改行IVM治疗12个周期(A组)或取部分小卵泡改行IVM治疗,同时保留部分卵泡继续行IVF - ET常规治疗18个周期(B组).小卵泡体外培养成熟后,通过卵胞浆内单精子注射(ICSI)获得受精卵并行胚胎移植或冷冻.统计分析未成熟卵的成熟率、卵母细胞的受精率、胚胎的发育情况及临床结局.结果:两组30个取卵周期,共获未成熟卵240个,经IVM、ICSI和体外培养后,成熟率、受精率、正常卵裂率及优质胚胎率分别为61.25% (147/240),77.55% (114/147),92.98% (106/114)和29.25%(31/106).A组8例行IVM新鲜胚胎移植(8周期)4例临床妊娠,A、B两组有8例行IVM解冻胚胎移植(9周期)3例临床妊娠,已有3例分娩.A组12例无OHSS发生,促性腺激素用量少于B组,B组18例中3例有OHSS风险而取消胚胎移植.结论:对常规IVF促排周期中卵巢高反应患者及时改行IVM,可以避免周期取消及OHSS的发生,减少促排卵药物的使用量,同时获得较好的临床妊娠率.%Objective ; To explore the clinical application of in vitro oocyte maturation (IVM) technique in in vitro fertilization - embryo transfer (IVF - ET). Methods; In the treatment of IVF - ET, women who canceled the treatment cycle due to high risk of developing ovarian hyperstimulation syndrome ( OHSS) were divided into two groups according to their choices. Women in group A (n = 12) underwent oocyte retrieval guided by transvaginal ultrasound for all small follicles and treated with IVM technique for 12 cycles. Women in group B (n = 18) were performed oocyte retrieval for some small follicles treated with IVM technique and kept on IVF - ET with some

  15. Effect of β-mercaptoethanol and buffalo follicular fluid on fertilization and subsequent embryonic development of water buffalo (Bubalus bubalis) oocytes derived from in vitro maturation

    OpenAIRE

    Qin, J.; X.F. Zhang; J.H. Shang; Huang, Y J; F.X. Huang

    2010-01-01

    This study investigated effects of β-mercaptoethanol (β-ME) and buffalo follicular fluid (buFF) on IVF and embryonic development of water buffalo oocytes derived from IVM. In Experiment 1, β-ME (50μM and 100μM, treatments) was added in the media during IVM and IVC but not IVF. A highly significant difference was obtained in rate of blastocyst (9.26% Vs 26.72% or 27.04%, P0.05) were observed in rates of cleavage, blastocyst and hatched blastocyst between two treatment grou...

  16. 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.

  17. Tretinoin-loaded lipid-core nanocapsules decrease reactive oxygen species levels and improve bovine embryonic development during in vitro oocyte maturation.

    Science.gov (United States)

    Lucas, Caroline Gomes; Remião, Mariana Härter; Komninou, Eliza Rossi; Domingues, William Borges; Haas, Cristina; Leon, Priscila Marques Moura de; Campos, Vinicius Farias; Ourique, Aline; Guterres, Silvia S; Pohlmann, Adriana R; Basso, Andrea Cristina; Seixas, Fabiana Kömmling; Beck, Ruy Carlos Ruver; Collares, Tiago

    2015-12-01

    In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25μM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates. PMID:26476360

  18. Tretinoin-loaded lipid-core nanocapsules decrease reactive oxygen species levels and improve bovine embryonic development during in vitro oocyte maturation.

    Science.gov (United States)

    Lucas, Caroline Gomes; Remião, Mariana Härter; Komninou, Eliza Rossi; Domingues, William Borges; Haas, Cristina; Leon, Priscila Marques Moura de; Campos, Vinicius Farias; Ourique, Aline; Guterres, Silvia S; Pohlmann, Adriana R; Basso, Andrea Cristina; Seixas, Fabiana Kömmling; Beck, Ruy Carlos Ruver; Collares, Tiago

    2015-12-01

    In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25μM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.

  19. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    OpenAIRE

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C...

  20. Effects of bone morphogenic protein 4 (BMP4 and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Fernandez-Martin Rafael

    2011-02-01

    Full Text Available Abstract Background BMP4 is a member of the transforming growth factor beta (TGFbeta superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments and embryos in vitro development (c experiments of bovine. Methods For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin. After 24 h, the nuclear stage of the oocytes was determined by staining with Hoechst 33342. In addition, RT-qPCR was performed on MII oocytes to study the relative concentration of ZAR1, GDF9, BAX, MATER and HSP70 transcripts. Treated oocytes were submitted to parthenogenic activation (PA or in vitro fertilization (IVF and cultured in CR2. For c experiments, non-treated matured oocytes were submitted to PA or IVF to generate embryos that were exposed to 100 ng/ml of BMP4 or Noggin in CR2 until day nine of culture. Cleavage, blastocyst and hatching rates, expression pattern of the transcription factor Oct-4 in blastocysts and embryo cell number at day two and nine post-activation or fertilization were evaluated. Results We found that Noggin, as BMP4, did not affect oocyte nuclear maturation. Noggin supplementation up-regulated the expression of HSP70 and MATER genes in matured oocytes. Moreover, BMP4 during maturation increased the proportion of Oct-4 positive cells in parthenogenic embryos. On the other hand, when Noggin was added to embryo culture medium, developmental rates of parthenogenic and in vitro fertilized embryos were reduced. However, BMP4 addition decreases the development only for in vitro fertilized embryos. BMP4 and Noggin during culture reduced the proportion of Oct-4-expressing cells. Conclusions Our results show that BMP4 is implicated in bovine oocytes maturation and embryo development. Moreover

  1. 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.

  2. 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.

  3. Relationship between the length of cell cycles, cleavage pattern and developmental competence in bovine embryos generated by in vitro fertilization or parthenogenesis.

    Science.gov (United States)

    Somfai, Tamás; Inaba, Yasushi; Aikawa, Yoshio; Ohtake, Masaki; Kobayashi, Shuji; Konishi, Kazuyuki; Imai, Kei

    2010-04-01

    This study was conducted to study the kinetics of initial cell divisions in relation with the cleavage patterns in viable (with the ability to develop to the blastocyst stage) and non-viable bovine embryos and parthenotes. The kinetics of in vitro development and cleavage patterns were observed by time lapse cinematography. The length of the first and second but not third cell cycle differed significantly between the viable and non-viable embryos after IVF or parthenogenesis. Viable embryos had significantly shorter first and second cell cycles than non-viable ones. The presence of fragments, protrusions and unequally-sized blastomeres was associated with an extended one-cell stage and reduced ability to develop to the blastocyst stage; however, the lengths of the second and third cell cycles were not altered. Oocytes showing direct division from one cell to 3 or 4 blastomeres showed similar developmental ability and embryonic cell numbers to those showing normal division, although, with a high frequency of chromosomal abnormalities. Our results suggest that the differences in the first cell cycles between viable and non-viable embryos were not sperm-related, whereas direct cleavage of 1-cell embryos to 3 or more blastomeres and protrusion formation are related to sperm-driven factors. The length of the first and second cell cycles and the cleavage pattern should be examined simultaneously to predict developmental competence of embryos at early cleavage stages. PMID:20035110

  4. Effect of aspiration pressure during oocyte harvesting on oocyte recovery and in vitro development of ovine oocytes.

    Science.gov (United States)

    Morton, K M; Maxwell, W M C; Evans, G

    2008-02-01

    Oocytes from abattoir-sourced ovine ovaries were aspirated from 2- to 4-mm follicles using 25, 50 or 100 mmHg pressure and an aspiration pump, or a needle (20-G) and syringe (2.5 ml) and subjected to in vitro maturation, fertilization and culture to determine the effect of aspiration pressure on the number and quality of oocytes recovered, and early embryonic development. Oocyte recovery rate was similar between groups (range: 57.1-73.1%; p > 0.05). The number and percentage of grade I and II oocytes recovered was reduced for 100 mm (24.5 +/- 3.6 and 31.1 +/- 6.1%) compared with 25 mm (51.4 +/- 7.0 and 60.2 +/- 7.8%) and 50 mm pressure (40.8 +/- 5.6 and 50.3 +/- 4.4%) and a syringe (40.3 +/- 12.0 and 45.2 +/- 2.1%; p < 0.05). Oocyte cleavage was similar for all groups at 24 (range: 30.9-49.6%) and 48 h post-insemination (49.7-65.5%), but blastocyst formation (% cleaved oocytes) was lower for oocytes aspirated with 25 mm (37.8%) than 50 (69.2%) or 100 mm (67.2%) pressure, and a syringe (72.0%; p < 0.05). Embryo production efficiency (% of oocytes cultured developing to the blastocyst stage) was higher for oocytes aspirated with 50 mm (45.4%) and 100 mm pressure (43.8%) and a syringe (45.0%) than 25 mm pressure (18.8%; p < 0.05). These results demonstrate that the aspiration of ovine oocytes with an aspiration pressure of 50 mm, or aspiration with a needle and syringe are equally efficacious for the in vitro production of embryos. PMID:18199266

  5. Cancer and fertility: strategies to preserve fertility.

    Science.gov (United States)

    Diedrich, K; Fauser, B C J M; Devroey, P

    2011-03-01

    Fertility preservation is a key component of cancer management in young people. The Fourth Evian Annual Reproduction Workshop Meeting was held in April 2009 to discuss cancer and fertility in young adults. Specialists in oncology, assisted reproduction, embryology and clinical genetics presented published data and ongoing research on cancer and fertility, with particular focus on strategies to preserve fertility. This report is based on the expert presentations and group discussions, supplemented with publications from literature searches and the authors' knowledge. Fertility preservation should be considered for all young people undergoing potentially gonadotoxic cancer treatment. A variety of options are required to facilitate safe and effective fertility preservation for individual patients. Sperm banking is a simple and low-cost intervention. Embryo cryopreservation is the only established method of female fertility preservation. Oocyte cryopreservation offers a useful option for women without a male partner. Emergency ovarian stimulation and cryopreservation of ovarian tissue (followed by tissue transplantation or in-vitro maturation of oocytes) are experimental techniques for women who require urgent cancer treatment. Further prospective studies are required to validate cryopreservation of oocytes and ovarian tissue, in-vitro maturation of oocytes and new vitrification techniques and to identify any long-term sequelae of slow freezing of embryos.

  6. Coagulansin-A has beneficial effects on the development of bovine embryos in vitro via HSP70 induction

    Science.gov (United States)

    Khan, Imran; Lee, Kyeong-Lim; Fakruzzaman, Md.; Song, Seok-Hwan; Ihsan-ul-Haq; Mirza, Bushra; Yan, Chang Guo; Kong, Il-Keun

    2016-01-01

    Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans which belong to Solanaceae family. The present study investigated the effects of coagulansin-A on bovine oocyte maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To determine whether coagulansin-A has beneficial effects on bovine oocyte maturation in vitro, 355 oocytes per group (control and treated) in seven replicates were subjected with different concentrations (1, 2.5, 5, 7.5 and 10 μM) of coagulansin-A. The coagulansin-A was added in the in vitro maturation (IVM) media followed by in vitro fertilization (IVF) and then in vitro culture (IVC). Only treatment with 5 μM coagulansin-A remarkably (P<0.05) improved embryos development (Day 8 blastocyst) having 27.30 and 40.01% for control and coagulansin-A treated groups respectively. Treatment with 5 μM coagulansin-A significantly induced activation of heat shock protein 70 (HSP70) (P<0.05). Immunofluorescence analysis revealed that 5 μM coagulansin-A treatment also significantly inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing 8-oxoguanosine (8-OxoG) (P<0.05) and nuclear factor-κB (NF-κB) (P<0.05). The expressions of HSP70 and NF-κB were also conformed through real-time PCR (RT-PCR). Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay confirmed that coagulansin-A treatment significantly improved the embryo quality and reduced bovine embryo DNA damage (P<0.05). The present study provides new information regarding the mechanisms by which coagulansin-A promotes bovine embryo development in vitro. PMID:26831738

  7. Slow freezing and vitrification differentially modify the gene expression profile of human metaphase II oocytes.

    OpenAIRE

    Monzo, Cécile; Haouzi, Delphine; Roman, K.; Assou, Said; Dechaud, Hervé; Hamamah, Samir

    2012-01-01

    BACKGROUND: Cryopreservation is now considered as an efficient way to store human oocytes to preserve fertility. However, little is known about the effects of this technology on oocyte gene expression. The aim of this study was to examine the effect of the two cryopreservation procedures, slow freezing and vitrification, on the gene expression profile of human metaphase II (MII) oocytes. METHODS: Unfertilized MII oocytes following ICSI failure were cryopreserved either by slow freezing or by ...

  8. Cysteamine, glutathione and ionomycin treatments improve IVF of prepubertal goat oocytes

    OpenAIRE

    Urdaneta Vargas, Aixa Efrailda; Izquierdo Tugas, Maria Dolors; Jiménez y de Macedo, Ana Raquel; Paramio Nieto, María Teresa

    2004-01-01

    The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by T1 studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1mM GSH added to fertilization medium of oocytes matured with 400 μ M cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertil...

  9. The effect of bovine viral diarrhoea virus on fertility in dairy cows: two case-control studies in the province of Styria, Austria.

    Science.gov (United States)

    Burgstaller, Johann; Obritzhauser, Walter; Kuchling, Sabrina; Kopacka, Ian; Pinior, Beate; Köfer, Josef

    2016-01-01

    Bovine viral diarrhoea (BVD) leads to substantial economic losses in beef and dairy herds worldwide. Two case-control studies were carried out using production data from 1996 to 2012 to analyse the impact of BVD virus (BVDV) on fertility in dairy herds in the province of Styria during an eradication programme. In study 1, herds in which at least one persistently BVDV-infected (PI) animal was detected (case herds) were compared to a group of control herds proven free from BVDV infection (contro herds). In study 2, within BVD infected herds the period during which P animals were present (exposed period) was compared to the period after successful BVD eradication (unexposed period). Calving interval (CAl) and the probability of a first service conception (FSC) were used as indicators in a mixed regression model to investigate the impact of BVD on reproductive performance. The model results indicated that BVD had a significant influence on CAl and FSC. Cows from control herds were 1.1 times more likely to conceive at first service compared to cows from case herds and cows served during the BVDV unexposed period were 1.3 times more likely to conceive at first service than those inseminated during the exposed period. In BVD-infected herds the CAI averaged seven days shorter in unexposed periods than in exposed periods. Besides BVD the animal breed and the parity substantially impact the analysed fertility indicators.

  10. The measurement of sperm motility by the fibre optic Doppler anemometer as a prediction of bovine fertility

    Science.gov (United States)

    Bullock, J. G.; Ross, D. A.

    The fibre optic Doppler anemometer (FODA) has been used to develop an accurate quantitative method of routinely assessing bull fertility. This method is of importance to the artificial insemination industry because the present qualitative estimation, performed by viewing semen using a microscope, can only set broad limits of quality. Laser light from the FODA was directed into diluted semen samples and the back scattered light was measured. A digital correlator was used to calculate the signal correlation of the back scattered light. The resultant data curves were interpreted in terms of the collective motility and swimming speed of the spermatozoa using a microcomputer. These two parameters are accepted as being indicative of fertility. The accuracy of this method is demonstrated by examination of results obtained in an experiment where enzymes, thought to alter fertility, were added to semen. The effect of the enzymes on the swimming speed and motility was clearly demonstrated.

  11. Ribosomal RNA and nucleolar proteins from the oocyte are to some degree used for embryonic nucleolar formation in cattle and pig

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul; Svarcova, Olga; Laurincik, Josef

    2007-01-01

    The nucleolus is the site of ribosomal RNA (rRNA) and ribosome production. In the bovine primordial follicle oocyte, this organelle is inactive, but in the secondary follicle an active fibrillo-granular nucleolus develops and proteins involved in rDNA transcription (topoisomerase I, RNA polymerase...... I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) localize to it. At the end of the oocyte growth phase, the nucleolus is inactivated again and transforms into a solid remnant. The nucleolar remnant is dissolved when meiosis is resumed. Upon...... fertilization, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities are engaged in the re-establishment of fibrilo-granular nucleoli at the major activation of the embryonic genome. This nucleolar formation can...

  12. Rendimento do inhame adubado com esterco bovino e biofertilizante no solo e na folha Yam yield fertilized with bovine manure and biofertilizers in the soil and leaf

    Directory of Open Access Journals (Sweden)

    Jandiê A. da Silva

    2012-01-01

    Full Text Available Neste trabalho objetivou-se avaliar o rendimento do inhame, cultivar Da Costa, adubado com doses de esterco bovino e biofertilizante. O delineamento experimental utilizado foi o de blocos casualizados, em parcelas subdivididas, 6 x 2 + 1 em três repetições. Nas parcelas foram testadas seis doses de esterco bovino (0; 6; 12; 18; 24 e 30 t ha-1, combinadas fatorialmente com a presença e ausência de biofertilizante e, nas subparcelas, duas formas de aplicação do biofertilizante no solo e na folha e um tratamento adicional com adubação convencional (esterco bovino e NPK. A dose de 30 t ha-1 de esterco bovino e o biofertilizante aplicado no solo e na folha produziram túberas de inhame com peso médio ideal para o comércio. O esterco bovino na dose de 19,2 t ha-1 e na ausência do biofertilizante proporcionou produtividade máxima de 20,3 t ha-1 de túberas comerciais. Nas subparcelas em que o biofertilizante foi aplicado no solo e na folha, a dose de 30 t ha-1 de esterco bovino foi responsável, respectivamente, pelas produtividades máximas de 22,8 e 24 t ha-1 de túberas comerciais. A adubação orgânica e a convencional não causaram alterações significativas no peso médio de túberas; porém, a adubação convencional aumentou a produtividade de túberas comerciais.The objective of this study was to evaluate the yam yield, cultivar Da Costa, fertilized with bovine manure doses and biofertilizer. The experimental design was randomized blocks, in subdivided plots 6 x 2 + 1 with three repetitions. In plots six doses of cattle manure (0; 6; 12; 18; 24 and 30 t ha-1 were tested, factorially combined with the presence and absence of biofertilizer and in subplots, two forms of application of biofertilizer in the soil and by spray on the leaf and an additional treatment with conventional fertilization (animal manure and NPK. The doses of 30 t ha-1 of bovine manure and the biofertilizer which was applied in the soil and leaf produced tubers

  13. Collection of oocytes through transvaginal ultrasound-guided aspiration of follicles in an Indian breed of cattle.

    Science.gov (United States)

    Manik, R S; Singla, S K; Palta, P

    2003-04-15

    The present study was undertaken in Karan Fries, an Indian breed of cattle to (1) determine the number of follicles available for puncture and (2) explore the potential of this breed as a donor of developmentally competent oocytes. Ovum pick-up (OPU) was performed using an ultrasound machine with a transvaginal convex transducer (5 MHz) with a needle guide, single lumen 19-gauge 60 cm long needle and a vacuum pressure of 90 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large (>/=10 mm). The oocytes recovered were classified by quality. They were matured in vitro, irrespective of their grade, in 50 microl droplets of the in vitro maturation (IVM) medium (TCM-199+10% fetal bovine serum(FBS)+5 microg/ml follicle stimulating hormone (folltropin)+1 microg/ml estradiol-17beta+0.2 mM sodium pyruvate), covered with paraffin oil, in 35 mm petridish for 24 h in a CO(2) incubator (5% CO(2) in air) at 38.5 degrees C. The cleavage rate was recorded at day 2 post-insemination after subjecting the oocytes to in vitro fertilization (IVF). The differences in follicular populations of all size categories among individual donors were not significant. A total of 92 oocytes were recovered by aspiration of 157 follicles, with an overall recovery rate of 59% (range 35-79%). Of these, 32% were of grades A and B and the rest of grades C and D. The mean numbers of total follicles and the oocytes recovered per session did not differ significantly among individual donors. Out of the 73 oocytes subjected to IVM and IVF, 24 reached 2-4 cell stage at day 2 post-fertilization, with a cleavage rate of 33%. The total number of oocytes recovered was correlated with the number of small (R=0.54, Pslaughter is not allowed for religious reasons. PMID:12586489

  14. Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth

    Directory of Open Access Journals (Sweden)

    Vanderhyden Barbara C

    2006-04-01

    Full Text Available Abstract Ovarian folliculogenesis is regulated by both endocrine and intraovarian mechanisms that coordinate the processes of oocyte growth and somatic cell proliferation and differentiation. Within the follicle, paracrine interactions between the oocyte and surrounding granulosa cells are critical for normal cell development and function. This review focuses on the role of paracrine interactions during early oocyte and follicular development that ensure proper coordination of oocyte and somatic cell function. Particular emphasis is given to granulosa cell-derived Kit Ligand (KitL, whose functional importance for oocyte growth has been demonstrated by a wide range of in vivo and in vitro studies. Reported interactions between KitL and oocyte-derived growth differentiation factor-9 (GDF9 and bone morphogenetic protein-15 (BMP15 suggest the molecular basis of oocyte-granulosa cell interactions, but also hint at the complexity of these communications. These paracrine interactions and the structure of the oocyte-granulosa cell interface are follicle stage-specific and regulated by FSH. Elucidation of the molecular mechanisms that promote the development of healthy oocytes with good developmental competence has potential applications for improving fertility and for in vitro growth systems for oocytes from domestic animals and humans.

  15. 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.

  16. 催乳素和牛卵泡液对胚胎体外成熟及未成熟的牛卵母细胞的影响%The effect of prolactin and bovine follicular fluid on the in vitro maturation and subsequent development of immature buffalo (Bubalus bubalis) oocytes

    Institute of Scientific and Technical Information of China (English)

    Abdul Rahman SESAY; 石德顺

    2004-01-01

    Buffalo oocytes with compact cumulus cells were collected from slaughterhouse ovaries and cultured in TCM 199 supplemented with 5% estrus cow serum (ECS) + 0.1 μg/mL follicle stimulating hormone (FSH), prolactin (PRL 0, 0.1, 1.0, 10 μg/mL, Experiment 1), bovine follicular fluid (BFF) (0% BFF + 1.0 μg/mL PRL, 5% BFF, 5% BFF + 1.0 μg/mL PRL, Experiment 2), incubated at 38.5 ℃ in φ = 5% CO2 in humidified air. After 24 to 26 hours of maturation, IVF was done with swim-up separated frozen-thawed buffalo spermatozoa at 1×106 mL-1 in modified Tyrodes medium (TALP). At 24 to 26 hours post insemination, the oocytes were co-cultured with granulosa cell monolayer in droplets containing culture medium. The proportion of cleaved oocytes that developed to blastocyst stage within 9 days after commencing co-culture with granulosa cell monolayer was then evaluated. In experiment 1, the proportion of cleaved oocytes that developed to blastocyst stage was higher (12.8%) at 1.0 μg/mL PRL though not significantly different from the control (9.1%). In experiment 2, addition of 5% BFF to the maturation medium had significant increase in the cleavage rate of oocytes compared to the control (30.7% vs. 21.7%, P<0.05), but did not influence the proportion of cleaved oocytes that developed into blastocysts;addition of 5% BFF+1.0 μg/mL PRL to the maturation medium had a cleavage rate of 38.1%, with 14.0% of the cleaved oocytes developing to blastocysts (P<0.05). In conclusion, the results indicate that the addition of appropriate amounts of prolactin and bovine follicular fluid to the maturation medium will enhance the maturation of immature buffalo oocytes with their subsequent development to the blastocyst stage.%对催乳素和牛卵泡液在水牛卵母细胞体外成熟中的作用进行了探讨. 来自屠宰场水牛卵巢的卵母细胞和卵丘细胞复合体,在含体积分数为5% CO2的培养箱中培养24~26 h,然后通过体外受精测定

  17. The functional role of insulin in fertility and embryonic development-What can we learn from the bovine model?

    Science.gov (United States)

    Laskowski, D; Sjunnesson, Y; Humblot, P; Andersson, G; Gustafsson, H; Båge, R

    2016-07-01

    Insulin is a key metabolic hormone that plays a crucial role in regulating energy homeostasis in the body. In addition, insulin-dependent signaling has important functions in reproduction and early embryo development. As metabolism and reproduction are closely linked, metabolic challenges may be the source of reproductive disorders and decreased fertility. This is known for the dairy cow and for other species including the human. Although metabolic disorders in the dairy cow often derive from a failure to adapt to a high milk production, the situation in the human is often linked to emerging conditions and associated diseases in our modern society such as obesity and diabetes, where an excess energy intake causes decreased fertility in women. Both energy excess and energy deficit are associated with a deviation of insulin concentrations in serum and follicular fluid from normal levels. Although many studies have shown that extreme variation in energy supply can negatively influence early embryo development by inducing changes in circulating concentrations of several metabolites or hormones like insulin, several in vitro culture media are still supplemented with insulin in high concentrations. In this review, direct and indirect effects of insulin on fertility will be described. Differences between the in vivo and in vitro situations will also be discussed. PMID:27177960

  18. 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.

  19. Nonesterified Fatty Acid-Induced Endoplasmic Reticulum Stress in Cattle Cumulus Oocyte Complexes Alters Cell Metabolism and Developmental Competence.

    Science.gov (United States)

    Sutton-McDowall, Melanie L; Wu, Linda L Y; Purdey, Malcolm; Abell, Andrew D; Goldys, Ewa M; MacMillan, Keith L; Thompson, Jeremy G; Robker, Rebecca L

    2016-01-01

    Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation. PMID:26658709

  20. Role of arachidonic acid cascade in Rhinella arenarum oocyte maturation.

    Science.gov (United States)

    Ortiz, Maria Eugenia; Arias-Torres, Ana Josefina; Zelarayán, Liliana Isabel

    2015-08-01

    There are no studies that document the production of prostaglandins (PGs) or their role in Rhinella arenarum oocyte maturation. In this study, we analysed the effect of arachidonic acid (AA) and prostaglandins (PGs) on maturation, activation and pronuclear formation in R. arenarum oocytes. Our results demonstrated that AA was capable of inducing maturation in time-dependent and dose-dependent manner. Arachidonic acid-induced maturation was inhibited by indomethacin. PGs from AA hydrolysis, such as prostaglandin F2α (PGF2α) and, to a lesser extent, PGE2, induced meiosis resumption. Oocyte maturation in response to PGF2α was similar to that produced by progesterone (P4). Oocyte response to PGE1 was scarce. Rhinella arenarum oocyte PGF2α-induced maturation showed seasonal variation. From February to June, oocytes presented low sensitivity to PGF2α. In following periods, this response increased until a maximum was reached during October to January, a close temporal correlation with oocyte response to P4 being observed. The effect of PGF2α on maturation was verified by analysing the capacity of oocytes to activate and form pronuclei after being injected with homologous sperm. The cytological analysis of activated oocytes demonstrated the absence of cortical granules in oocytes, suggesting that PGF2α induces germinal vesicle breakdown (GVBD) and meiosis resumption up to metaphase II. In turn, oocytes matured by the action of PGF2α were able to form pronuclei after fertilization in a similar way to oocyte maturated by P4. In microinjection of mature cytoplasm experiments, the transformation of pre-maturation promoting factor (pre-MPF) to MPF was observed when oocytes were treated with PGF2α. In summary, our results illustrated the participation of the AA cascade and its metabolites in maturation, activation and pronuclei formation in R. arenarum. PMID:24964276

  1. Early luteal phase endocrine profile is affected by the mode of triggering final oocyte maturation and the luteal phase support used in recombinant follicle-stimulating hormone-gonadotropin-releasing hormone antagonist in vitro fertilization cycles

    DEFF Research Database (Denmark)

    Fatemi, Human M; Polyzos, Nikolaos P; van Vaerenbergh, Inge;

    2013-01-01

    To assess endocrine differences during early luteal phase according to mode of triggering final oocyte maturation with or without luteal phase support (LPS).......To assess endocrine differences during early luteal phase according to mode of triggering final oocyte maturation with or without luteal phase support (LPS)....

  2. Porcine oocyte mtDNA copy number is high or low depending on the donor.

    Science.gov (United States)

    Pedersen, Hanne Skovsgaard; Løvendahl, Peter; Larsen, Knud; Madsen, Lone Bruhn; Callesen, Henrik

    2016-08-01

    Oocyte capacity is relevant in understanding decreasing female fertility and in the use of assisted reproductive technologies in human and farm animals. Mitochondria are important to the development of a functionally good oocyte and the oocyte mtDNA copy number has been introduced as a useful parameter for prediction of oocyte competence. The aim of this study was to investigate: (i) if the oocyte donor has an influence on its oocyte's mtDNA copy number; and (ii) the relation between oocyte size and mtDNA copy number using pre- and postpubertal pig oocytes. Cumulus-oocyte complexes were collected from individual donor pigs. The oocytes were allocated into different size-groups, snap-frozen and single-oocyte mtDNA copy number was estimated by quantitative real-time PCR using the genes ND1 and COX1. Results showed that mean mtDNA copy number in oocytes from any individual donor could be categorized as either 'high' (≥100,000) or 'low' (difference in threshold between pre- and postpubertal oocytes. No linear correlation was detected between oocyte size and mtDNA copy number within pre- and postpubertal oocytes. This study demonstrates the importance of the oocyte donor in relation to oocyte mtDNA copy number, irrespectively of the donor's puberty status and the oocyte's growth stage. Observations from this study facilitate both further investigations of the importance of mtDNA copy number and the unravelling of relations between different mitochondrial parameters and oocyte competence. PMID:26679989

  3. Avaliação da aplicabilidade da técnica de maturação in vitro de oócitos humanos e posterior fertilização Evaluation of the usefulness of the in vitro maturation technique of human oocyte and subsequent fertilization

    Directory of Open Access Journals (Sweden)

    Maria Clara Magalhães dos Santos Amaral

    2003-08-01

    Full Text Available OBJETIVO: avaliar a aplicabilidade da técnica de maturação in vitro de oócitos humanos e posterior fertilização. MÉTODOS: estudo prospectivo não randomizado descritivo realizado no período de novembro de 1999 a março de 2001 no qual foram incluídas 15 pacientes com infertilidade tubária e 20 ciclos de fertilização in vitro. Todas assinaram o termo de consentimento livre e esclarecido antes de iniciar o estudo. As pacientes tinham idade entre 18 e 32 anos incompletos, obstrução tubária como causa exclusiva de infertilidade e índice de massa corporal inferior a 25 kg/m². As pacientes receberam 300 UI de hormônio folículo estimulante (FSH recombinante por via intramuscular no segundo dia do ciclo e doses adicionais de 150 UI no quarto e no sexto dia do ciclo. A coleta ovular foi realizada no sétimo dia do ciclo. Os oócitos foram colocados em meio TCM 199 acrescido de antibióticos, piruvato, FSH, gonadotrofina coriônica humana e soro (Serum Substitute Supplement - Irvine Scientific®. Após 48 h de cultivo, os oócitos que atingiram o estágio de metáfase II foram inseminados e os fertilizados foram transferidos. RESULTADOS: foram puncionados 144 folículos com a coleta de 67 oócitos imaturos (46,5%. Quarenta e três oócitos atingiram o estágio de metáfase II (64,2% e foram inseminados. Destes, 30 fertilizaram e 25 embriões foram transferidos para 10 pacientes. Houve uma gravidez com nascimento de um bebê. CONCLUSÃO: concluiu-se que a técnica de maturar oócitos humanos in vitro previamente à fertilização in vitro é técnica exeqüível, capaz de gerar gravidez.PURPOSE: to evaluate the usefulness of the in vitro maturation technique of human oocyte and subsequent fertilization. METHODS: this is a prospective nonrandomized, descriptive study, carried out during the period of November 1999 to March 2001, with 20 cycles of in vitro fertilization of 15 patients with tubal infertility. All signed the written informed

  4. On-chip enucleation of an oocyte by untethered microrobots

    Science.gov (United States)

    Ichikawa, Akihiko; Sakuma, Shinya; Sugita, Masakuni; Shoda, Tatsuro; Tamakoshi, Takahiro; Akagi, Satoshi; Arai, Fumihito

    2014-09-01

    We propose a novel on-chip enucleation of an oocyte with zona pellucida by using a combination of untethered microrobots. To achieve enucleation within the closed space of a microfluidic chip, two microrobots, a microknife and a microgripper were integrated into the microfluidic chip. These microrobots were actuated by an external magnetic force produced by permanent magnets placed on the robotic stage. The tip of the microknife was designed by considering the biological geometric feature of an oocyte, i.e. the oocyte has a polar body in maturation stage II. Moreover, the microknife was fabricated by using grayscale lithography, which allows fabrication of three-dimensional microstructures. The microgripper has a gripping function that is independent of the driving mechanism. On-chip enucleation was demonstrated, and the enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm successful enucleation using this method, we investigated the viability of oocytes after enucleation. The results show that the production rate, i.e. the ratio between the number of oocytes that reach the blastocyst stage and the number of bovine oocytes after nucleus transfer, is 100%. The technique will contribute to complex cell manipulation such as cell surgery in lab-on-a-chip devices.

  5. Studies on the in vitro fertilization in cattle

    Institute of Scientific and Technical Information of China (English)

    YangQinzhang; YeXinghua; 等

    1994-01-01

    The in vitro maturation,fertilization and development of bovine ovary oocytes in two different cultural systems A and B were studied under the conditions of 38.5℃,5%CO2,95%air and 100% humidity.The maturation rates were 94.5% and 91.3%,respectively,and the difference was extremely significant.Frozen semen were thawed and sperm wrer capacitated with three kinds of capacitation agents for fertilization.The pronucleus rates were 76%,65%-68%and 62%respectively.The rates of embryos developed to morula and blastocyst were 19%,16%and 17% espectively.The developmental rates of embryos cocultured with bovine oviductal epithelium cells and bovine granulosa cells were 25%and 23.4% respectively,with no significant difference.Fresh embryos were transplanted into 15 recipiens,and three of them were pregnant and calves were born in 1990 and 1991,The pregnant rate was 20%,The emryos developod faster before 8-cell stage and slower after 8-cell stage ,in vitro than in vivo.

  6. Dimethyl sulfoxide inhibits spontaneous oocyte fragmentation and delays inactivation of maturation promoting factor (MPF) during the prolonged culture of ovulated murine oocytes in vitro

    OpenAIRE

    Choi, Taesaeng

    2011-01-01

    In this study, the effects of dimethyl sulfoxide (DMSO) on the spontaneous aging of ovulated murine oocyte were evaluated in vitro. When ovulated oocytes were cultured continuously in vitro without fertilization stimulation, they underwent several phenotypic changes, including non-activation, activation, fragmentation, and lysis. To investigate the effects of DMSO on these changes, I cultured ovulated oocytes with various concentrations of DMSO and evaluated the phenotypic changes for up to 3...

  7. 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

  8. Cryopreservation of Oocytes and Embryos in Human Assisted Reproduction

    Directory of Open Access Journals (Sweden)

    Konc J

    2005-01-01

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

  9. 309 proteomic analysis of the blastocoel fluid and remaining cells of bovine blastocysts

    DEFF Research Database (Denmark)

    Jensen, P L; Groendahl, M L; Beck, Helle;

    2012-01-01

    Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions...... such as Parkinson's disease, diabetes, and spinal cord injury. One of today's challenges in regenerative medicine is to define proper culture conditions for hESC. The natural milieu in the blastocyst may provide clues on how to improve culture conditions, and the aim of the present study was to determine...... the proteome of the blastocoel fluid and the remaining cells of bovine blastocysts. Bovine blastocysts were produced by in vitro fertilization of oocytes retrieved from slaughterhouse ovaries. The blastocoel from 195 blastocysts (1-8nL per blastocyst) were isolated by micromanipulation and analysed by nano...

  10. Efficient delivery of DNA into bovine preimplantation embryos by multiwall carbon nanotubes

    Science.gov (United States)

    Munk, Michele; Ladeira, Luiz O.; Carvalho, Bruno C.; Camargo, Luiz S. A.; Raposo, Nádia R. B.; Serapião, Raquel V.; Quintão, Carolina C. R.; Silva, Saulo R.; Soares, Jaqueline S.; Jorio, Ado; Brandão, Humberto M.

    2016-01-01

    The pellucid zone (PZ) is a protective embryonic cells barrier against chemical, physical or biological substances. This put, usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells. Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection, however its ability to cross the PZ and mediated gene transfer is unknown. Our data confirm that multiwall carbon nanotubes (MWNTs) can cross the PZ and delivery of pDNA into in vitro-fertilized bovine embryos. The degeneration rate and the expression of genes associated to cell viability were not affected in embryos exposed to MWNTs. Those embryos, however, had lower cell number and higher apoptotic cell index, but this did not impair the embryonic development. This study shows the potential utility of the MWNT for the development of new method for delivery of DNA into bovine embryos. PMID:27642034

  11. Cytoskeletal alterations associated with donor age and culture interval for equine oocytes and potential zygotes that failed to cleave after ICSI

    Science.gov (United States)

    Ruggeri, Elena; DeLuca, Keith F; Galli, Cesare; Lazzari, Giovanna; DeLuca, Jennifer G; Carnevale, Elaine M

    2016-01-01

    Intracytoplasmic sperm injection (ICSI) is an established method to fertilize equine oocytes, but not all oocytes cleave after ICSI. The aims of the present study were to examine cytoskeleton patterns in oocytes after aging in vitro for 0, 24 or 48 h (Experiment 1) and in potential zygotes that failed to cleave after ICSI of oocytes from donors of different ages (Experiment 2). Cytoplasmic multiasters were observed after oocyte aging for 48 h (P more frequently in sperm-injected oocytes from old than young mares. In the present study, multiasters appeared to be associated with cell aging, whereas actin vesicles were associated with aging of the oocyte donor. PMID:25798646

  12. Oocyte cryopreservation: is it time to remove its experimental label?

    OpenAIRE

    Noyes, Nicole; Boldt, Jeffrey; Nagy, Zsolt Peter

    2010-01-01

    As more reproductive-age women survive cancer at the expense of gonadotoxic therapy, the need for viable fertility preservation options has become paramount. Embryo cryopreservation, often using donor sperm, has been the standard offered these women over the past 20 years. Preservation of unfertilized oocytes now represents an acceptable and often equally viable alternative, particularly for single women, due to technologic advances made in the past decade. Given such, oocyte cryopreservation...

  13. 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

    Directory of Open Access Journals (Sweden)

    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

  14. DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Christine Couldrey

    Full Text Available Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT. Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5 during development of cattle generated either by artificial insemination (AI or in vitro fertilization (IVF and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic

  15. Expression Profile of Genes as Indicators of Developmental Competence and Quality of In Vitro Fertilization and Somatic Cell Nuclear Transfer Bovine Embryos

    Science.gov (United States)

    Monteleone, Melisa Carolina; Mucci, Nicolas; Kaiser, German Gustavo; Brocco, Marcela; Mutto, Adrián

    2014-01-01

    Reproductive biotechnologies such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA) of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70), endoplasmic reticulum (ER) stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5) and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3) in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A) + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip) was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART). PMID:25269019

  16. Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

    Directory of Open Access Journals (Sweden)

    Maria Jesús Cánepa

    Full Text Available Reproductive biotechnologies such as in vitro fertilization (IVF and somatic cell nuclear transfer (SCNT enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70, endoplasmic reticulum (ER stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5 and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3 in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART.

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

    Science.gov (United States)

    Lord, Tessa; Aitken, R John

    2013-12-01

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

  18. In Vitro Oocyte Maturation in Polycystic Ovarian Syndrome Patients

    Directory of Open Access Journals (Sweden)

    Fatemeh Ramazanzadeh

    2007-01-01

    Full Text Available Prevalence of Polycystic Ovarian Syndrome(PCOS in Iran is more than 6%. Therefore we encounter with many PCOS patients. In Vitro Maturation (IVM of oocytes as an attractive method in ART is considered. Studies show that changes in culture conditions should be administered to make IVM protocol more successful .For this purpose in this study we have set up the beneficial cultures for IVM procedure. Fourteen PCOS patients received FSH, 75 IU or 150 IU per day for 3 days initiating on day 3 of menstruation. Oocyte retrieval was performed transvaginally using an ultrasound-guided 17-gauge single lumen needle and filtered through a 70 micron gauge filter. Viable oocytes were put to maturation in TCM-199 supplemented with 10% Patient serum, recombinant FSH, pyruvate, penicillin, streptomycin sulphate and human chorionic gonadotropin..Oocytes were then inseminated by ICSI. The results indicated that 43.4% of oocytes matured to metaphase II. After 48 hours 47.5 % of M II oocytes fertilized by ICSI and cleaved to 2- and 4-cell stage. No pregnancy observed in PCOS patients. The oocytes maturation rate (43.4% and embryo formation (47.5 % from immature oocytes obtained in our IVM and ICSI culture system indicate that the present system may be nearly good, even though the number of patients were too small to draw significant conclusions.

  19. 不同化学激活方法及卵丘细胞层数对牛体外成熟卵母细胞孤雌发育的影响%Effects of different chemical activation methods and number of cumulus cell layers on in vitro parthenogenetic development of matured bovine oocytes

    Institute of Scientific and Technical Information of China (English)

    乔利敏; 乔富强; 姚华; 李向臣; 关伟军; 马月辉

    2011-01-01

    To explore the suitable activation methods for in vitro parthenogenetic development of matured bovine oo-cytes and increase the efficiency of nuclear transfer for embryonic parthenogenesis, we have studied the effects of activation by ethanol ( EH), ionomycin (Ion), A23187, 6-dimethyl-aminopurine (6-DMAP; 2 mmol·L-1 ) or cytoch-alasin B ( CCB) on the development of bovine oocytes. Meanwhile, we have analyzed the effects of different numbers of cumulus cell layers on parthenogenetic development of bovine oocytes in the same condition. The results showed that (1) Ion(5 μmol-L-1 ) , A23I87( 10 μmol-L-1 ) or ethanol(7% ) applied together with 6-DMAP(2 mmol· L -1 ) and cytochalasin B (CCB) significantly activated the growth of bovine parthenogenetic embryo, with treatmentof Ion + 6-DMAP + CCB showing the strongest effects as compared to other groups( P 0. 05 ), but were significantly higher than the group containing 1-2 lays and the mixed group ( P < 0. 05). Therefore, cells of these two groups were the optimal research material for experiment of parthenogenetic activation of in vitro embryo development in bovine.%为探索适宜的牛体外成熟卵母细胞孤雌激活方式,提高核移植效率及研究胚胎孤雌发育.研究比较了不同浓度的乙醇(EH)、离子霉素( ionomycin)、钙离子载体A23187与蛋白激酶抑制剂6-二甲氨基嘌呤(6-DMAP)、细胞松驰素(cytochalasin B,CCB)对牛卵母细胞激活发育的影响,并在相同条件下,比较了不同卵丘细胞层数对卵母细胞激活后胚胎发育能力的影响.结果表明:(1)5 μmol· L-1 Ionomycin,10 μmol·L-1A23187,7% EH分别联合2 mmol· L-1 6-DMAP,CCB均可以有效地激活牛孤雌胚,其中以lonomycin+6-DMAP+CCB组囊胚发育率极显著高于其他各组(P<0.01),并且激活液中CCB的存在对牛孤雌胚胎的发育有利;(2)卵母细胞包被的卵丘细胞层数不同对卵母细胞成熟激活有显著的影响,卵丘细胞层数3~5层和多于6层的

  20. Oocytes transport across the oviduct of Murrah and Nelore cows

    Directory of Open Access Journals (Sweden)

    P.S. Baruselli

    2010-02-01

    Full Text Available In order to verify the causes of the low embryo recovery rate in superovulated buffaloes, the effect of species and of estradiol-17β (E2 treatment were evaluated on oocyte transport across the oviduct in Murrah and Nelore. The females were synchronised with progesterone plus estradiol benzoate followed by an injection of PGF2α and eCG. The ovulation was induced with GnRH and 48hs after the animals were slaughtered and the oviducts removed. The oviducts were washed with HBSS and oocytes of both species were inserted into infundibulum portion. The oviducts were put in a dish with HBSS with or without E2 and incubated for 24 h. The oviduct were then flushed with DPBS, in order to recover and count the oocytes. Data were analyzed by ANOVA. There was no effect of interaction. The total number of oocytes and the recovery rate were higher for Nelore than Murrah (P<0.05 oviducts. There was no effect of treatment on these variables. The number of oocytes from buffaloes and bovine recovered was similar. These results indicate that oocytes transport across the oviduct of Murrah or Nelore does not depend on the oocyte species and is not influenced by E2.

  1. The Effects of Voluntary Exercise on Oocyte Quality in a Diet-Induced Obese Murine Model

    OpenAIRE

    Boudoures, Anna L.; Chi, Maggie; Thompson, Alysha; Zhang, Wendy; Moley, Kelle H.

    2015-01-01

    Obesity negatively affects many aspects of the human body, including reproductive function. In females, the root of the decline in fertility is linked to problems in the oocyte. Problems seen in oocytes that positively correlate with increasing BMI include changes to the metabolism, lipid accumulation, meiosis, and metaphase II (MII) spindle structure. Studies in mice indicate dietary interventions fail to reverse these problems [4]. How exercise affects the oocytes has not been addressed. Th...

  2. 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

  3. From fresh heterologous oocyte donation to autologous oocyte banking

    OpenAIRE

    Stoop, D.

    2012-01-01

    Introduction: Today, oocyte donation has become well established, giving rise to thousands of children born worldwide annually. The introduction of oocyte cryopreservation through vitrification allows the introduction of egg banking, improving the efficiency and comfort of oocyte donation. Moreover, the vitrification technique can now enable autologous donation of oocytes to prevent future infertility. Methods: We evaluated fresh heterologous oocyte donation in terms of obstetrical and perina...

  4. 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.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  6. RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo- and in vitro-derived bovine blastocysts

    Directory of Open Access Journals (Sweden)

    Driver Ashley M

    2012-03-01

    Full Text Available Abstract Background A valuable tool for both research and industry, in vitro fertilization (IVF has applications range from gamete selection and preservation of traits to cloning. Although IVF has achieved worldwide use, with approximately 339,685 bovine embryos transferred in 2010 alone, there are still continuing difficulties with efficiency. It is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day 8 of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. To investigate potential influences in-vitro fertilization (IVF has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade (expanded, excellent quality to determine the degree of transcriptomic variation beyond morphology using RNA-Seq. Results A total of 26,906,451 and 38,184,547 fragments were sequenced for in vitro and in vivo embryo pools, respectively. We detected expression for a total of 17,634 genes, with 793 genes showing differential expression between the two embryo populations with false discovery rate (FDR Conclusions Thus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos.

  7. 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

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    Seddigheh Esmaeilzadeh

    2009-03-01

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

  10. Effect of concentration and exposure period to butyrolactone I on meiosis progression in bovine oocytes Efeito de concentração e tempo de exposição à butirolactona I na progressão da meiose de oócitos bovinos

    Directory of Open Access Journals (Sweden)

    P.R. Adona

    2006-06-01

    Full Text Available The effect of concentration and exposure period of bovine oocytes to butyrolactone I (BLI on meiotic block and in vitro maturation (IVM kinetics was studied. In experiment 1, all oocytes were at germinal vesicle stage (GV, after 6h in culture with 0, 50 and 100µM BLI. After 12h, all oocytes cultured with 50 and 100µM BLI remained in GV. After 24h, less oocytes were in GV with 50µM (82% than with 100µM BLI (99%, P0.05. After 18h IVM, metaphase II (MII rates were similar for all groups (76-81%. In experiment 3, after 6h IVM, 74% of treated oocytes (50 or 100µM BLI for 12h were in GV. This rate was lower than for control oocytes (97.3%, P0.05 were in MII with BLI than for control (73%, PEstudou-se o efeito da concentração e do tempo de exposição à butirolactona I (BLI no bloqueio meiótico e na cinética da maturação in vitro (MIV de oócitos bovinos. No experimento 1, todos os oócitos encontravam-se em vesícula germinativa (VG após 6h de cultivo nas concentrações de 0,50 e 100µM BLI. Após 12h, somente oócitos cultivados com BLI (50 e 100µM estavam em VG. Após 24h, menos oócitos tratados com 50µM (82% estavam em VG em relação a 100µM (99%, P0,05. A taxa de metáfase II (MII, 76-81% foi similar para todos os tempos de exposição, após 18h de MIV. No experimento 3, após 6h de MIV, menos oócitos tratados (74% para 50 ou 100µM BLI por 12h estavam em VG comparados aos controles (97%, P0,05 do que os controles (73%, P<0.05. Conclui-se que para cultivos mais curtos, a concentração mais baixa de BLI bloqueia a meiose a cinética da maturação nuclear é acelerada em oócitos expostos à BLI e isso é afetado pelo tempo de cultivo, mas não pela concentração da droga.

  11. Mammalian Oocyte Cryopreservation - Review

    OpenAIRE

    Ada Cean; Alexandru T. Bogdan; Nicolae Păcală; Alexandra Ivan; Daniela E. Ilie

    2011-01-01

    Although oocyte cryopreservation represents one of the main objectives of the reproductive medicine and cryobiology in the last years, until now, regardless of the studied specie, there is no freezing protocol that will assure satisfactory survival rates after thawing. Oocyte cryopreservation is now one of the most problematic issues in cryobiology aria, especially because they are extremely sensitive to low temperatures, and the maintaining of a normal development potential after thawing is ...

  12. Exceptional material requirement for reproduction in mouse oocytes.

    Science.gov (United States)

    Yu, L; Wang, S F; Zhai, Q Z; Yao, Y Q; Jiang, F; Lu, Y X

    2015-01-01

    Limited information on oocytes and fertilization prevents the efficient therapy of patients with infertility. The most important reason for this lack of understanding is a deficiency in research dedicated to oocytes and fertilization. Currently, we are concerned with the role of nutrition in the process of oocyte development to better understand the relationship between nutrition and infertility. The aim of this study was to explore the relationship between some exceptional materials and infertility to elucidate the role of these materials in oocyte development. We used proteomic analysis to identify numerous nutrition-related proteins in three developmental stages: the germinal vesicle stage, the metaphase II-arrested stage, and the fertilized oocyte-zygote stage. Specific proteins were abundantly expressed during the three stages. These proteins included astacin-like metalloendopeptidase, selenium-binding proteins, and other proteins involved in metabolic and signaling pathways. Other proteins were involved in the citrate cycle, the electron transport chain, the urea cycle, fatty acid metabolism, and the insulin signaling pathway. Almost all these proteins exhibited different expression levels in the three stages. The results of the present study provide a better understanding of the molecular mechanisms of early embryonic development and suggest new treatment methods for infertility. PMID:26600495

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

    Indian Academy of Sciences (India)

    Bernd Rosenbusch

    2012-12-01

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

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Naoko Ohnami

    2012-05-01

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

  16. 组织型纤溶酶原激活剂基因在牛卵丘—卵母复合体体外成熟进程中的表达%Expression of Tissue-type Plasminogen Activator in Bovine Cumulus-oocytes during in vitro Maturation

    Institute of Scientific and Technical Information of China (English)

    关洪敏; 米焱; 张伶俐; 李海军; 曹贵方

    2012-01-01

    In the present study,four experiments were conducted to investigate the potential effects of tPA on bovine cumulus and oocytes during in vitro maturation (IVM) (0,8,16,24 h). The expressions of tPA were detected by relative Real-time RT-PCR experiments in bovine cumulus and oocytes both after 0,8,16,24 h of IVM and under different FSH concentrations after 16 h of IVM separately. Meanwhile,the degree of bovine cumulus cell expansion and the percentages of first polar body in oocyte were assessed respectively. TPA mRNA levels were higher in cumulus and oocytes at 16 h and 24 h than those at 0 h and 8 h of IVM significantly (P<0. 05). Among those groups that under different FSH concentrations (0,0. 01,0. 1 and 1 IU/mL) after 16 h of IVM.tPA mRNA levels of cumulus was increased following the elevated FSH concentrations,as well as the cumulus expansion,tPA mRNA of cumulus cells in 1 IU/mL FSH group was significantly higher than others. The results indicated that the expression of tPA in bovine cumulus was regulated positively by FSH.tPA could facilitate the cumulus expansion during IVM. However.it needed to be further investigated that tPA expression might be involved in first polar body excrusion of bovine oocytes.%为了初步了解组织型纤溶酶原激活剂( tissue-plasminogen activator,tPA)在牛卵丘—卵母复合体体外成熟进程中的潜在作用,试验运用实时定量RT-PCR技术分别检测体外成熟过程中不同时间段(0、8、16、24 h)以及添加不同浓度FSH成熟培养16 h后的牛卵丘细胞和卵母细胞中tPA基因相对表达变化,观察添加不同浓度FSH成熟培养16h后卵丘细胞膨胀程度差异,并统计卵母细胞第一极体排出情况.结果显示,卵丘—卵母复合体在体外成熟初期,tPA mRNA相对表达水平在体外成熟16和24 h的卵丘细胞和卵母细胞中显著高于0和8h组;在添加不同浓度FSH(0、0.01、0.1和1 IU/mL)体外成熟16h的各处理组,卵丘细胞中tPA mRNA相对表达

  17. Ultrastructural analysis of bovine oocytes from ovarian follicles with different diametersAnálise ultra-estrutural de oócitos bovinos provenientes de folículos ovarianos com diferentes diâmetros

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    Cesar Roberto Esper

    2011-10-01

    Full Text Available In vitro embryo production is an important technique for facilitating the reproduction of animals with high genetic merit. The greatest challenge for the reproducibility of this technique is the quality of the oocyte that is submitted for in vitro maturation. The aim of this work was to evaluate the ultrastructural characteristics of oocytes from follicles of different diameters using transmission electron microscopy. The animals were divided into 2 groups and were given a single i.m. injection of 250 IU FSH (Pluset, Serono, Italy. To synchronize the follicular growth, all follicles > 2 mm were aspirated during the estrous cycle, which was considered day zero (D0. Group 1 (G1; n = 4 received FSH on day 1 (D1, and the 2- to 5-mm follicles were aspirated on day 2 (D2. The animals in group 2 (G2; n = 5 received FSH on day 2 (D2, and their 10- to 15-mm follicles were aspirated on day 5 (D5. After aspiration, the oocytes from both groups were fixed and prepared for ultrastructural analysis. The oocytes analyzed from both groups had similar ultrastructural characteristics. The presence and distribution of organelles in the cytoplasm of the oocytes did not differ between groups, suggesting that, in relation to the ultrastructural characteristics, oocytes from 2 to 5 mm and 10 to 15 mm follicles are similar.A produção in vitro de embriões é uma técnica importante para facilitar a reprodução de animais com elevado mérito genético. O maior desafio para a reprodutibilidade desta técnica é a qualidade do oócito destinado à maturação in vitro. O objetivo deste trabalho foi avaliar as características ultra-estruturais de oócitos provenientes de folículos com diferentes diâmetros por microscopia eletrônica de transmissão. Os animais foram divididos em dois grupos e receberam uma única injeção im de 250 UI de FSH (Pluset, Serono, Itália. Para sincronizar o crescimento folicular, todos os folículos > 2 mm foram aspirados durante o ciclo

  18. Cryopreservation of Embryos and Oocytes in Human Assisted Reproduction

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    János Konc

    2014-01-01

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

  19. An Update on Oxidative Damage to Spermatozoa and Oocytes

    OpenAIRE

    Opuwari, Chinyerum S.; Henkel, Ralf R.

    2016-01-01

    On the one hand, reactive oxygen species (ROS) are mandatory mediators for essential cellular functions including the function of germ cells (oocytes and spermatozoa) and thereby the fertilization process. However, the exposure of these cells to excessive levels of oxidative stress by too high levels of ROS or too low levels of antioxidative protection will render these cells dysfunctional thereby failing the fertilization process and causing couples to be infertile. Numerous causes are respo...

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  1. Storage of bovine isolated follicles: a new alternative way to improve the recovery rate of viable embryos from ovarian follicles of slaughtered cows.

    Science.gov (United States)

    Pavlok, A; Cech, S; Kubelka, M; Lopatárová, M; Holý, L; Jindra, M

    2006-11-01

    The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The

  2. Dietary sugar in healthy female primates perturbs oocyte maturation and in vitro preimplantation embryo development.

    Science.gov (United States)

    Chaffin, Charles L; Latham, Keith E; Mtango, Namdori R; Midic, Uros; VandeVoort, Catherine A

    2014-07-01

    The consumption of refined sugars continues to pose a significant health risk. However, nearly nothing is known about the effects of sugar intake by healthy women on the oocyte or embryo. Using rhesus monkeys, we show that low-dose sucrose intake over a 6-month period has an impact on the oocyte with subsequent effects on the early embryo. The ability of oocytes to resume meiosis was significantly impaired, although the differentiation of the somatic component of the ovarian follicle into progesterone-producing cells was not altered. Although the small subset of oocytes that did mature were able to be fertilized in vitro and develop into preimplantation blastocysts, there were >1100 changes in blastocyst gene expression. Because sucrose treatment ended before fertilization, the effects of sugar intake by healthy primates are concluded to be epigenetic modifications to the immature oocyte that are manifest in the preimplantation embryo. PMID:24731100

  3. Female Fertility: Is it Safe to "Freeze?"

    Directory of Open Access Journals (Sweden)

    Lu Zhang

    2015-01-01

    Full Text Available Objective: To evaluate the safety and risk of cryopreservation in female fertility preservation. Data sources: The data analyzed in this review were the English articles from 1980 to 2013 from journal databases, primarily PubMed and Google scholar. The criteria used in the literature search show as following: (1 human; embryo; cryopreservation/freezing/vitrification, (2 human; oocyte/immature oocyte; cryopreservation/ freezing/vitrification, (3 human; ovarian tissue transplantation; cryopreservation/freezing/vitrification, (4 human; aneuploidy/DNA damage/epigenetic; cryopreservation/freezing/vitrification, and (5 human; fertility preservation; maternal age. Study selection: The risk ratios based on survival rate, maturation rate, fertilization rate, cleavage rate, implantation rate, pregnancy rate, and clinical risk rate were acquired from relevant meta-analysis studies. These studies included randomized controlled trials or studies with one of the primary outcome measures covering cryopreservation of human mature oocytes, embryos, and ovarian tissues within the last 7 years (from 2006 to 2013, since the pregnancy rates of oocyte vitrification were significantly increased due to the improved techniques. The data involving immature oocyte cryopreservation obtained from individual studies was also reviewed by the authors. Results: Vitrifications of mature oocytes and embryos obtained better clinical outcomes and did not increase the risks of DNA damage, spindle configuration, embryonic aneuploidy, and genomic imprinting as compared with fresh and slow-freezing procedures, respectively. Conclusions: Both embryo and oocyte vitrifications are safe applications in female fertility preservation.

  4. Adubação orgânica da batata com esterco bovino no município de Esperança - PB Potato organic fertilization with bovine manure in Esperança county - PB

    Directory of Open Access Journals (Sweden)

    Lucas Borchartt

    2011-06-01

    Full Text Available Objetivou-se avaliar a eficiência do esterco bovino na adubação orgânica na cultura da batata. O experimento foi realizado no município de Esperança/PB, em Neossolo Regolítico. A cultivar utilizada foi a Monalisa, onde o delineamento utilizado foi de blocos casualizados, com três repetições. Os tratamentos foram constituídos de aplicação de esterco bovino em quantidades de 0; 3; 6; 9; 12; 15; 18; 21 e 24 t ha-1 e 10 t ha-1 com a complementação de NPK com percentagens de 0; 12,5; 25; 37,5; 50; 62,5; 75; 87,5 e 100% da fórmula 120-80-90 (N-P2O5-K2O, calculada conforme a análise de solo. As variáveis avaliadas foram: número e peso médio de tubérculos por planta, produção total, produção comercial, perdas de produção e matéria seca de tubérculos. O uso de esterco bovino e esterco bovino à adição de NPK proporcionaram aumento no peso de tubérculos por planta, produção total e produção comercial de tubérculos de batata. Desta forma, observa-se que o esterco bovino é uma boa alternativa como adubação orgânica para os produtores de batata.In the Paraíba state the potato shows a low yield, due to the rain seasonality allied to the low soil fertility. This study aimed to evaluate the bovine manure efficiency in the organic fertilization of potato. The experiment was carried out in Esperança county, in a Regolithic neossoil. The design used was randomized blocks, with bovine manure application in quantities of 0; 3; 6; 9; 12; 15; 18; 21 and 24 t ha -1 and 10 t ha -1 of bovine manure + NPK with percentages of 0; 12.5; 25; 37.5; 50; 62.5; 75; 87.5; and 100% of the formula 120-80-90 ( N-P2O5-K2O , calculated by the soil analysis. The variables evaluated were: number and weight of tubers per plant, total yield and commercial; yield losses and dry matter of tubers. The use of bovine manure and bovine manure + NPK provided an increase in weight of tubers per plant, total yield and commercial potato tubers. This way, it

  5. Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

    Science.gov (United States)

    Paes, V M; Vieira, L A; Correia, H H V; Sa, N A R; Moura, A A A; Sales, A D; Rodrigues, A P R; Magalhães-Padilha, D M; Santos, F W; Apgar, G A; Campello, C C; Camargo, L S A; Figueiredo, J R

    2016-09-01

    The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P  0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase

  6. THE PREDICTORS OF CLINICAL FERTILIZATION IN IVF CYCLES

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    S. Esmailzadeh

    2008-05-01

    Full Text Available We examined the demographic characteristics of couples, ovarian response and sperm quality to determine the predictors of clinical fertilization in IVF cycles. A descriptive and analytic study was conducted using data and biologic specimens obtained Fatemeh-Alzahra of Babol infertility center from 2004 to 2005. Only data from 315 women who had medical indication for conventional IVF treatment were included in the analysis. Treatment using ICSI was excluded. In the univariate analysis, the following variables affected on fertilization rate: the length of infertility, the number of IVF cycle, basal LH serum on 2 days, the number of administration of hMG, the duration of ovarian stimulation, the number of follicles, the number of oocytes retrieved, the number of oocytes stage II and III, sperm count, sperm motility, sperm grading III and IV. In the multivariate analysis, the strongest predictor of positive fertilization was the mean number of oocytes retrieved. Also, the mean number of oocytes stage II and stage III were positive predictors of fertilization. The mean of basal LH serum on day 2 and the mean duration of ovarian stimulation were negative predictors of fertilization Ovarian response to gonadotropins and the quality of oocytes were main predictors of fertilization. Although some parameters of sperm quality were significant variables of fertilization rate in univariate analysis, in multivariate analysis one's effects were negligible. This information should be used when selecting couples for IVF cycles or oocytes for fertilization to raise the rate of clinical fertilization.

  7. 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

  8. WASH complex regulates Arp2/3 complex for actin-based polar body extrusion in mouse oocytes

    OpenAIRE

    Wang, Fei; Zhang, Liang; Zhang, Guang-Li; Wang, Zhen-Bo; CUI, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2014-01-01

    Prior to their fertilization, oocytes undergo asymmetric division, which is regulated by actin filaments. Recently, WASH complex were identified as actin nucleation promoting factors (NPF) that activated Arp2/3 complex. However, the roles of WASH complex remain uncertain, particularly for oocyte polarization and asymmetric division. Here, we examined the functions of two important subunits of a WASH complex, WASH1 and Strumpellin, during mouse oocyte meiosis. Depleting WASH1 or disrupting Str...

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

    OpenAIRE

    Trounson Alan; Lacham-Kaplan Orly

    2008-01-01

    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 ma...

  10. Ethical issues in paying for long-distance travel and accommodation expenses of oocyte donors.

    Science.gov (United States)

    Heng, Boon Chin

    2005-11-01

    In many countries where the sale and purchase of donor oocytes is banned, a legal loophole often exploited is the use of free air tickets and hotel stay to entice prospective oocyte donors, in lieu of monetary payment. Such a means of procuring much-needed donor oocytes is ethically unsound. There is a lack of transparency and the personal motivation of the oocyte donor may be clouded by the desire for a 'free' holiday. Moreover, such a system is open to abuse by medical professionals. Private fertility clinics may source for oocyte donors to attract patients. The oocyte donor is paid nothing (except free travel and hotel stay), while the medical professional makes a handsome profit from treating infertile patients, which is not equitable. Medical professionals can also easily make a profit by marking up the price of air tickets and hotel stay to the patient (oocyte recipient). This would be thoroughly unprofessional, since the money earned is not directly related to the medical skills and expertise of the fertility specialist. Hence, it is imperative that various regulatory authorities should critically re-examine the giving of free travel and accommodation to oocyte donors, instead of monetary compensation. PMID:16409701

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

    Directory of Open Access Journals (Sweden)

    NURBARIAH

    2011-12-01

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

  12. Oocyte banking for anticipated gamete exhaustion (AGE) is a preventive intervention, neither social nor nonmedical.

    Science.gov (United States)

    Stoop, Dominic; van der Veen, Fulco; Deneyer, Michel; Nekkebroeck, Julie; Tournaye, Herman

    2014-05-01

    The scope of female fertility preservation through cryopreservation of oocytes or ovarian cortex has widened from mainly oncological indications to a variety of fertility-threatening conditions. So far, no specific universally accepted denomination name has been given to cryopreservation of oocytes or ovarian cortex for the prevention of age-related fertility decline. We argue that the commonly used phrases 'social' and 'nonmedical freezing' to denote the indication for cryopreservation are not entirely correct. We suggest 'AGE banking', as this has not only the advantage of being catchy but also depicts the exact indication for the strategy, anticipated gamete exhaustion.

  13. The molecular basis of fertilization (Review)

    Science.gov (United States)

    Georgadaki, Katerina; Khoury, Nikolas; Spandidos, Demetrios A.; Zoumpourlis, Vasilis

    2016-01-01

    Fertilization is the fusion of the male and female gamete. The process involves the fusion of an oocyte with a sperm, creating a single diploid cell, the zygote, from which a new individual organism will develop. The elucidation of the molecular mechanisms of fertilization has fascinated researchers for many years. In this review, we focus on this intriguing process at the molecular level. Several molecules have been identified to play a key role in each step of this intriguing process (the sperm attraction from the oocyte, the sperm maturation, the sperm and oocyte fusion and the two gamete pronuclei fusion leading to the zygote). Understanding the molecular mechanisms of the cell-cell interactions will provide a better understanding of the causes of fertility issues due to fertilization defects. PMID:27599669

  14. Phenotypes of the ovarian follicular basal lamina predict developmental competence of oocytes

    OpenAIRE

    Irving-Rodgers, Helen F.; Morris, Stephanie; Collett, Rachael A.; Peura, Teija T.; Davy, Margaret; Jeremy G. Thompson; Mason, Helen D; Rodgers, Raymond J.

    2008-01-01

    BACKGROUND The ovarian follicular basal lamina underlies the epithelial membrana granulosa and maintains the avascular intra-follicular compartment. Additional layers of basal lamina occur in a number of pathologies, including pili annulati and diabetes. We previously found additional layers of follicular basal lamina in a significant percentage of healthy bovine follicles. We wished to determine if this phenomenon existed in humans, and if it was related to oocyte function in the bovine. MET...

  15. Anandamide levels fluctuate in the bovine oviduct during the oestrous cycle.

    Directory of Open Access Journals (Sweden)

    Maria Gracia Gervasi

    Full Text Available Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization.

  16. Dickkopf-related protein 1 inhibits the WNT signaling pathway and improves pig oocyte maturation.

    Directory of Open Access Journals (Sweden)

    Lee D Spate

    Full Text Available The ability to mature oocytes in vitro provides a tool for creating embryos by parthenogenesis, fertilization, and cloning. Unfortunately the quality of oocytes matured in vitro falls behind that of in vivo matured oocytes. To address this difference, transcriptional profiling by deep sequencing was conducted on pig oocytes that were either matured in vitro or in vivo. Alignment of over 18 million reads identified 1,316 transcripts that were differentially represented. One pathway that was overrepresented in the oocytes matured in vitro was for Wingless-type MMTV integration site (WNT signaling. In an attempt to inhibit the WNT pathway, Dickkopf-related protein 1 was added to the in vitro maturation medium. Addition of Dickkopf-related protein 1 improved the percentage of oocytes that matured to the metaphase II stage, increased the number of nuclei in the resulting blastocyst stage embryos, and reduced the amount of disheveled segment polarity protein 1 protein in oocytes. It is concluded that transcriptional profiling is a powerful method for detecting differences between in vitro and in vivo matured oocytes, and that the WNT signaling pathway is important for proper oocyte maturation.

  17. EFFECT OF FETAL SERUM AND FOLLICULAR LIQUOR SUPPLEMENTATION ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

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    Clara Larocca

    2012-01-01

    Full Text Available To optimize In Vitro Fertilization (IVF results in bovines by lowering costs, comparing the efficiency of Fetal Calf Serum (FCS (high cost with respect to bovine Follicular Fluid (bFF (low cost and by quantifying embryo production. Cumulus-Oocyte-Complexes (COC obtained from a slaughter house, transported at 37°C in saline solution, were classified according to their quality in A, B, C and D. A and B oocytes were washed with modified Phosphate-Buffered Saline (m-PBS and three groups were randomly formed (GC; G1; G2 cultured in drops of the respective media (10 COC/drop of 100 μL, covered with mineral oil and placed in an incubator (38,5°C, 5% CO2 y 95% humidity. Maturation was done in Tissue Culture Medium (TCM-199 and antibiotics, for 22h and developed in CR1aa medium and antibiotics. The Control Group (CG was supplemented during both maturation and development stages with 5% FCS and 10% bFF; G1 with 5% FCS during maturation and development and G2 with 10% bFF during maturation and development. We obtained bFF from follicles larger than 15 mm, it was centrifuged (800G inactivating it. At fertilization Bracket and Oliphant (BO medium was used. Zygotes were evaluated every 48 h for 8 days since insemination, counting the Division Rate (DR and the Embryo Development (ED of compacted morulae. Χ2 Test was applied. For RD, G2 differs from G1 and CG (p0.05. During development no significant differences were found between groups (p>0.05. Results show that when using FCS, bFF or a combination of both, development results are similar. It is assumed that it is possible to substitute FCS with bFF."

  18. 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.

  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. Microinjection of Xenopus oocyte

    OpenAIRE

    sprotocols

    2014-01-01

    ### Equipment 1. Parafilm - Petri dishes/100mm tissue culture dishes - Marker pen - Microscope slides - Glass capillaries - Very fine tweezers - MBS solution* - Plastic transfer pipettes - Mineral oil - Samples ### Method: Frogs are killed by destruction of the head, ovaries are then dissected and put into MBS solution. **Preparation of the oocytes** 1. Wash the ovary as soon as possible with MBS solution to remove all traces of blood and debris....

  1. Fertility preservation in young patients′ with cancer

    Directory of Open Access Journals (Sweden)

    Sharmila Dudani

    2014-01-01

    Full Text Available Preservation of fertility is an important issue in the management of young cancer patients. Though embryo cryostorage is a well-established procedure, it can only be availed by couples. Recent studies have indicated increasing success rates with mature and immature oocyte cryopreservation. Cryostorage induces injuries on the human oocytes which can be minimized by slow freezing and vitrification. Selection of candiidates is crucial so that the most suitable technique can be offered without any delay in initiation of cancer therapy. Factors affecting suitability are age of patient, assessment of ovarian reserve, hormonal status and type and stage of neoplastic disease. Encouraging results have been obtained with oocyte in vitro maturation (IVM followed by vitrification for cryostorage. Data on the use of vitrified eggs in routine in vitro fertilization (IVF show that pregnancy rates can be comparable to those achieved with fresh oocytes.

  2. 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.

  3. Analysis of 232 total fertilization failure cycles during intracytoplasmic sperm injection

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    Esma Sarikaya

    2011-01-01

    Full Text Available Background: The intracytoplasmic sperm injection procedure ending with total fertilization failure is very distressfull event for both the clinician and the patient.Objective: The aim of this study was to identify independent factors which could be used to identify total fertilization failure before the day of intracytoplasmic sperm injection.Materials and Methods: This was a retrospective study of 232 patients who were admitted to a tertiary-care hospital IVF Unit and showed total fertilization failure during intracytoplasmic sperm injection cycles. To sort out the interwined effects of female age, basal FSH, sperm quality, antral follicle count, starting dose of gonadotrophine, sperm extraction technique, cycle length, >14 mm follicle number, oocyte number after oocyte pick up, estradiol and progesterone level on the day of hCG and the MI, MII and GV oocyte number on the fertilization, multiple logistic regression analysis was used.Results: The total fertilization failure rate was 6% and the recurrance rate was 23%. The original model illustrated that the presence of GV oocytes, total oocyte number less than six, <2000 pg/mL E2 concentration on the day of hCG and testicular sperm extraction increases the total fertilization failure risk.Conclusion: It is very difficult to predict total fertilization failure. Sometimes even with one good quality oocyte and sperm and in the case of globozoospermia fertilization can be achieved. Not only azoospermia but also low oocyte numbers increase the chance of total fertilization failure even after intracytoplasmic sperm injection

  4. Adenosine Modulates the Oocyte Developmental Competence by Exposing Stages and Synthetic Blocking during In Vitro Maturation.

    Science.gov (United States)

    Cheon, Yong-Pil

    2016-06-01

    Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. By the inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage caused of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence.. PMID:27660830

  5. Effects of upregulation of Hsp27 expression on oocyte development and maturation derived from polycystic ovary syndrome.

    Directory of Open Access Journals (Sweden)

    Lingbo Cai

    Full Text Available Heat shock protein 27 (Hsp27 is a heat shock protein family member which can inhibit apoptosis. Our previous studies reported down-regulated Hsp27 in ovarian tissue derived from women with polycystic ovary syndrome (PCOS however, the exact effect of Hsp27 on oocyte maturation and developmental competence in PCOS is unclear. The effect of Hsp27 over-expression was studied in vitro using oocytes derived from PCOS patients. An artificial GFP-plasmid was injected into human oocyte to increase Hsp27 protein level. Oocyte maturation was evaluated by morphological observation. Mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI and embryonic developmental competence was evaluated. Critical apoptotic factors and cytokines were measured at both the mRNA and protein level. Our results revealed that Overexpression of HSP27 lowered the maturation rate of oocytes derived from PCOS patients. Meanwhile, fertilization rate and high quality embryo rate were similar between the Hsp27 overexpressing group and controls; however, the blastocyst formation rate in this group was significantly higher than control. Expression analysis revealed that the oocyte-secreted factors, BMP15 and GDF9, and the apoptotic-related regulators, Caspase 3, 8 and 9, were all significantly decreased in Hsp27 overexpressing oocytes. In conclusion, upregulation of Hsp27 inhibits oocyte maturation from PCOS patients, but improves embryonic developmental potential.

  6. Roles of Calmodulin-dependent Protein Kinase Ⅱ in Meiotic Maturation and Fertilization of Oocytes%钙调蛋白依赖的蛋白激酶Ⅱ在卵母细胞减数分裂和受精中的作用

    Institute of Scientific and Technical Information of China (English)

    范衡宇; 霍立军; 孙青原

    2003-01-01

    钙调蛋白依赖的蛋白激酶(CaMK)是一类分布广泛的丝/苏氨酸蛋白激酶家族,在钙离子和钙调蛋白存在的条件下发生自磷酸化而被激活,在细胞内对于钙信号的传递具有重要的介导作用.近年来的研究表明CaMKⅡ是参与调节卵母细胞减数分裂的重要分子,在卵母细胞成熟、极体排放、受精和活化等过程中发挥作用.CaMKⅡ作为Ca2+的下游信号分子,在受精后促进成熟促进因子(MPF)和细胞静止因子(CSF)的失活,并调节纺锤体微管的组装和中心体的复制过程.虽然CaMKⅡ在减数分裂中的作用广泛而关键,但目前的研究主要集中于低等动物和小鼠,今后有待进一步阐明该蛋白激酶在其他哺乳动物中的作用和调节机制.%Calmodulin-dependent protein kinase (CaMK), activated by auto-phosphorylation at the presence of calcium and calmodulin, is widely distributed in eukaryotes. CaMKs are important mediators of calcium signal in eukaryotes. Recent researches have suggested that CaMKⅡ is involved in the regulation of meiotic cell cycle of oocytes. It plays functional roles in meiotic maturation, polar body extrusion, fertilization and egg activation. As one of the down-stream signaling molecules of calcium, CaMKⅡ facilitates the inactivation of maturation promoting factor (MPF) and cytostatic factor (CSF) following fertilization, as well as the spindle microtubule organization and centrosome duplication. Although the functions of CaMKⅡ in oocyte meiosis are versatile and essential, the present results are primarily obtained from low vertebrates and mouse. In future studies, the function and regulation of this kinase in other mammals should be stressed.

  7. CD9 Expression by Human Granulosa Cells and Platelets as a Predictor of Fertilization Success during IVF

    Directory of Open Access Journals (Sweden)

    Carolyn R. Jaslow

    2010-01-01

    Full Text Available Objective. To determine whether CD9 expression on human granulosa cells (GCs and platelets could predict the success of conventional fertilization of human oocytes during in vitro fertilization (IVF. Methods. Thirty women undergoing IVF for nonmale factor infertility participated. Platelets from venous blood and GCs separated from retrieved oocytes were prepared for immunofluorescence. Flow cytometry quantified the percent of GCs expressing CD9, and CD9 surface density on GCs and platelets. Fertilization rate was determined for the total number of oocytes, and the number of mature oocytes per patient. Correlations tested for significant relationships (P<.05 between fertilization rates and CD9 expression. Results. CD9 surface density on human GCs is inversely correlated with fertilization rate of oocytes (P=.04, but the relationship was weak. Conclusion. More studies are needed to determine if CD9 expression on GCs would be useful for predicting conventional fertilization success during IVF.

  8. Evaluation of Protective Effects of Crocin Onembyo Developing Process in in Vitro Fertilization (IVFin Cyclophosphamide Treated Mice

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    F Khan Mohammadi

    2014-05-01

    Conclusion: The co-administration of crocin with CP chemotherapy caused a significant improvement in fertilizing potential and promoted the embryo development. Key words: Cyclophosphamide, Crocin, Mice, Oocyte, In vitro fertilizing

  9. Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos Melatonina no meio de maturação não melhorou as taxas de maturação dos ovócitos, de desenvolvimento embrionário e a fragmentação do DNA dos embriões bovinos

    Directory of Open Access Journals (Sweden)

    Luciana Takada

    2010-08-01

    Full Text Available Melatonin (MEL acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM on bovine embryos. This study tested the addition of MEL to maturation medium (MM with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum - FCS at 39ºC and 5% CO2 in air. After 24 hours of culture in MM with 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH; 10-9 M MEL or 10-9 M MEL, 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10-9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM.Melatonin (MEL atua como um potente redutor de radicais livres. Efeito direto da MEL na função gonadal também foi observado. Existem poucos estudos relacionados ao efeito da MEL durante a maturação no desenvolvimento embrionário in vitro. Avaliou-se a adição de MEL no meio de maturação (sem gonadotrofinas nas taxas de maturação nuclear e de desenvolvimento embrionário e na incidência de fragmentação do DNA nos embriões produzidos in vitro

  10. The role of progesterone in oocyte acquisition of developmental competence.

    Science.gov (United States)

    Fair, T; Lonergan, P

    2012-08-01

    It is generally accepted that progesterone (P4) is a key regulator of reproductive function in mammals. In cattle, the primary focus of P4's actions has been uterine receptivity and maintenance of pregnancy. Studies in mammalian laboratory species and ovarian derived cell lines also highlight their role in ovarian function. Extensive research in non-mammalian species has elucidated a critical role for P4 and both its nuclear and membrane-bound receptors in oocyte maturation and ovulation. Until recently, such a role in mammalian oocytes has been disputed. However, as oestrous synchronization regimes are constantly tweaked and revised to maximize pregnancy rates to artificial insemination in cattle, the importance of P4 priming of the dominant ingfollicle is once again tak centre stage. Sequencing of the bovine genome and the development of multiple transcriptomic data mining tools have facilitated an explosion in global transcriptome profiling of immature and matured oocytes and their surrounding cumulus cells. Many of the differentially regulated genes and their associated preferentially populated pathways appear to be P4 regulated in other tissues. Therefore, attention is once again turning to a potential role for P4 in ovulatory follicle development and oocyte maturation in cattle. The current review summarizes the most recent findings in these areas. PMID:22827363

  11. 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.

  12. N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle.

    Science.gov (United States)

    Oseikria, Mouhamad; Elis, Sébastien; Maillard, Virginie; Corbin, Emilie; Uzbekova, Svetlana

    2016-06-01

    The positive effect of n-3 polyunsaturated fatty acids (FAs) on fertility in ruminants seems to be partly mediated through direct effects on the oocyte developmental potential. We aimed to investigate whether supplementation with physiological levels of docosahexaenoic acid (DHA, C22:6 n-3 polyunsaturated fatty acids) during IVM has an effect on oocyte maturation and in vitro embryo development in cattle. We reported that DHA (0, 1, 10, or 100 μM) had no effect on oocyte viability or maturation rate after 22-hour IVM. Incubation of oocyte-cumulus complexes with 1-μM DHA during IVM significantly increased (P reaction, negative effects of 100-μM DHA were associated with significant increase of progesterone synthesis by oocyte-cumulus complexes, a three-fold increase in expression level of FA transporter CD36 and a two-fold decrease of FA synthase FASN genes in cumulus cells (CCs) of corresponding oocytes. Docosahexaenoic acid at 1 and 10 μM had no effect on expression of those and other key lipid metabolism-related genes in CC. In conclusion, administration of a low physiological dose of DHA (1 μM) during IVM may have beneficial effects on oocyte developmental competence in vitro without affecting lipid metabolism gene expression in surrounding CCs, contrarily to 100 μM DHA which diminished oocyte quality associated with perturbation of lipid and steroid metabolism in CC. PMID:26898414

  13. Comparative analysis of the oocytes and early development of two species of curimatidae teleost fish.

    Science.gov (United States)

    Perini, V R; Sato, Y; Rizzo, E; Bazzoli, N

    2013-02-01

    Curimatella lepidura and Steindachnerina elegans are small forage fish, constituting an important link in the food chain, serving as food for larger commercial fish. In this study, characteristics of the eggs, of the oocyte's surface ultrastructure and of the embryogenesis are first described for these species. Absolute fecundity was 40864 ± 8769 oocytes for C. lepidura and 22089 ± 8710 oocytes for S. elegans. Oocytes of both species are yellowish, weakly adhesive and with a post-fertilization diameter of 1019.5 ± 20.6 μm and 978.75 ± 29.16 μm for C. lepidura and S. elegans, respectively. The ultrastructural analysis, using scanning electron microscopy, showed that the oocyte's surface of both species has pore canals over the entire surface and a funnel-shaped micropyle. At 24°C, the embryonic development of C. lepidura was completed 25 h after fertilization, and blastopore closure occurred in 7 h 30 min. In S. elegans, larvae hatched 20 h after fertilization, and blastopore closure occurred in 7 h 15 min. The fertilization rate was 74.5 ± 7.96 and 71.2 ± 10.8% for C. lepidura and S. elegans, respectively. This study provides important support for clarifying phylogenetic relationships and in ecological and zoological understanding of Neotropical Curimatidae fish. PMID:22583005

  14. RESEARCHES REGARDING THE INFLUENCE OF THE NUMBER OF CUMULAR CELLS LAYER OVER THE OOCYTE MATURATION EFFICIENCY

    Directory of Open Access Journals (Sweden)

    V. CARABĂ

    2009-05-01

    Full Text Available During the experiments we have carried out with imature oocyte collected from the ovarian follicles, wefound a variety of oocyte-cumulus complexes. We got the following experiment in order to understand therole of cumular cells on the achievement of the cytoplasma and oocyte nucleus maturation. We select theoocyte-cumulus complexes collected both from cows and sows according to the number of cumular celllayers and we watched their development to the blastocyst stade. Thus, we achieved three groups of COC(oocyte-cumulus complexes.One group was made of oocyte without cumular cells, the second group had a layer of cumular cells andthe third group had many layers of cumular cells. we performed an incubation of all these types of COCin TCM-199 enriched with 20% of bovine fetal serum. Because only 1,2 oocyte of the ones who lack thecumular cells layer had maturation signs during cultivation in the thermostat versus 55 and 115,respectively, of the ones that had many cellular layers, presents a solid evidence that cumular cells areindispensable for the maturation and even to the fecundation process. The cumular cells perform adecisive role on the cytoplasma and oocyte nucleus maturation process.

  15. RESEARCHES REGARDING THE INFLUENCE OF THE NUMBER OF CUMULAR CELLS LAYER OVER THE OOCYTE MATURATION EFFICIENCY

    Directory of Open Access Journals (Sweden)

    V. CARABĂ

    2013-07-01

    Full Text Available During the experiments we have carried out with imature oocyte collected from the ovarian follicles, we found a variety of oocyte-cumulus complexes. We got the following experiment in order to understand the role of cumular cells on the achievement of the cytoplasma and oocyte nucleus maturation. We select the oocytecumulus complexes collected both from cows and sows according to the number of cumular cell layers and we watched their development to the blastocyst stade. Thus, we achieved three groups of COC (oocyte-cumulus complexes. One group was made of oocyte without cumular cells, the second group had a layer of cumular cells and the third group had many layers of cumular cells. we performed an incubation of all these types of COC in TCM-199 enriched with 20% of bovine fetal serum. Because only 1,2 oocyte of the ones who lack the cumular cells layer had maturation signs during cultivation in the thermostat versus 55 and 115, respectively, of the ones that had many cellular layers, presents a solid evidence that cumular cells are indispensable for the maturation and even to the fecundation process. The cumular cells perform a decisive role on the cytoplasma and oocyte nucleus maturation process.

  16. Ultrastructural studies on the nematode Xiphinema diversicaudatum: Oogenesis and fertilization.

    Science.gov (United States)

    Bleve-Zacheo, T; Melillo, M T; Zacheo, G

    1993-06-01

    Oogenesis and fertilization in longidorid nematodes has been examined for the first time at electron microscope level in Xiphinema diversicaudatum. Oogonia in the germinative zone of the ovary are irregularly shaped and lie adjacent to each other or separated by processes of the epithelial cells of the ovary. Developing oocytes pass in single file up to the growth zone and fibrogranular formation occurs around their nucleus. The perinuclear deposits remain until the oocyte is fully grown. Oocytes increase rapidly in volume because of the production of secretory granules. Three types of granules are recognizable. Type 1 granules are spherical, amorphous in structure and delimited by a lighter area, probably consisting of lipoprotein. Type 2 granules, electron lucent, arranged in groups, are lipid inclusions. Type 3 are dense spheres and may represent yolk bodies. The two last are then utilized by the developing embryo. Mature oocytes assume a smooth, cylindrical configuration as they traverse the oviduct. A cone of fertilization seems to be formed at the distal pole of the oocyte, where the sperm penetrates. The sperm totally penetrates the oocyte, through an invagination formed at the oocyte surface. The oocyte continues to undergo two unequal cytoplasmic divisions, resulting in the formation of a female pronucleus and two polar bodies. Under the stimulus of fertilization, a new egg cell membrane is produced, the first one becoming the vitelline envelope.

  17. Coagulansin-A has beneficial effects on the development of bovine embryos in vitro via HSP70 induction

    OpenAIRE

    Khan, Imran; Lee, Kyeong-Lim; Fakruzzaman, Md.; Song, Seok-Hwan; Ihsan-ul-Haq,; Mirza, Bushra; Yan, Chang Guo; Kong, Il-Keun

    2016-01-01

    Treatment with the steroidal lactone, coagulansin-A, improves bovine oocyte maturation and embryo development in vitro by inducing heat shock protein 70 (HSP70), which reduces the levels of reactive oxygen species (ROS), DNA damage and inflammation.

  18. 精卵孵育过程中活性氧对体外受精-胚胎移植结局的影响%Impact of ROS on the outcome of in vitro fertilization and embryo transfer in the incubation of oocytes and sperm

    Institute of Scientific and Technical Information of China (English)

    张娜; 张轶; 张耀恒; 赵世彬; 乜照燕; 甄秀丽; 李亚丽

    2014-01-01

    and 20h after insemination to detect of the content of H2O2 and CTA. On the oocyte retrieval day, the OCCCs which retrieved from the same patient were divided into short-term fertilization group (group A) and long-term fertilization group (group B) according to incubation time. The concentrations of H2O2 and CTA in insemination medium were detected respectively. On the embryo transfer day,the patients were divided into two groups: one group of patients chose short-term fertilized embryo to transfer, one group of patients chose long fertilized embryo to transfer. Results The levels of hydrogen peroxide and catalase at 5h after insemination were positively correlated with those of group A(r=0.477, 0.518, P0.05). The polyspermy rate in group B was significantly higher than that in group A(P0.05). Conclusion Shorten the incubation time of oocytes and spermcould protect oocytes from the high reactive oxygen environment so as to improve the quality of embryos.

  19. Fertilization in the cestode Echinococcus multilocularis (Cyclophyllidea, Taeniidae).

    Science.gov (United States)

    Świderski, Zdzisław; Miquel, Jordi; Azzouz-Maache, Samira; Pétavy, Anne-Françoise

    2016-01-01

    Fertilization in the taeniid cestode Echinococcus multilocularis with uniflagellate spermatozoa was examined by means of transmission electron microscopy (TEM). Fertilization in this species occurs in the oviduct lumen or in the fertilization canal proximal to the ootype, where the formation of the embryonic capsule precludes sperm contact with the oocytes. Cortical granules are not present in the cytoplasm of the oocytes of this species, however, several large bodies containing granular material where frequently observed. Spermatozoa coil spirally around the oocytes and syngamy occurs by lateral fusion of oocyte and sperm plasma membranes. In the ootype, one vitellocyte associates with fertilized oocyte, forming a membranous capsule which encloses both cell types. In this stage, the spirally coiled sperm body adheres partly to the external oocyte surface, and partially enters into the perinuclear cytoplasm. The electron-dense sperm nucleus becomes progressively electron-lucent within the oocyte cytoplasm after penetration. Simultaneously with chromatin decondensation, the elongated sperm pronucleus changes shape, forming a spherical male pronucleus, which attains the size of the female pronucleus. Cleavage begins immediately after pronuclear fusion. PMID:26751876

  20. Effect of GPAG Supplement on Porcine Oocyte Maturation and Its Following Embryonic Development by Parthenogenetic Activated

    Institute of Scientific and Technical Information of China (English)

    YUE Shunli; ZHU Jiawei; WANG Zhongwei; ZHANG Jie; ZHOU Jiabo

    2008-01-01

    The aim of this study was to compare the effect of GPAG and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after the fertilization.COCs were aspirated from follicles and cultured for 16, 24, 32, 40 and 48 h in TCM-199 medium either with GPAG or FCS.After 24 h with GPAG,89.4% of oocytes reached M 1 stage while in the medium supplemented with FCS,only 27.7% of oocytes reached the same stage (P<0.05).Prolonged incubation for up to 32 h clearly demonstrated that some of oocytes cultured in GPAG medium were at M Ⅱ stage (35.7%),few of oocytes from FCS medium were at M II stage (7.5%) (P<0.05).Both groups of oocytes reached the same stage of maturation within 48 h. After 48 h of culture,the oocytes with extruded polar bodies were inseminated.Fertilized oocytes were cultured in PZM3 medium supplemented with 3 mg·mL-1 of BSA. After 7 days,the development and the quality of embryos were evaluated.The results showed that the maturation of oocytes in the presence of GPAG significantly increased their subsequent developmental ability when compared with FCS supplementation (29.2%:18.9% of blastocysts,P<0.05).However,differential staining revealed that once blastocysts were formed in either group, they had the same total cell number (39:38) and the ICM/total cell ratio (0.26:0.28).

  1. Sperm competition in the absence of fertilization in Caenorhabditis elegans.

    OpenAIRE

    Singson, A; Hill, K L; L'Hernault, S. W.

    1999-01-01

    Hermaphrodite self-fertilization is the primary mode of reproduction in the nematode Caenorhabditis elegans. However, when a hermaphrodite is crossed with a male, nearly all of the oocytes are fertilized by male-derived sperm. This sperm precedence during reproduction is due to the competitive superiority of male-derived sperm and results in a functional suppression of hermaphrodite self-fertility. In this study, mutant males that inseminate fertilization-defective sperm were used to reveal t...

  2. Female Fertility: Is it Safe to "Freeze?"

    Institute of Scientific and Technical Information of China (English)

    Lu Zhang; Li-Ying Yan; Xu Zhi; Jie Yan; Jie Qiao

    2015-01-01

    Objective:To evaluate the safety and risk of cryopreservation in female fertility preservation.Data sources:The data analyzed in this review were the English articles from 1980 to 2013 from journal databases,primarily PubMed and Google scholar.The criteria used in the literature search show as following:(1) human; embryo; cryopreservation/freezing/vitrification,(2) human; oocyte/immature oocyte; cryopreservation/freezing/vitrification,(3) human; ovarian tissue transplantation; cryopreservation/ freezing/vitrification,(4) human; aneuploidy/DNA damage/epigenetic; cryopreservation/freezing/vitrification,and (5) human; fertility preservation; maternal age.Study selection:The risk ratios based on survival rate,maturation rate,fertilization rate,cleavage rate,implantation rate,pregnancy rate,and clinical risk rate were acquired from relevant meta-analysis studies.These studies included randomized controlled trials or studies with one of the primary outcome measures covering cryopreservation of human mature oocytes,embryos,and ovarian tissues within the last 7 years (from 2006 to 2013,since the pregnancy rates of oocyte vitrification were significantly increased due to the improved techniques).The data involving immature oocyte cryopreservation obtained from individual studies was also reviewed by the authors.Results:Vitrifications of mature oocytes and embryos obtained better clinical outcomes and did not increase the risks of DNA damage,spindle configuration,embryonic aneuploidy,and genomic imprinting as compared with fresh and slow-freezing procedures,respectively.Conclusions:Both embryo and oocyte vitrifications are safe applications in female fertility preservation.

  3. Risk assessment of transmission of bovine viral diarrhea virus (BVDV) in abattoir-derived in vitro produced embryos.

    Science.gov (United States)

    Perry, G H

    2007-07-01

    Bovine virus diarrhea virus (BVDV) is a pathogen of the bovine reproductive system causing reduced conception rates, abortions and persistently infected calves. Most if not all strains of BVDV are transmissible by natural mating and AI. For international trade, it is recommended that in vitro fertilized embryos be washed according to the IETS Manual. However, BVDV may not be entirely washed out, resulting in possible transmission risks to recipients. Donor cows, donor bulls and biological agents are all possible sources of contamination. The process for producing in vitro produced (IVP) embryos is complex and non-standard, and some procedures can contribute to spread of BVDV to uninfected embryos. The structure of the zone pellucida (ZP) of IVP embryos permits adherence of BVDV to the ZP. To estimate the risk of producing infected recipients and persistently infected calves from abattoir-derived IVP embryos, a quantitative risk assessment model using Microsoft Excel and Palisade @Risk was developed. Assumptions simplified some of the complexities of the IVP process. Uncertainties due to incomplete or variable data were addressed by incorporating probability distributions in the model. Model variables included: disease prevalence; the number of donor cows slaughtered for ovaries; the number of oocytes collected, selected and cultured; the BVDV status of ovaries, semen, biological compounds and its behavior in the IVP embryo process. The model used the Monte Carlo method to simulate the IVP process. When co-culture cells derived from donor cows of unknown health status were used for in vitro culture (IVC), the probability of a recipient cow at risk of infection to BVDV per oocyte selected for IVP processing averaged 0.0006. However, when co-culture free from BVDV was used, the probability was 1.2 x 10(-5). Thus, for safe international trade in bovine IVP embryos (i.e. negligible risks of transmission of BVDV), co-culture cells, if used during IVC for producing IVP

  4. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    Science.gov (United States)

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2016-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p quality and the cleavage stage of the embryo produced. PMID:26858565

  5. BPA exposure during in vitro oocyte maturation results in dose-dependent alterations to embryo development rates, apoptosis rate, sex ratio and gene expression.

    Science.gov (United States)

    Ferris, Jacqueline; Mahboubi, Kiana; MacLusky, Neil; King, W Allan; Favetta, Laura A

    2016-01-01

    Alterations in the oocyte's environment can negatively affect embryo development. Oocyte quality, which can determine embryonic viability, is easily perturbed, thus factors affecting normal oocyte maturation are a concern. Bisphenol A (BPA) is an endocrine disrupting chemical that elicits a variety of reproductive effects. BPA has previously been found to disrupt meiosis, however the embryonic effects in mammals are not well documented. Here, bovine oocytes were matured in vitro with and without BPA treatment. Resulting embryos exhibited decreased embryonic development rates, increased apoptosis, and a skewed sex ratio. Gene expression in blastocysts was not altered, whereas treatment with 15ng/mL BPA resulted in increased expression of several of the genes studies, however this increase was largely due to a vehicle effect. BPA exposure during oocyte maturation in vitro can therefore, in a dose-dependent way, decrease oocyte and embryo quality and developmental potential and affect gene expression of developmentally important transcripts. PMID:26686065

  6. Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection.

    Science.gov (United States)

    Heindryckx, Björn; De Gheselle, Stefanie; Gerris, Jan; Dhont, Marc; De Sutter, Petra

    2008-11-01

    Failed fertilization after intracytoplasmic sperm injection (ICSI) can occur due to an oocyte activation defect. In these cases assisted oocyte activation (AOA) may help but efficiency is still unknown. Prior to AOA, the mouse oocyte activation test (MOAT) can be carried out by injecting human spermatozoa into mouse oocytes to evaluate their activating capacity. According to the MOAT activation percentage achieved, patients were classified into three groups: 0-20% (16 patients); 20-85% (seven patients); 85-100% (seven patients). For AOA, CaCl(2) was injected together with spermatozoa followed by a double Ca(2+) ionophore treatment. The fertilization rates before application of AOA in 50 cycles were 6%, 22% and 14% in, respectively, groups 1, 2 and 3 without any pregnancy. Fertilization and pregnancy rates after AOA in 61 cycles were significantly increased to 75% and 34% for group 1, 73% and 43% for group 2, and 75% and 17% for group 3 (P < 0.0001 and P < 0.004, respectively). Application of AOA results in normal fertilization and pregnancy rates in patients whose spermatozoa show deficient activation. When MOAT reveals no activation deficiency in spermatozoa, AOA still allows for high fertilization and acceptable pregnancy rates. The obstetric and neonatal outcomes after AOA were normal as no malformations were observed.

  7. A genetic marker test for brachyspina and fertility in cattle

    DEFF Research Database (Denmark)

    2010-01-01

    This invention relates to methods for the detection of bovine that are affected by or carriers of Brachyspina (BS). The present invention provides a method for determining whether a bovine is affected by or earner of BS by analyzing its genomic DNA or its RNA. The method can be used to perform...... marker assisted selection or genomic selection for increased fertility in said bovine....

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    Fatma Simsek-Duran

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

  10. 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.

  11. Development of frozen-thawed embryos derived from immature human oocytes following in-vitro maturation and fertilization in women with PCOS%多囊卵巢综合征妇女体外成熟卵母细胞胚胎慢速冷冻复苏移植结局分析

    Institute of Scientific and Technical Information of China (English)

    郑晓英; 刘平; 王丽娜; 廉颖; 吴昱琪; 陈媛

    2013-01-01

    Objective To evaluate the development of frozen - thawed embryos derived from immature human oocytes following in - vitro maturation and fertilization in women with PCOS. Methods 385 PCOS women underwent frozen- thawed embryo transfer cycles between Jan. 2006 to Dec. 2010 in Peking University Third Hospital. The frozen embryos derived from in vitro maturation of immature oocytes (IVM group) or mature oocytes of routine IVF cycles (IVF group). All embryos were cryopreserved using slow- freeze protocol. The frozen embryos were thawed and transferred for assessment the survival, clinical pregnancy and implantation rates. Results 243 embryos were thawed and 162 embryos were survived in IVM group, The survival rate of IVM group (66. 67%) was comparable to IVF group (67.41%, P>0. 05). However, the clinical pregnancy and implantation rates of embryos cryopreserved in IVM group were significantly lower than that in IVF group (19. 30% vs 45. 45%, 10. 61% vs 26.14%; P<0. 05)). Conclusion Cleavage embryo slow - freeze programe may not be as efficient in IVM as in that in standard IVF. It implies the inferior development potential of IVM derived embryos before frozen.%目的 评价未成熟卵母细胞体外成熟(IVM)后形成的卵裂期胚胎经慢速冷冻-解冻后的发育能力.方法 将2006年1月至2010年12月北京大学第三医院因多囊卵巢综合征(PCOS)合并不孕症行卵裂期胚胎复苏移植的385例患者分为两组:复苏胚胎来源于体外成熟的卵母细胞组(IVM组,46例)和复苏胚胎来源于常规体内成熟的卵母细胞组(IVF组,339例).采用慢冻速溶法解冻移植后比较两组患者的临床结局.结果 IVM组复苏胚胎243枚,复苏后存活162枚,复苏率为66.67%;IVF组复苏胚胎1 605枚,复苏后存活1 082枚,复苏率为67.41%,两组比较,差异无统计学意义(P>0.05).IVM组患者的临床妊娠率和着床率分别为19.30% (11/57)和10.61% (14/132),明显低于IVF组临床妊娠率(45.45%,175

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    Spitzer D

    2015-01-01

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

  14. 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.

  15. 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.

  16. Molecular aspects of mammalian fertilization

    Institute of Scientific and Technical Information of China (English)

    Hector Serrano; Dolores Garcia-Suarez

    2001-01-01

    Mammalian fertilization is a highly regulated process, much of which are not clearly understood. Here we present some information in order to elaborate a working hypothesis for this process, beginning with the sperm modifications in the epidydimis up to sperm and egg plasmalemma interaction and fusion. We also discuss the still poorly understood capacitation process, the phenomenon of sperm chemo-attraction that brings the capacitated sperm to interact with the oocyte vestments and certain aspects of the acrosome reaction.

  17. Basal follicle stimulating hormone and leptin on the day of hCG administration predict successful fertilization in in vitro fertilization

    Directory of Open Access Journals (Sweden)

    Andon Hestiantoro

    2016-04-01

    Full Text Available Background: Successful pregnancy in in vitro fertilization (IVF program depends on multiple factors. This study aimed to determine whether age, body mass index (BMI, basal follicle stimulating hormone (FSH, estradiol, and leptin on the day of trigger ovulation with human chorionic gonadotropin (hCG might be used as predictor for successful oocyte fertilization in in vitro fertilization (IVF program.Methods: This is a cross sectional study conducted in Yasmin Fertility Clinic, Cipto Mangunkusumo Hospital, Jakarta, Indonesia. Forty participating patients underwent IVF program, excluding smokers, patients with diabetic, morbid obesity, and severe oligospermia or azoospermia. Age, BMI, basal FSH, estradiol, leptin on the day of hCG administration, oocyte count on oocyte retrieval, the number of mature oocyte, and fertility rate were analyzed using bivariate and multivariate analysis to determine which eligible factors play role in predicting the successful of fertilization.Results: Significant correlation was found between basal FSH level and serum leptin/oocyte ratio on the day of hCG administration with successful fertilization. We found probability formula as follows: 1/(1+exp –(6.2 - 0.4(leptin serum/oocyte ratio - 0.8(basal FSH, with 77.8% sensitivity, 77.8% specificity, and AUC levels of 85.6% indicating strong predictability. Probability of successful fertilization related to basal FSH level of 5.90 mIU/mL and leptin serum/oocyte ratio of 3.98.Conclusion: The formula consisting of basal FSH and leptin serum/oocyte ratio on the day of trigger ovulation was capable in predicting the probability of successful fertilization in IVF procedure.

  18. Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro.

    Science.gov (United States)

    Ohboshi, S; Etoh, T; Sakamoto, K; Fujihara, N; Yoshida, T; Tomogane, H

    1997-04-15

    Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar. PMID:16728072

  19. Seasonal variations in developmental competence and relative abundance of gene transcripts in buffalo (Bubalus bubalis) oocytes.

    Science.gov (United States)

    Abdoon, Ahmed S; Gabler, Christoph; Holder, Christoph; Kandil, Omaima M; Einspanier, Ralf

    2014-11-01

    Hot season is a major constraint to production and reproduction in buffaloes. The present work aimed to investigate the effect of season on ovarian function, developmental competence, and the relative abundance of gene expression in buffalo oocytes. Three experiments were conducted. In experiment 1, pairs of buffalo ovaries were collected during cold season (CS, autumn and winter) and hot season (HS, spring and summer), and the number of antral follicles was recorded. Cumulus oocyte complexes (COCs) were aspirated and evaluated according to their morphology into four Grades. In experiment 2, Grade A and B COCs collected during CS and HS were in vitro matured (IVM) for 24 hours under standard conditions at 38.5 °C in a humidified air of 5% CO2. After IVM, cumulus cells were removed and oocytes were fixed, stained with 1% aceto-orcein, and evaluated for nuclear configuration. In vitro matured buffalo oocytes harvested during CS or HS were in vitro fertilized (IVF) using frozen-thawed buffalo semen and cultured in vitro to the blastocyst stage. In experiment 3, buffalo COCs and in vitro matured oocytes were collected during CS and HS, and then snap frozen in liquid nitrogen for gene expression analysis. Total RNA was extracted from COCs and in vitro matured oocytes, and complementary DNA was synthesized; quantitative Reverse Transcription-Polymerase Chain Reaction was performed for eight candidate genes including GAPDH, ACTB, B2M, GDF9, BMP15, HSP70, and SOD2. The results indicated that HS significantly (P ovary. The number of Grade A, B, and C COCs was lower (P quality. In vitro maturation of buffalo oocytes during HS impairs their nuclear and cytoplasmic maturation, fertilization, and subsequent embryo development to the morula and blastocyst stages. This could be in part because of the altered gene expression found in COCs and in vitro matured oocytes. PMID:25156970

  20. Mitofusin 2 regulates the oocytes development and quality by modulating meiosis and mitochondrial function

    Science.gov (United States)

    Liu, Qun; Kang, Lina; Wang, Lingjuan; Zhang, Ling; Xiang, Wenpei

    2016-01-01

    Mitofusin-2 (Mfn2), one of the mitochondrial dynamic proteins plays a key role in maintaining the integrity of mitochondrial morphology and function. However, it is unknown if Mfn2 influences the quality of oocytes in the process of development by modulating mitochondrial function in vitro. In this study, immature oocytes were transfected with Mfn2-siRNA for 16 h. We found that the expression level of the Mfn2 gene was significantly lower than those of the control group. The rates of maturation and fertility were also found to have declined. Moreover, mitochondrial structure and function, especially the morphogenesis of spindles, were observed as abnormal during meiosis. Thus, the above findings indicate that down-regulation of Mfn2 may have an impact on the maturation and fertilization of immature oocytes in vitro by modulating meiosis and mitochondrial function. PMID:27469431

  1. Mitofusin 2 regulates the oocytes development and quality by modulating meiosis and mitochondrial function.

    Science.gov (United States)

    Liu, Qun; Kang, Lina; Wang, Lingjuan; Zhang, Ling; Xiang, Wenpei

    2016-01-01

    Mitofusin-2 (Mfn2), one of the mitochondrial dynamic proteins plays a key role in maintaining the integrity of mitochondrial morphology and function. However, it is unknown if Mfn2 influences the quality of oocytes in the process of development by modulating mitochondrial function in vitro. In this study, immature oocytes were transfected with Mfn2-siRNA for 16 h. We found that the expression level of the Mfn2 gene was significantly lower than those of the control group. The rates of maturation and fertility were also found to have declined. Moreover, mitochondrial structure and function, especially the morphogenesis of spindles, were observed as abnormal during meiosis. Thus, the above findings indicate that down-regulation of Mfn2 may have an impact on the maturation and fertilization of immature oocytes in vitro by modulating meiosis and mitochondrial function. PMID:27469431

  2. Psychological Counseling of Female Fertility Preservation Patients.

    Science.gov (United States)

    Lawson, Angela K; Klock, Susan C; Pavone, Mary Ellen; Hirshfeld-Cytron, Jennifer; Smith, Kristin N; Kazer, Ralph R

    2015-01-01

    Young cancer patients are increasingly interested in preserving their fertility prior to undergoing gonadotoxic therapies. Although the medical safety and treatment protocols for fertility preservation have been well documented, limited research has addressed the emotional issues that arise in fertility preservation patients. We briefly review the literature on the psychosocial issues in adult female fertility preservation treatment and describe our experiences within this patient population. Our findings suggest that several important issues to be addressed during the psychological counseling of adult female fertility preservation patients include: (1) preexisting psychological distress in patients undergoing treatment, (2) choice of fertility preservation strategy in the face of an uncertain relationship future, (3) decision making regarding use of third-party reproduction (e.g., sperm/egg donation, gestational surrogacy), (4) treatment expectations regarding pregnancy and miscarriage, (5) ethical issues related to treatment including the creation, cryopreservation, and disposition of embryos/oocytes, and (6) decision regret from patients who declined fertility preservation. PMID:25996581

  3. Intrauterine fertilization capsules--a clinical trial

    DEFF Research Database (Denmark)

    Lenz, S; Lindenberg, S; Sundberg, K;

    1991-01-01

    Treatment of 26 women with tubal infertility was attempted using intrauterine capsules loaded with oocytes and spermatozoa. The stimulation protocol was as used for in vitro fertilization and embryo transfer and consisted of short-term use of Buserelin, human menopausal gonadotropin, and human ch...

  4. CDC2/SPDY transiently associates with endoplasmic reticulum exit sites during oocyte maturation

    NARCIS (Netherlands)

    Holzenspies, Jurriaan J.; Stoorvogel, Willem; Colenbrander, Ben; Roelen, Bernard A. J.; Gutknecht, Dagmar R.; van Haeften, Theo

    2009-01-01

    Background: Mammalian oocytes acquire competence to be fertilized during meiotic maturation. The protein kinase CDC2 plays a pivotal role in several key maturation events, in part through controlled changes in CDC2 localization. Although CDC2 is involved in initiation of maturation, a detailed analy

  5. High Serum FSH is Associated with Brown Oocyte Formation and a Lower Pregnacy Rate in Human IVF Parctice

    Directory of Open Access Journals (Sweden)

    Hongyi Xu

    2016-07-01

    Full Text Available Background/Aims: To investigate whether brown zona pellucida (ZP of oocytes affects the outcome of fertilization, embryo quality and pregnancy rate in in vitro fertilization-embryo transfer (IVF-ET. Methods: Based on the ZP color of their oocytes, a total number of 703 patients dated from 2012 to 2014 were divided into a normal egg group (group A and a brown oocyte group (group B, with 629 and 74 cases, respectively. Clinical characteristics, gonadotropin (Gn days, Gn dosage, serum hormone levels on the day of human chorionic gonadotropin (HCG injection, ZP thickness (ZPT of the eggs, fertilization rate, rescue intracytoplasmic sperm injection (rICSI rate, good-quality embryo rate and pregnancy rate were compared between the two groups. Results: No significant differences were found in the duration and the causes of infertility, and their basal level of endocrine hormone before IVF-ET between normal egg group and brown egg group. The level of serum hormone including estradiol, progesterone and luteinizing hormone on the day of HCG injection were again similar. Moreover, there were no differences in number of mature oocytes, oocyte fertilization rates and rICSI rates after IVF between the two groups. However, we observed that the ZPT of brown oocytes (group B was higher than that of normal oocytes (group A. Moreover, the Gn dosage and FSH levels on the day of HCG injection were significantly higher in group B than in group A and the good-quality embryo rate and pregnancy rate in group B were lower than those in group A. Conclusion: Compared with normal eggs, oocytes with a brown ZP were found to have a higher ZPT, lower embryo quality and lower pregnancy rate, which might be due to a high Gn dosage injection and high serum FSH levels during IVT-ET cycles.

  6. The fertilization—induced Ca2+ oscillation in mouse oocytes is cytoplasmic maturation dependent

    Institute of Scientific and Technical Information of China (English)

    DENGMANQI; FANGZHENSUN

    1996-01-01

    Mature eggs (at metaphase II stage) produce a series of Ca2+ oscillation at fertilization.To define whether the fertilization-induced Ca2+ oscillation is restrict to the metaphase II eggs and cell cycle dependent,mouse oocytes at prophase I (arrested at germinal vesicle stage),metaphase I,metaphase II,as well as the pronuclear embryos at interphase of the first mitotic division derived from fertilization of parthenogenetic activation were inseminated after removal of zona pellucida,The results show that the fertilization-induced Ca2+ oscillation is not specific to metaphase II eggs.This is supported by the fact that immature oocytes generated the Ca2+ oscillations at fertilization regardless of their nuclear progression from prophase I to metaphase I (in vitro matured) stage.More interestingly,it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca2+ oscillations in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca2+ oscillations in MII eggs.This suggests that the ability of oocytes to generate Ca2+ oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cytoplasmic maturation.

  7. Combination effects of epidermal growth factor and glial cell line-derived neurotrophic factor on the in vitro developmental potential of porcine oocytes.

    Science.gov (United States)

    Valleh, Mehdi Vafaye; Rasmussen, Mikkel Aabech; Hyttel, Poul

    2016-06-01

    The developmental potential of in vitro matured porcine oocytes is still lower than that of oocytes matured and fertilized in vivo. Major problems that account for the lower efficiency of in vitro production include the improper nuclear and cytoplasmic maturation of oocytes. With the aim of improving this issue, the single and combined effects of epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) on oocyte developmental competence were investigated. Porcine cumulus-oocyte cell complexes (COCs) were matured in serum-free medium supplemented with EGF (0, 10 or 50 ng/ml) and/or GDNF (0, 10 or 50 ng/ml) for 44 h, and subsequently subjected to fertilization and cultured for 7 days in vitro. The in vitro-formed blastocysts derived from selected growth factor groups (i.e. EGF = 50 ng/ml; GDNF = 50 ng/ml; EGF = 50 ng/ml + GDNF = 50 ng/ml) were also used for mRNA expression analysis, or were subjected to Hoechst staining. The results showed that the addition of EGF and/or GDNF during oocyte maturation dose dependently enhanced oocyte developmental competence. Compared with the embryos obtained from control or single growth factor-treated oocytes, treatment with the combination of EGF and GDNF was shown to significantly improve oocyte competence in terms of blastocyst formation, blastocyst cell number and blastocyst hatching rate (P competency and blastocyst quality. PMID:26350562

  8. Kinetics of Cryptosporidium parvum sporozoite neutralization by monoclonal antibodies, immune bovine serum, and immune bovine colostrum.

    Science.gov (United States)

    Perryman, L E; Riggs, M W; Mason, P H; Fayer, R

    1990-01-01

    Monoclonal antibodies, immune bovine serum, and immune bovine colostral whey neutralized infectivity of Cryptosporidium parvum sporozoites for mice in a time-dependent manner. Immune colostral whey neutralized sporozoites more rapidly and completely than immune serum, monoclonal antibody (MAb) 18.44, or a combination of MAb 18.44 and MAb 17.41. Mice were partially protected against oral challenge with C. parvum oocytes when treated with immune colostral whey, MAb 17.41, or a combination of MAb 17.41 and MAb 18.44. PMID:2294054

  9. Control of polyspermic fertilization in short-term insemination

    Institute of Scientific and Technical Information of China (English)

    Jin Hai-xia; Sun Ying-pu

    2011-01-01

    In conventional in vitro fertilization (IVF),since sperm metabolites,granular cells and death spermatozoa may consume lots of energy in the culture medium due to longer co-incubation of oocytes and high concentration spermatozoa.The oocytes are in innutritive environment,which leads to the hardening of oocyte plasma membrane.At the same time,the high levels of estradiol (E2) and progestone (P) produced by granular cells have direct toxic effects to affect embryo cleavage,development and implantation.Therefore,short-term insemination is adopted in more and more reproductive centers.1.Short-term insemination may increase oocyte-utilization rate,high-quality embryo rate and embryo-utilization rate.2.Retention of cumulus cells may reduce polyspermic fertilization rate.Studies have indicated that the polyspermic fertilization rate is significantly higher in cumulus cell-free group than in cumulus cell group.In shortterm insemination,the remaining oocytes should retain cumulus cells to reduce polyspermic fertilization under the circumstance of successful fertilization.3.There is no significant difference in 2PN embryo chromosome abnormality between conventional IVF group and short-term insemination group.4.Short-term insemination may significantly decrease ICSI rate and partial ICSI rate.5.Complete fertilization failure rate significantly decreases in short-term insemination.Short-term insemination reduces unfavourable factors for embryo development,therefore increases high quality embryo rate.If short-term insemination is adopted in IVF,under the circumstance of successful fertilization,the remaining oocytes should retain cumulus cells as much as possible to reduce polyspermic fertilization,improve oocyteutilization rate and optimize IVF outcomes.

  10. 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.

  11. 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.

  12. Effect of Kuntai Capsule on the Number of Retrieved Oocytes, High-quality Oocytes and Embryos in in Vitro Fertilization of Poor Ovarian Response Patients%坤泰胶囊对体外受精卵巢低反应患者获卵数、卵细胞及胚胎质量的影响

    Institute of Scientific and Technical Information of China (English)

    连方; 姜晓媛

    2014-01-01

    目的 观察坤泰胶囊对体外受精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)卵巢低反应(poor ovarian response,POR)患者获卵数、卵细胞和胚胎质量的影响.方法 70例将行IVF-ET的POR患者随机分为观察组和对照组,每组35例.观察组在IVF-ET超排卵周期的前3个月经周期(简称准备周期)以及超排卵过程中联合应用坤泰胶囊,对照组在此过程中联合应用安慰剂.观察两组用药前后肾阴虚证候改善情况;基础促卵泡刺激素(basal follicle-stimulating hormone,bFSH)、促黄体生成激素(luteinizing hormone,LH)、雌激素(estradiol,E2)、抗苗勒氏管激素(anti-Müllerian hormone,AMH)、FSH/LH、窦卵泡数(antral follicle count,AFC)情况;两组HCG日单个卵E2水平、获卵数、优质卵率、优质胚胎率.结果 观察组肾阴虚证候改善情况、bFSH、LH水平下降、AFC增加数目、HCG日单个卵E2水平、获卵数、优质卵率、优质胚胎率均优于对照组,差异有统计学意义(P <0.05);FSH/LH水平下降两组比较差异无统计学意义(P>0.05);E2 、AMH水平观察组服药后比服药前升高,对照组服药后比服药前下降,两组E2、AMH差值比较,差异有统计学意义(P<0.05).结论 坤泰胶囊可增加IVF-ET中POR患者的获卵数,提高卵细胞和胚胎质量.

  13. [New strategies for fertility preservation in young women with cancer].

    Science.gov (United States)

    Moffat, Rebecca; De Geyter, Christian; von Wolff, Michael

    2009-12-01

    Especially young women with cancer face rising survival rates due to remarkable progress in oncologic therapies. Preserving fertility is a major concern for both patients and their next of kin. Well established reproductive technologies such as cryopreservation of fertilized oocytes after in vitro fertilization already make fertility preservation possible for some patients. This review is dedicated to the emerging techniques that are becoming widely accepted for fertility preservation in young women and girls with cancer, such as auto transplantation of ovarian tissue cryopreservation and in vitro maturation (IVM) of either oocytes or follicles. First results are encouraging. But some challenges still have to be tackled in order for these novel technologies to be routinely employed with the aim of successful fertility preservation.

  14. 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.

  15. The dormant and the fully competent oocyte

    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...... identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P...

  16. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    Science.gov (United States)

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

  17. 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.

  18. 不同促精子活动剂对水牛卵母细胞体外受精及随后胚胎发育的影响%The effect of sperm-motility enhancing agents on the in vitro fertilization of Bubalus bubalis oocytes and their subsequent development to blastocyst

    Institute of Scientific and Technical Information of China (English)

    Abdul Rahman SESAY; 石德顺

    2005-01-01

    主要探讨精子活动促进剂对水牛Bubalus bubalis卵母细胞体外受精及随后胚胎发育的影响. 来自屠宰场水牛卵巢的卵母细胞和卵丘细胞复合体,在体积分数为5%CO2 的培养箱中培养24~26 h,然后通过体外受精测定其受精和胚胎发育能力. 试验1:解冻的精子用50 g/mL的胰蛋白酶处理30 min,然后进行受精;试验2:受精在含不同浓度咖啡因 (0, 2.5, 5.0 和 10.0 mmol/L)的受精液中进行;试验3:在含不同浓度的钙离子载体A23187 (0, 0.1, 1.0 和 2.5 μmol/L)的受精液中进行体外受精;试验4:在PHE、咖啡因和A23187不同组合(空白, PHE, PHE+2.5 mmol/L咖啡因+2.5 μmol/L A23187和PHE+2.5 μmol/L A23187) 的受精液中进行体外受精. 试验结果表明,精子用胰蛋白酶处理后的受精卵囊胚发育率显著下降 (12.5%和2.6%,P<0.05),而用咖啡因和钙离子载体A23187处理,对卵母细胞的卵裂率和囊胚发育率没有明显影响. 在体外受精中PHE和钙离子载体的组合使用能提高受精卵的胚胎发育率,而高浓度的咖啡因则降低卵母细胞的卵裂率和胚胎发育率.%Effects of sperm-motility enhancing agents on the in vitro fertilization of buffalo oocytes and their subsequent development in vitro were examined in this study. Buffalo oocytes from ovaries taken at slaughter were matured in vitro for 24-26 h at 38.5 ℃ under a humidified 5% CO2 in air, and then fertilized in vitro using buffalo spermatozoa washed with 50 μg/mL trypsin in experiment 1, treated with different concentration of caffeine (0, 2.5, 5.0, and 10.0 mmol/L) in experiment 2, calcium-ionophore A23187 (0, 0.1, 1.0 and 2.5 μmol/L) in experiment 3 or PHE only, PHE+2.5 mmol/L caffeine+2.5 μmol/L A23187, and PHE+2.5 μmol/L A23187 in experiment 4. At 24 to 26 h post insemination, the oocytes were co-cultured with granulosa cell monolayer in droplets containing culture medium to evaluate their embryonic development. There was a significant

  19. Fertility preservation in female cancer patients: An overview

    OpenAIRE

    Nalini Mahajan

    2015-01-01

    Fertility preservation is becoming increasingly important to improve the quality of life in cancer survivors. Despite guidelines suggesting that discussion of fertility preservation should be done prior to starting cancer therapies, there is a lack of implementation in this area. A number of techniques are available for fertility preservation, and they can be used individually or together in the same patient to maximize efficiency. Oocyte and embryo cryopreservation are now established techni...

  20. Are there optimal numbers of oocytes, spermatozoa and embryos in assisted reproduction?

    Science.gov (United States)

    Milachich, Tanya; Shterev, Atanas

    2016-01-01

    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 (strategy suggests the vitrification of good embryos, therefore quality is prior to number and patient recruitment for this strategy should be performed cautiously. PMID:27584608

  1. The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

    Directory of Open Access Journals (Sweden)

    Canepa Sylvie

    2009-11-01

    Full Text Available Abstract Background Oviduct epithelial cells (OEC co-culture promotes in vitro fertilization (IVF in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. Methods & results In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1 gametes co-incubated with equine vs porcine OEC, 2 intact cumulus-oocyte complexes vs denuded oocytes, 3 OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4 in vivo vs in vitro matured oocytes. In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though

  2. Desorption electrospray ionization mass spectrometry reveals lipid metabolism of individual oocytes and embryos.

    Directory of Open Access Journals (Sweden)

    Andrés Felipe González-Serrano

    Full Text Available Alteration of maternal lipid metabolism early in development has been shown to trigger obesity, insulin resistance, type 2 diabetes and cardiovascular diseases later in life in humans and animal models. Here, we set out to determine (i lipid composition dynamics in single oocytes and preimplantation embryos by high mass resolution desorption electrospray ionization mass spectrometry (DESI-MS, using the bovine species as biological model, (ii the metabolically most relevant lipid compounds by multivariate data analysis and (iii lipid upstream metabolism by quantitative real-time PCR (qRT-PCR analysis of several target genes (ACAT1, CPT 1b, FASN, SREBP1 and SCAP. Bovine oocytes and blastocysts were individually analyzed by DESI-MS in both positive and negative ion modes, without lipid extraction and under ambient conditions, and were profiled for free fatty acids (FFA, phospholipids (PL, cholesterol-related molecules, and triacylglycerols (TAG. Principal component analysis (PCA and linear discriminant analysis (LDA, performed for the first time on DESI-MS fused data, allowed unequivocal discrimination between oocytes and blastocysts based on specific lipid profiles. This analytical approach resulted in broad and detailed lipid annotation of single oocytes and blastocysts. Results of DESI-MS and transcript regulation analysis demonstrate that blastocysts produced in vitro and their in vivo counterparts differed significantly in the homeostasis of cholesterol and FFA metabolism. These results should assist in the production of viable and healthy embryos by elucidating in vivo embryonic lipid metabolism.

  3. Culture of bovine embryos in polyester mesh sections: the effect of pore size and oxygen tension on in vitro development.

    Science.gov (United States)

    Somfai, T; Inaba, Y; Aikawa, Y; Ohtake, M; Kobayashi, S; Akai, T; Hattori, H; Konishi, K; Imai, K

    2010-12-01

    The purpose of this study was to assess the feasibility of polyester mesh culture for the in vitro production of bovine embryos, as polyester mesh is an alternative way for tracking individual embryos throughout culture using time-lapse cinematography (TLC). Bovine embryos were isolated during in vitro culture using sections of three different polyethylene terephthalate (PET) mesh products. In vitro matured and fertilized bovine oocytes were cultured in the 217 × 217, 230 × 230 or 238 × 238-μm openings of PET mesh sections or in simple micro-drops (control) for 7 days under either 20% or 5% O(2) tensions. No difference in embryo developmental rates was found between the culture groups in terms of cleavage, blastocyst formation and blastocyst expansion irrespective of O(2) tension. In contrast, under 20% O(2) tension, blastocysts that developed in PET mesh with 217 × 217-μm opening had significantly higher numbers of total and trophectoderm (TE) cells than control embryos; however, the numbers and proportions of inner cell mass (ICM) cells did not differ. Under 5% O(2) tension, no difference was found among the culture groups in the numbers of total, ICM and TE cells in embryos. All three PET mesh products investigated in this study were proven to be effective to prevent embryo movement. The results demonstrate that bovine embryos can be cultured in PET mesh sections without negative side-effects and suggest that embryo distance determined by the mesh affects embryo quality at atmospheric oxygen tension. Polyethylene terephthalate mesh with 217 × 217-μm openings was found to be the most suitable for further application in TLC. PMID:19845884

  4. 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

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

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    Pera Renee

    2010-01-01

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

  6. 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

  7. Scrambled and fried: Cigarette smoke exposure causes antral follicle destruction and oocyte dysfunction through oxidative stress

    International Nuclear Information System (INIS)

    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

  8. 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.

  9. In-vitro maturation and cryopreservation of oocytes at the time of oophorectomy

    Directory of Open Access Journals (Sweden)

    Melanie L. Walls

    2015-08-01

    Full Text Available A 27 year old female presented for fertility preservation prior to undergoing pelvic radiotherapy. She had previously undergone a radical laparoscopic hysterectomy for cervical carcinoma seven months earlier. A trans-vaginal oocyte aspiration was not advisable due to a vaginal recurrence of the disease. Due to a polycystic ovarian morphology (PCO, follicle stimulating hormone (FSH priming with no human chorionic gonadotrophin (hCG trigger was performed prior to oophorectomy followed by ex-vivo oocyte aspiration and in vitro maturation (IVM. All visualized follicles were punctured and follicular fluid aspirated. There were 22 immature oocytes identified and placed into maturation culture for 24 h. After this time, 15 oocytes were deemed to be mature and suitable for vitrification. Following an additional 24 h in maturation culture of the remaining 7 oocytes, three more were suitable for cryopreservation. The patient recovered well and progressed to radiotherapy three days later. This report demonstrates the use of IVM treatment to store oocytes for oncology patients in time-limited circumstances.

  10. Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes.

    Science.gov (United States)

    Vanhoutte, Leen; Nogueira, Daniela; Gerris, Jan; Dhont, Marc; De Sutter, Petra

    2008-06-01

    The present study aimed to analyze detailed morphological and functional characteristics of mouse in vitro matured oocytes after a pre-maturation culture (PMC) by temporary nuclear arrest with the specific phosphodiesterase 3-inhibitor (PDE3-I) Cilostamide. In a first experiment the lowest effective dose of Cilostamide was determined. Cumulus-oocyte complexes (COCs), isolated from small antral follicles, were exposed to different concentrations of Cilostamide (ranging from 0 (control) to 10 microM) for 24 hr. Afterwards, oocytes were removed from PDE3-I-containing medium and underwent in vitro maturation (IVM) for 16-18 hr. A concentration of 1 microM Cilostamide was the lowest effective dose for maximum level of inhibition and reversibility of meiosis inhibition. This concentration was used in further experiments to evaluate oocyte quality following IVM in relation to different parameters: kinetics of meiotic progression, metaphase II (MII) spindle morphology, aneuploidy rate, fertilization, and embryonic developmental rates. The results were compared to nonarrested (in vitro control) and in vivo matured oocytes (in vivo control). Following withdrawal of the inhibitor, the progression of meiosis was more synchronous and accelerated in arrested when compared to nonarrested oocytes. A PMC resulted in a significant increase in the number of oocytes constituting a MII spindle of normal morphology. None of the oocytes exposed to PDE3-I showed numerical chromosome alterations. In addition, fertilization and embryonic developmental rates were higher in the PMC group compared to in vitro controls, but lower than in vivo controls. These results provide evidence that induced nuclear arrest by PDE3-I is a safe and reliable method to improve oocyte quality after IVM.

  11. A simple mathematical model of the bovine estrous cycle: Follicle development and endocrine interactions

    NARCIS (Netherlands)

    Boer, H.M.T.; Stötzel, C.; Röblitz, S.; Deuflhard, P.; Veerkamp, R.F.; Woelders, H.

    2011-01-01

    Bovine fertility is the subject of extensive research in animal sciences, especially because fertility of dairy cows has declined during the last decades. The regulation of estrus is controlled by the complex interplay of various organs and hormones. Mathematical modeling of the bovine estrous cycle

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  14. Maturation and fertilisation of sheep oocytes cultured in serum-free medium containing silk protein sericin.

    Science.gov (United States)

    Yasmin, Cut; Otoi, Takeshige; Setiadi, Mohamad Agus; Karja, Ni Wayan Kurniani

    2015-03-01

    Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation in a maturation medium on the meiotic competence and fertilisability of sheep oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM199 supplemented with sericin at various concentrations of 0 (control), 0.1, 0.25 and 0.5%, either with or without bovine serum albumin (BSA). When the COCs were matured without BSA, the supplementation of 0.1% sericin significantly increased the rates of maturation to metaphase II and the total fertilisation of oocytes compared with the other concentrations of sericin. When the COCs were matured with BSA, the beneficial effects of 0.1% sericin supplementation on the maturation and fertilisation of oocytes were not observed. Our findings indicate that supplementation with 0.1% sericin during maturation culture may improve the nuclear maturation and fertilisability of sheep oocytes. Moreover, it may be possible to replace BSA with sericin in chemically defined media without the risk of disease transmission. PMID:25655418

  15. Antibody microarray analyses of signal transduction protein expression and phosphorylation during porcine oocyte maturation.

    Science.gov (United States)

    Pelech, Steven; Jelinkova, Lucie; Susor, Andrej; Zhang, Hong; Shi, Xiaoqing; Pavlok, Antonin; Kubelka, Michal; Kovarova, Hana

    2008-07-01

    Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.

  16. Reduced fertilization after ICSI and abnormal phospholipase C zeta presence in spermatozoa from the wobbler mouse.

    Science.gov (United States)

    Heytens, Elke; Schmitt-John, Thomas; Moser, Jakob M; Jensen, Nanna Mandøe; Soleimani, Reza; Young, Claire; Coward, Kevin; Parrington, John; De Sutter, Petra

    2010-12-01

    Failed fertilization after intracytoplasmic sperm injection (ICSI) can be due to a reduced oocyte-activation capacity caused by reduced concentrations and abnormal localization of the oocyte-activation factor phospholipase C (PLC) zeta. Patients with this condition can be helped to conceive by artificial activation of oocytes after ICSI with calcium ionophore (assisted oocyte activation; AOA). However some concern still exists about this approach. Mouse models could help to identify potential oocyte-activation strategies and evaluate their safety. In this study, the fertilizing capacity of wobbler sperm cells was tested and the efficiency of AOA with two exposures to ionomycin to restore fertilization and embryo development was studied. The quality of the obtained blastocysts was assessed and embryo transfer was performed to evaluate post-implantation development. The presence of PLCzeta in the spermatozoa and testis of the wobbler mouse was evaluated by PLCzeta immunostaining and quantitative reverse-transcription polymerase chain reaction. Sperm cells from wobbler mice had reduced fertilizing capacity and abnormalities in PLCzeta localization, but not in its expression. Artificially activating the oocytes restored fertilization and embryo development. Therefore, the wobbler mouse can be a model for failed fertilization after ICSI to study PLCzeta dynamics and aid in optimization of the AOA method.

  17. Holding equine oocytes in a commercial embryo-holding medium: New perspective on holding temperature and maturation time.

    Science.gov (United States)

    Dini, Pouya; Bogado Pascottini, Osvaldo; Ducheyne, Kaatje; Hostens, Miel; Daels, Peter

    2016-09-15

    In the present study, we examined the effect of holding equine oocytes in Syngro embryo holding medium (EHM) overnight at either 4 °C, 17 °C, or 22 °C to 25 °C, on the time to maturation and developmental competence. We also examined the effect of placing denuded oocyte without extruded polar body back in maturation condition on subsequent maturation rate. In experiment 1, cumulus-oocyte complexes (COCs) were recovered postmortem and placed in EHM at 22 °C to 25 °C for 18 to 20 hours (OH) or placed directly in maturation (DM). The maturation rate was assessed after 22, 24, or 28 hours of culture. After denuding cumulus cells at 22 or 24 hours, oocytes without obvious polar body were placed back into culture and reassessed at subsequent time points. At 22 hours, a higher proportion of oocytes placed in OH achieved nuclear maturation than those placed in DM (63% and 37%, respectively, P = 0.008). At 24 and 28 hours, no significant differences in the % MII stage oocytes were observed between OH and DM. The nuclear maturation rate for OH oocytes was similar at 22, 24, and 28 hours, indicating that the maximum maturation rate was reached at an earlier time than that in DM. Oocytes fertilized by intracytoplasmic sperm injection resulted in a 7.1% and 6.3% blastocyst rate for OH and DM, respectively. Denuding oocytes after 22 hours or more of culture did not have an adverse effect on the final nuclear maturation rate. After 28 hours of culture, the same nuclear maturation rate (MII) was reached for nondenuded oocytes and oocytes denuded after 22 hours of 24 hours of culture. In experiment 2, COCs were held overnight at room temperature in EHM, then placed in maturation for 20, 22, and 28 hours. Nuclear maturation rate was significantly lower at 20 hours than 22 and 28 hours of culture and was similar at 22 and 28 hours, suggesting that at least 22 hours of culture is required to reach maximal maturation rate for stored oocytes (43%, 62%, and 65% at 20, 22

  18. In vitro embryo production through modification of time and gonadotropin hormone during oocytes maturation

    Directory of Open Access Journals (Sweden)

    Endang Triwulaninngsih

    2001-10-01

    Full Text Available This research has been conducted at the laboratory of in vitro fertilization of the University of Wisconsin, USA. These embryos can be used for improving genetic value of Indonesian cattle. There is transportation constraint in importing oocytes from USA. It takes more than 24 hours to bring it to Indonesia. In fact, oocytes maturation and ready to be fertilized normally requires only 24 hours in 5% CO2 incubator at 38.5°C. Therefore, this research is needed to study the effect of gonadotropin hormone and time for oocyte maturity and ready to be fertilized at a period more than 24 hours. If this problem could be solved then the importation of oocytes could be cheaper and easier than importation of life animals or embryos. Ovaries were collected from slaughterhouse in Wisconsin. Oocytes were matured in TCM-199 medium in 5% CO2 incubator and at 30°C enriched with FSH 10 μl/ml, oestradiol 17 β 1μl/ml and 10 % FCS as control of gonadotropin hormone treatment (A; with FSH 10 μl/ml (B; with oestradiol 17 β 1μl/ml (C and without gonadotropin hormone (D for 24 hours, 30 hours and 36 hours as time of maturation treatment I, II and III respectively. The oocytes were fertilized in vitro with motile sperm selection by Percoll gradient and incubation between sperm and oocytes in fertilization media (TALP for 20 hours. All zygotes were cultured in modification of KSOM medium up to blastocyst and were fed serum 5 μl/50 μl medium on day 6. Data were analyzed by SAS program. Percentage of cleavage between time of maturation were significant (p0.05. Percentage of blastocyst between time of maturation were not significant (p>0.05, but between gonadotropin hormone treatment A vs B and A vs C and B vs D and C vs D were significant (p0.05. Percentage of cleavage, morula, blastocyst, expanded blastocyst and unertilized ova on this study are 66.73%, 22.43%, 40.33%, 0.81% and 32.51 for 24 hours incubation (I; 61.55%, 25.69%, 32.69%, 0.54% and 27.61% for 30

  19. DNA double strand breaks but not interstrand crosslinks prevent progress through meiosis in fully grown mouse oocytes.

    Directory of Open Access Journals (Sweden)

    Wai Shan Yuen

    Full Text Available There is some interest in how mammalian oocytes respond to different types of DNA damage because of the increasing expectation of fertility preservation in women undergoing chemotherapy. Double strand breaks (DSBs induced by ionizing radiation and agents such as neocarzinostatin (NCS, and interstrand crosslinks (ICLs induced by alkylating agents such as mitomycin C (MMC, are toxic DNA lesions that need to be repaired for cell survival. Here we examined the effects of NCS and MMC treatment on oocytes collected from antral follicles in mice, because potentially such oocytes are readily collected from ovaries and do not need to be in vitro grown to achieve meiotic competency. We found that oocytes were sensitive to NCS, such that this ionizing radiation mimetic blocked meiosis I and caused fragmented DNA. In contrast, MMC had no impact on the completion of either meiosis I or II, even at extremely high doses. However, oocytes treated with MMC did show γ-H2AX foci and following their in vitro maturation and parthenogenetic activation the development of the subsequent embryos was severely compromised. Addition of MMC to 1-cell embryos caused a similarly poor level of development, demonstrating oocytes have eventual sensitivity to this ICL-inducing agent but this does not occur during their meiotic division. In oocytes, the association of Fanconi Anemia protein, FANCD2, with sites of ICL lesions was not apparent until entry into the embryonic cell cycle. In conclusion, meiotic maturation of oocytes is sensitive to DSBs but not ICLs. The ability of oocytes to tolerate severe ICL damage and yet complete meiosis, means that this type of DNA lesion goes unrepaired in oocytes but impacts on subsequent embryo quality.

  20. 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.

  1. In Vitro Embryo Production and Transfer of Bubaline Embryos using Oocytes Derived from Transvaginal Ultrasound-Guided Follicular Aspiration (TUFA

    Directory of Open Access Journals (Sweden)

    FP Aquino

    2014-06-01

    Full Text Available Transvaginal ultrasound-guided follicular aspiration (TUFA has become a popular tool for embryo production in vitro due to its high degree of repeatability in terms of recovering oocytes from live animals. In Study 1, the quantity and quality of oocytes from Bulgarian Murrah buffalo cows (n=10 of varying ages (Group 1, 8-12; and Group 2, 13-17 years were assessed. Group 1 buffalo donor cows yielded significantly higher (P<0.05 number of oocytes vs Group 2 buffalo donor cows (71 vs 29 oocytes, respectively, though in terms of oocyte quality, no difference was observed. In Study 2, oocytes collected (n=100 in Study 1 were matured, fertilized in vitro and the resulting zygotes were cultured which developed to blastocyst stage embryos. The maturation, fertilization and blastocyst development rates obtained were 53.0%, 40.0% and 32.5%, respectively. In Study 3, the viability of resulting blastocyst stage embryos was determined by transferring to recipient cows. Of 10 recipients 1 got pregnant and delivered a 35 kg male calf after 310 days gestation period. Overall, the results of the studies conducted demonstrated the potential of TUFA technology in the in vitro production of embryos which eventually could be used in the production of live offspring.

  2. Feed restriction as a biostimulant of the production of oocyte, their quality and GDF-9 gene expression in rabbit oocytes.

    Science.gov (United States)

    Daoud, Niveen M; Mahrous, Karima F; Ezzo, Omaima H

    2012-12-01

    The use of short-term feed restriction (R) without or with subsequent refeeding (F) as biostimulant of rabbit fertility was examined in this study. A total of 40 mature, non-pregnant, non-lactating New Zealand white female rabbits were allocated to five treatments. The rabbits were individually caged and fed a complete pelleted diet (16.7% CP; 13.1 CF; 2490kcalDE/kg). Rabbits on the control (C) treatment received 150g/d of the diet. Two groups of 8 rabbits received 70% of the control daily feed intake (105g/d; moderate restriction; M) and the other two groups received 50% of the control feed intake (75g/d; severe restriction; S) for 21d. At the end of this period, one group each of M and S fed rabbits were slaughtered for oocyte recovery. Rabbits in the remaining three groups (C, MF and SF) were retained for a further 8d before slaughter and fed the control level of the diet during this period. The effects on body weight, oocyte number and quality, GDF-9 gene expression in oocytes, and changes in serum levels of leptin and IGF-1 were recorded. Initial mean body weights were not significantly different ranging from 2.50±0.33kg (S) to 2.58±0.24kg (C). After 3wk on treatment the C rabbits were significantly heavier (2.65±0.32kg; Pgrade A oocytes recovered per ovary was significantly lower for control (3.3±0.35) than the refed treatments (MF 4.0±0.30; SF 4.5±0.39; Pgrade A oocytes had significantly lower levels of expression (1.27±0.20; P<0.05) than those of refed rabbits (MF 1.60±0.10; SF 1.39±0.01). Leptin and IGF-1 values for refed rabbits were significantly higher (P<0.05) than at the end of feed restriction and the start point. It was concluded that this biostimulant method has the potential to improve the fertility of rabbits. PMID:23084759

  3. Effect of Ca Ionophore On Blastocyst Production Following Intracytoplasmic Sperm Injection in Caprine Oocytes.

    Science.gov (United States)

    Kharche, S D; Pathak, J; Agarwal, S; Kushwah, B; Sikarwar, Aks

    2016-08-01

    The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro-matured Caprine oocytes. A total of 470 in vitro-matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48-72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro-matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm-injected in vitro-matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non-activated oocytes. PMID:27170442

  4. 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.

  5. Diagnosing cellular defects in an unexplained case of total fertilization failure.

    Science.gov (United States)

    Combelles, Catherine M H; Morozumi, Kazuto; Yanagimachi, Ryuzo; Zhu, Liben; Fox, Janis H; Racowsky, Catherine

    2010-07-01

    Despite the advent of ICSI, cases of total fertilization failure (TFF) often lead to cycle cancellation with limited diagnostic and therapeutic strategies currently available. We report on the case of an infertile couple who failed to conceive after repeated IVF and ICSI. Sperm of the husband were morphologically normal and passed a functional test assessing their ability to activate mouse oocytes. Whether oocytes were activated artificially with calcium ionophore after injection of husband's or with donor sperm, all oocytes failed to fertilize. Multiple polar bodies and two disorganized spindle structures were predominantly observed, pointing towards a cytoplasmic defect in the oocytes as the primary cause of the couple's infertility. In fact, injection of husband's sperm into donor oocytes resulted in the delivery of healthy twins. This report describes a course of action that may be applied for couples with TFF after both IVF and ICSI.

  6. In silico identification and molecular characterization of genes predominantly expressed in the fish oocyte

    Directory of Open Access Journals (Sweden)

    Mahé Sophie

    2008-10-01

    Full Text Available Abstract Background In fish, molecular mechanisms that control follicle-enclosed oocyte progression throughout oogenesis and oocyte developmental competence acquisition remain poorly understood. Existing data in mammals have indicated that the so called "oocyte-specific" genes play an important role in oogenesis, fertilization, and early embryo development. In teleost species, very little is known about "oocyte-specific" genes. The present study therefore aimed at identifying and characterizing oocyte-specific genes in fish. Results Using digital differential display PCR, mouse ESTs exhibiting an oocyte-predominant expression were identified. Those murine ESTs were subsequently used to identify cognate rainbow trout (Oncorhynchus mykiss ESTs using a reciprocal Blast search strategy. In the present study we report the identification of five previously uncharacterized rainbow trout cDNAs exhibiting a oocyte-specific, oocyte-predominant, or gonad-specific expression: zygote arrest 1 (zar1, v-mos Moloney murine sarcoma viral oncogene-like protein (mos, B-cell translocation gene (btg3, growth differentiation factor 9 (gdf9, and mutS homolog 4 (msh4. The orthology relationship of each of these genes with vertebrate counterparts was verified by phylogenetic analysis. Among those five genes, three had never been characterized in any fish species. In addition, we report the oocyte-predominant expression of btg3 for the first time in any vertebrate species. Finally, those five genes are present in unfertilized eggs as maternally-inherited mRNAs thus suggesting that they could participate in ovarian folliculogenesis as well as early embryonic development. Conclusion The expression patterns of zar1, mos, btg3, gdf9 and msh4 in rainbow trout and the functions of their orthologs in higher vertebrates strongly suggest that they might play an important role in follicle-enclosed oocyte development, meiosis control and early embryonic development in fish. Future

  7. 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.

  8. FOLLICULAR DEVELOPMENT, OOCYTE MATURITY AND FERTILIZATION IN VITRO

    Institute of Scientific and Technical Information of China (English)

    ZHANGLi-Zhu; ZHAOWen-Xin

    1989-01-01

    From February to December, 1987, 44 infertile women in 44 cycles were stiraulated by cc/hMG / hCG (39 cycles) and by ce/hCG (5 cycles) fot the purpose of IVF (37 cases with 35 transfers) and GIFT (7 cases). Intraoperative ovum pick-up was performed 32-36

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

    Directory of Open Access Journals (Sweden)

    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

  10. 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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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

  14. mRNA translation during oocyte maturation plays a key role in development of primordial germ cells in Xenopus embryos

    Indian Academy of Sciences (India)

    Bahman Zeynali; Keith E Dixon

    2004-09-01

    It is believed that cytoplasmic localization in the egg is necessary for development of primordial germ cells (PGCs) in Xenopus embryos. In this study, we sought to determine if translation of maternal mRNA during oocyte maturation is involved in the development of PGCs. Donor oocytes were collected from both stimulated (those who receive gonadotropin) and unstimulated females, artificially matured and fertilized using a host transfer technique. Using chloramphenicol (50 M and 500 M RNA), RNA translation was inhibited during oocyte maturation. Our results showed that in unstimulated embryos treated with 50 M chloramphenicol, there was a significant reduction in the number of PGCs reaching genital ridges. In stimulated embryos, however, the number of PGCs was unchanged unless a higher concentration (500 M) of chloramphenicol was used. From these results it is suggested that maternal mRNA translation during oocyte maturation plays a key role in development of PGCs.

  15. Female fertility preservation: a clinical perspective.

    Science.gov (United States)

    Pavone, Mary E; Confino, Rafael; Steinberg, Marissa

    2016-08-01

    For patients with cancer, preserving the ability to start a family at a time of their choosing is especially important and may influence decisions pertaining to cancer treatment. For other women who have delayed childbearing for personal or professional reasons, fertility preservation offers the possibility of having a biological child regardless of age. Though these women may be interested in or benefit from fertility preservation, fertility preservation services remain underutilized. While embryo and oocyte cryopreservation remain the standard strategies for female fertility preservation recommended by the American Society of Reproductive Medicine, the American Society of Clinical Oncology and the European Society of Medical Oncology, other strategies (e.g. pharmacological protection of the ovaries and ovarian tissue cryopreservation) are the subject of increasing research. This review will present new data that have become available over the past few years pertaining to all available methods of fertility preservation. PMID:26847846

  16. Distinct subtypes of zona pellucida morphology reflect canine oocyte viability and cumulus-oocyte complex quality.

    Science.gov (United States)

    Lunn, Matthew O; Wright, Shirley J

    2013-09-15

    The aim of this study was to analyze surface morphology of the zona pellucida (ZP) and assess its relationship with oocyte viability, cumulus-oocyte complex (COC) quality, and oocyte donor age in dogs. Canine ovaries were sliced to release COCs for use in three experiments. In Experiment 1, oocytes from high-quality (grade I) COCs were viewed with scanning electron microscopy to visualize the zona surface. Four zonae, classified as types I, II, III, and IV, were detectable on high-quality oocytes. Most (95.5%) dog donors had oocytes with two or three ZP types. The ZP type I had a smooth compact surface with few pores. The ZP type II was less compact with many distinct circular or elliptical pores. The ZP type III had a rough surface with folds and many irregular shaped pores and hollows. The ZP type IV also had a rough surface with folds, but in addition, stringy filaments obscured the pores and hollows. The frequency of ZP type I in the oocyte population was low (2.7%), whereas ZP types II, III, and IV each occurred in approximately one-third of the oocyte population. In Experiment 2, oocytes from high-quality COCs were stained with propidium iodide (PI) before scanning electron microscopy to investigate the relationship of oocyte viability with ZP morphology. In Experiment 3, oocytes were collected from low-quality (grade 2) and high-quality (grade 1) COCs to investigate the role of COC quality on zona structure. Zonae types I and II were characteristic of PI-positive (dead) oocytes and oocytes from low-quality COCs, whereas ZP types III and IV were prevalent on PI-negative (living) oocytes and oocytes from high-quality COCs. We concluded that the heterogeneous ZP surface underwent structural rearrangements related to oocyte viability and COC quality. This warrants further investigation into ZP structure and may be useful for canine-assisted reproduction. PMID:23790239

  17. 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.

  18. Fertility preservation options in breast cancer patients.

    Science.gov (United States)

    Kasum, Miro; von Wolff, Michael; Franulić, Daniela; Čehić, Ermin; Klepac-Pulanić, Tajana; Orešković, Slavko; Juras, Josip

    2015-01-01

    The purpose of this review is to analyse current options for fertility preservation in young women with breast cancer (BC). Considering an increasing number of BC survivors, owing to improvements in cancer treatment and delaying of childbearing, fertility preservation appears to be an important issue. Current fertility preservation options in BC survivors range from well-established standard techniques to experimental or investigational interventions. Among the standard options, random-start ovarian stimulation protocol represents a new technique, which significantly decreases the total time of the in vitro fertilisation cycle. However, in patients with oestrogen-sensitive tumours, stimulation protocols using aromatase inhibitors are currently preferred over tamoxifen regimens. Cryopreservation of embryos and oocytes are nowadays deemed the most successful techniques for fertility preservation in BC patients. GnRH agonists during chemotherapy represent an experimental method for fertility preservation due to conflicting long-term outcome results regarding its safety and efficacy. Cryopreservation of ovarian tissue, in vitro maturation of immature oocytes and other strategies are considered experimental and should only be offered within the context of a clinical trial. An early pretreatment referral to reproductive endocrinologists and oncologists should be suggested to young BC women at risk of infertility, concerning the risks and benefits of fertility preservation options.

  19. Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: A randomized controlled trial

    NARCIS (Netherlands)

    E.B. Baart (Esther); E. Martini (Elena); M.J.C. Eijkemans (René); D. van Opstal (Diane); N.G.M. Beckers (Nicole); A. Verhoeff (Arie); N.S. Macklon (Nick); B.C.J.M. Fauser (Bart)

    2007-01-01

    textabstractBACKGROUND: To test whether ovarian stimulation for in-vitro fertilization (IVF) affects oocyte quality and thus chromosome segregation behaviour during meiosis and early embryo development, preimplantation genetic screening of embryos was employed in a prospective, randomized controlled

  20. Fertilization success in marine invertebrates: the influence of gamete age.

    Science.gov (United States)

    Williams, Mark Elliott; Bentley, Matthew Graeme

    2002-02-01

    Gamete age has been postulated to be unimportant to the fertilization ecology of marine invertebrates. However, recent research suggests that, for some species at least, it may have a direct impact upon fertilization success. We present comparative data on the influence of gamete age on fertilization and development success in several marine invertebrates: the polychaetes Arenicola marina and Nereis virens and the asteroid echinoderm Asterias rubens. Oocytes are much longer lived in the polychaetes than in the echinoderm, with A. marina oocytes still capable of fertilizing and developing normally 96 h post-spawning. Developmental abnormalities and failure to reach blastula tend to occur well before the fertilizable life of the oocytes has expired. Sperm are similarly longer lived in the polychaetes; however, fertilizing capacity is markedly reduced following incubation in conspecific egg-conditioned seawater. These results are discussed in terms of the fertilization strategies of the three species. We further suggest that, for A. marina at least, longer-lived sperm and eggs are central to the fertilization strategy of this species.

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

    Directory of Open Access Journals (Sweden)

    Loro L Kujjo

    Full Text Available BACKGROUND: Therapeutic approaches to preserve fertility in females undergoing cancer treatments are currently ineffective. This is partly due to limited knowledge of the molecular mechanisms that injured germ cells elicit to repair damage and survive or to abort repair and activate biochemical pathways leading to death. So far, we know that following spontaneously occurring or drug-induced DNA damage, the efficiency of DNA repair is a critical determinant of the cell's fate. The protein encoded by the Rad51 gene is one of several components recruited for homologous recombination-dependent DNA double-strand break repair in both somatic cells and germ cells. Recently, we showed that microinjection of recombinant Rad51 into AKR/J mouse oocytes decreased the extent of spontaneous DNA double-strand breaks, suppressed apoptosis, and restored the developmental competence in AKR/J embryos. Herein we characterized the nature of chemotherapy-induced lesions in oocytes, and the associated individual components of the DNA damage sensor and repair apparatus. For comparison, we also assessed parallel spontaneous changes in aging oocytes. METHODS: Data collected were derived from: analysis of apoptosis; immunodepletion; oocyte microinjections; immunocytochemistry; immunofluorescence; and CHIP-like assays. RESULTS: Our data show that: (i DNA damage in oocytes can be induced by both chemotherapy and spontaneously by the aging process; (ii oocytes possess the machinery and capability for repairing such DNA damage; (iii Rad51 is a critical player in the repair of both chemotherapy-induced and spontaneously-sustained DNA damage; and (iv in response to damage, oocytes exhibit an inverse functional relationship between presence of Bax and activity of Rad51. CONCLUSION/SIGNIFICANCE: Our results establish Rad51 and/or Bax as potential candidates that can be targeted for development of individualized chemotherapeutic interventions that are effective, but minimal in

  2. Driving folliculogenesis by the oocyte-somatic cell dialog: Lessons from genetic models.

    Science.gov (United States)

    Monniaux, Danielle

    2016-07-01

    This review focuses on the role of the dialog between the oocyte and its companion somatic cells in driving folliculogenesis from the primordial to the preovulatory follicle stage. Mouse and sheep genetic models have brought complementary evidence of these cell interactions and their consequences for ovarian function. In mouse, the deletion of genes encoding connexins has shown that functional gap junction channels between oocytes and granulosa cells and between granulosa cells themselves maintain the follicle in a functionally integrated state. Targeted deletions in oocytes or granulosa cells have revealed the cell- and stage-specific role of ubiquist factors belonging to the phosphatidylinositol 3 kinase signaling pathway in primordial follicle activation, oocyte growth and follicle survival. Various models of transgenic mice and sheep carrying natural loss-of-function mutations associated with sterility have established that the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor 9 orchestrate follicle development, support cumulus metabolism and maturation and participate in oocyte meiosis arrest. Unexpectedly in sheep, mutations resulting in the attenuation of BMP signaling lead to enhanced ovulation rate, likely resulting from a lowered follicular atresia rate and the enhancement of FSH-regulated follicular maturation. Both the activation level of BMP signaling and an adequate equilibrium between BMP15 and growth differentiation factor 9 determine follicle survival, maturation, and development toward ovulation. The physiological approaches which were implemented on genetic animal models during the last 20 years have opened up new perspectives for female fertility by identifying the main signaling pathways of the oocyte-somatic cell dialog. PMID:27155734

  3. In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine.

    Science.gov (United States)

    Crocomo, L F; Ariu, F; Bogliolo, L; Bebbere, D; Ledda, S; Bicudo, S D

    2016-04-01

    The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos. PMID:26890275

  4. Ultrastructural evaluation of recurrent and in-vitro maturation resistant metaphase I arrested oocytes.

    Science.gov (United States)

    Windt, M L; Coetzee, K; Kruger, T F; Marino, H; Kitshoff, M S; Sousa, M

    2001-11-01

    An infertile couple whose female partner showed recurrent retrieval of immature metaphase I (MI) oocytes that were resistant to in-vitro maturation, was studied. Four spermiograms revealed teratozoospermia. Consistent non-fertilization and negative pregnancy outcomes were obtained after intrauterine insemination, gamete intra-Fallopian transfer and IVF. Two intracytoplasmic sperm injection (ICSI) cycles were finally performed. All oocytes (n = 17) in both cycles were arrested at MI and failed to mature after 48 h culture. ICSI also resulted in total non-fertilization. In the last cycle, two oocytes were analysed by transmission electron microscopy and showed almost identical results. All organelles showed normal characteristics of an MI oocyte. The main abnormality found was related to the MI spindle, with absence of microtubules and dispersion of the female chromosomes. Minor abnormalities were observed (immature fibrous appearance of the zona pellucida; the presence of small vesicle aggregates which formed a foam-like body). The injected sperm nucleus was arrested in the middle of the chromatin decondensation process, with no visible nuclear envelope reformation. Normal disruption of sperm acrosomal and flagellar components were observed. Only a partial cortical reaction was observed. This represents the first documented case of developmental arrest due to complete absence of spindle formation in association with an otherwise mature ooplasm. PMID:11679527

  5. Comparisons between nulliparous heifers and cows as oocyte donors for embryo production in vitro.

    Science.gov (United States)

    Rizos, Dimitrios; Burke, Lisa; Duffy, Patrick; Wade, Mary; Mee, John F; O'Farrell, Kevin J; Macsiurtain, Mairtin; Boland, Maurice P; Lonergan, Patrick

    2005-02-01

    The aims of the present study were to compare (1) Holstein-Friesian heifers versus early postpartum lactating cows, and (2) different age categories of crossbred beef heifers versus cows, in terms of oocyte yield, morphological quality and developmental competence. Four experiments were designed to test the associated hypotheses. In Experiment 1, ovum pick up was carried out twice weekly for a period of 5 weeks on Holstein-Friesian heifers (n = 8) and early postpartum cows (n = 8). Oocytes were submitted to in vitro maturation (IVM), fertilization and culture. Significantly more follicles were punctured on the ovaries of heifers than cows (10.4 versus 7.8, P ovaries of slaughtered crossbred beef heifers (under 30 months, n = 1241) and cows (over 4 years old, n = 1125), and processed in vitro as above. No significant difference was observed between the two groups in terms of the number of aspirated follicles or oocytes recovered. A significantly higher proportion (P ovaries were separated according to age of heifer into three groups: (1) 12-18 months, (2) 19-24 months and (3) 25-30 months, and compared with cow oocytes. There was no significant difference in the blastocyst yield between the different age groups of heifers. Irrespective of heifer age, the blastocyst yield on Day 8 was significantly lower than that from cow oocytes (35.0, 35.2, 36.5 and 48.3%, respectively, P development. PMID:15629810

  6. 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.

  7. Fertility Awareness

    Science.gov (United States)

    ... Can I Help a Friend Who Cuts? Fertility Awareness KidsHealth > For Teens > Fertility Awareness Print A A A Text Size What's in ... español Monitoreo de fertilidad What Is It? Fertility awareness is a way to prevent pregnancy by not ...

  8. Preovulatory Aging In Vivo and In Vitro Affects Maturation Rates, Abundance of Selected Proteins, Histone Methylation Pattern and Spindle Integrity in Murine Oocytes.

    Science.gov (United States)

    Demond, Hannah; Trapphoff, Tom; Dankert, Deborah; Heiligentag, Martyna; Grümmer, Ruth; Horsthemke, Bernhard; Eichenlaub-Ritter, Ursula

    2016-01-01

    Delayed ovulation and delayed fertilization can lead to reduced developmental competence of the oocyte. In contrast to the consequences of postovulatory aging of the oocyte, hardly anything is known about the molecular processes occurring during oocyte maturation if ovulation is delayed (preovulatory aging). We investigated several aspects of oocyte maturation in two models of preovulatory aging: an in vitro follicle culture and an in vivo mouse model in which ovulation was postponed using the GnRH antagonist cetrorelix. Both models showed significantly reduced oocyte maturation rates after aging. Furthermore, in vitro preovulatory aging deregulated the protein abundance of the maternal effect genes Smarca4 and Nlrp5, decreased the levels of histone H3K9 trimethylation and caused major deterioration of chromosome alignment and spindle conformation. Protein abundance of YBX2, an important regulator of mRNA stability, storage and recruitment in the oocyte, was not affected by in vitro aging. In contrast, in vivo preovulatory aging led to reduction in Ybx2 transcript and YBX2 protein abundance. Taken together, preovulatory aging seems to affect various processes in the oocyte, which could explain the low maturation rates and the previously described failures in fertilization and embryonic development. PMID:27611906

  9. Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation

    Directory of Open Access Journals (Sweden)

    Karre Inga

    2012-08-01

    Full Text Available Abstract Background Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation. Methods Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6 and four days later (group 2, n = 7. The oviduct and uterine horn of one side were flushed separately and the flushing’s were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution. Results The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P Conclusions Oocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus

  10. In vitro culture of oocytes and granulosa cells collected from normal, obese, emaciated and metabolically stressed ewes.

    Science.gov (United States)

    Tripathi, S K; Farman, M; Nandi, S; Mondal, S; Gupta, Psp; Kumar, V Girish

    2016-07-01

    The present study was undertaken to investigate the oocyte morphology, its fertilizing capacity and granulosa cell functions in ewes (obese, normal, metabolic stressed and emaciated). Ewes (Ovis aries) of approximately 3 years of age (Bellary breed) from a local village were screened, chosen and categorized into a) normal b) obese but not metabolically stressed, c) Emaciated but not metabolically stressed d) Metabolically stressed based on body condition scoring and blood markers. Oocytes and granulosa cells were collected from ovaries of the ewes of all categories after slaughter and were classified into good (oocytes with more than three layers of cumulus cells and homogenous ooplasm), fair (oocytes one or two layers of cumulus cells and homogenous ooplasm) and poor (denuded oocytes or with dark ooplasm). The good and fair quality oocytes were in vitro matured and cultured with fresh semen present and the fertilization, cleavage and blastocyst development were observed. The granulosa cells were cultured for evaluation of metabolic activity by use of the MTT assay, and cell viability, cell number as well as estrogen and progesterone production were assessed. It was observed that the good and fair quality oocytes had greater metabolic activity when collected from normal and obese ewes compared with those from emaciated and metabolically stressed ewes. No significant difference was observed in oocyte quality and maturation amongst the oocytes collected from normal and obese ewes. The cleavage and blastocyst production rates were different for the various body condition classifications and when ranked were: normal>obese>metabolically stressed>emaciated. Lesser metabolic activity was observed in granulosa cells obtained from ovaries of emaciated ewes. However, no changes were observed in viability and cell number of granulosa cells obtained from ewes with the different body condition categories. Estrogen and progesterone production from cultured granulosa cells were

  11. Effects of canine serum collected from dogs at different estrous cycle stages on in vitro nuclear maturation of canine oocytes.

    Science.gov (United States)

    Oh, Hyun Ju; Fibrianto, Yuda Heru; Kim, Min Kyu; Jang, Goo; Hossein, M Shamim; Kim, Hye Jin; Kang, Sung Keun; Lee, Byeong Chun; Hwang, Woo Suk

    2005-08-01

    Canine oocytes are ovulated at prophase of the first meiotic division and undergo maturation in the distal part of the oviduct for at least 48-72 h. Because of these differences from other domestic mammals, the efficiency of in vitro maturation (IVM) of canine oocyte is very low. The present study was conducted to evaluate the effects of canine serum on IVM of canine oocytes recovered from ovaries in various reproductive states (follicular, luteal or anestrous stages). Oocytes were recovered by mincing ovaries from bitches presented for ovariohysterectomy at various stages of the estrous cycle. Heat-inactivated canine serum was prepared with blood taken from dogs at the anestrous, estrous or diestrous stage of the estrous cycle as determined by progesterone concentration and vaginal cytology. Oocytes were cultured for 72 h in tissue culture medium (TCM)-199 supplemented with 10% canine anestrous, estrous or diestrous serum or fetal bovine serum (FBS) (experiment 1), or supplemented with 0 (control), 5%, 10% or 20% canine estrous serum (experiment 2). In experiment 1, IVM of oocytes collected at the follicular stage of the estrous cycle to metaphase II (MII) stage was higher (p < 0.05) with canine estrous serum (14.2%) than with canine anestrous (5.2%) or diestrous serum (6.3%), FBS (2.2%) or in the control (2.2%). In experiment 2, oocytes collected at the follicular stage of the estrous cycle cultured in TCM-199 with 10% canine estrous serum showed a higher maturation rate to MII stage (13.5%, p < 0.05) compared with those cultured with 5% (1.3% MII) or 20% canine estrous serum (5.1% MII) or the control (2.7% MII). In conclusion, our results demonstrate that supplementing culture medium with 10% canine estrous serum improves IVM of canine follicular stage oocytes. PMID:16261767

  12. In-vitro maturation and cryopreservation of oocytes at the time of oophorectomy

    OpenAIRE

    Walls, Melanie L.; Kirsty Douglas; Ryan, John P.; Jason Tan; Roger Hart

    2015-01-01

    A 27 year old female presented for fertility preservation prior to undergoing pelvic radiotherapy. She had previously undergone a radical laparoscopic hysterectomy for cervical carcinoma seven months earlier. A trans-vaginal oocyte aspiration was not advisable due to a vaginal recurrence of the disease. Due to a polycystic ovarian morphology (PCO), follicle stimulating hormone (FSH) priming with no human chorionic gonadotrophin (hCG) trigger was performed prior to oophorectomy followed by ex-...

  13. Could oxidative stress influence the in-vitro maturation of oocytes?

    OpenAIRE

    Combelles, Catherine MH; Gupta, Sajal; Agarwal, Ashok

    2009-01-01

    In the efforts aimed at improving the quality of in-vitro-matured human oocytes, the dynamic balance and roles of pro-/antioxidants merit further consideration. In-vitro maturation (IVM) is emerging as a popular technology at the forefront of fertility treatment and preservation. However, standard in-vitro culture conditions exert oxidative stress or an imbalance between oxidants and antioxidants. Reactive oxygen species (ROS) are oxygen-derived molecules formed as intermediary products of ce...

  14. Control of Oocyte Reawakening by Kit.

    Science.gov (United States)

    Saatcioglu, Hatice Duygu; Cuevas, Ileana; Castrillon, Diego H

    2016-08-01

    In mammals, females are born with finite numbers of oocytes stockpiled as primordial follicles. Oocytes are "reawakened" via an ovarian-intrinsic process that initiates their growth. The forkhead transcription factor Foxo3 controls reawakening downstream of PI3K-AKT signaling. However, the identity of the presumptive upstream cell surface receptor controlling the PI3K-AKT-Foxo3 axis has been questioned. Here we show that the receptor tyrosine kinase Kit controls reawakening. Oocyte-specific expression of a novel constitutively-active KitD818V allele resulted in female sterility and ovarian failure due to global oocyte reawakening. To confirm this result, we engineered a novel loss-of-function allele, KitL. Kit inactivation within oocytes also led to premature ovarian failure, albeit via a contrasting phenotype. Despite normal initial complements of primordial follicles, oocytes remained dormant with arrested oocyte maturation. Foxo3 protein localization in the nucleus versus cytoplasm explained both mutant phenotypes. These genetic studies provide formal genetic proof that Kit controls oocyte reawakening, focusing future investigations into the causes of primary ovarian insufficiency and ovarian aging. PMID:27500836

  15. In vitro fertility rate of 129 strain is improved by Buserelin (GnRH) administration prior to superovulation

    OpenAIRE

    Vasudevan, K; Sztein, J M

    2012-01-01

    The 129 mice are well recognized for their low fertility and it is speculated that this lack of fertility may be due to oocyte condition. In this study we investigated superovulation regimens for 129S1/SvImJ mouse strain to improve the oocyte quality and fertility rate of in vitro fertilization (IVF). Female mice were divided into four groups based on hormone and timing of injection. Group 1 received pregnant mare serum gonatotropin (PMSG) and 48 hours later human chorionic gonadotropin (hCG)...

  16. Effect of oil overlay on inhibition potential of roscovitine in sheep cumulus-oocyte complexes.

    Science.gov (United States)

    Crocomo, L F; Marques Filho, W C; Ulian, C M V; Branchini, N S; Silva, D T; Ackermann, C L; Landim-Alvarenga, F C; Bicudo, S D

    2015-06-01

    Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.

  17. 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.

  18. Are Patients with Polycystic Ovarian Syndrome Ideal Candidates for Oocyte Donation?

    Science.gov (United States)

    Vaz, George Queiroz; Evangelista, Alessandra Viviane; Sartorio, Cassio Alessandro Paganoti; Cardoso, Maria Cecilia Almeida; Erthal, Maria Cecilia; Gallo, Paulo; Oliveira, Marco Aurelio Pinho

    2016-01-01

    Background. The use of donated oocytes for in vitro fertilization treatment in patients with ovarian failure is universally recognized. But would patients with polycystic ovarian syndrome (PCOS) be a good choice for egg donation programs? Objective. Comparing the pregnancy rates of egg receptors from donor patients diagnosed with PCOS to receptors from donors without PCOS. Design. Retrospective cohort study. Methods. A total of 234 patients who had undergone egg reception program were separated into two groups: Group I, receptors from PCOS donors (n = 36); Group II, receptors from donors without PCOS (n = 198). Medical records were reviewed and the fertilization, implantation, and pregnancy rates were calculated. Results. PCOS patients had an average of 3.23 more oocytes retrieved, but there were no differences in the number of mature oocytes that were used for donation between the groups. We also observed that the number of transferred embryos was also not significantly different, as well as the fertilization and implantation rates. The clinical pregnancy rates were not significantly different: 28% and 26% in Group I and Group II, respectively. Conclusions. Women with PCOS should not be excluded from egg donation programs. PMID:27190994

  19. Does Unilateral Oocyte Retrieval due to Transvaginally Inaccessible Ovaries, Contrary to Common Beliefs, Affect IVF/ICSI Treatment Outcomes That Much?

    Science.gov (United States)

    Olgan, Safak; Mumusoglu, Sezcan; Bozdag, Gurkan

    2016-01-01

    Objective. To investigate in vitro fertilization (IVF) treatment outcomes of unilateral oocyte retrieval in patients with transvaginally inaccessible ovaries. Study Design. Ninety-two women who underwent unilateral oocyte retrieval were retrospectively matched for age, antral follicle count, and body mass index with 184 women who underwent bilateral oocyte retrieval. Each patient in bilateral oocyte retrieval group had the same number of cumulus oophorus complexes (COCs) from single ovary and had comparable number of follicles (±2) on contralateral site where follicular aspiration was performed. Results. The number of COCs, metaphase-2 oocytes, 2-pronuclei, and top-quality embryos was significantly lower in unilateral oocyte retrieval group. However, proportion of patients with an embryo transfer of at least one top-quality embryo was found to be comparable between unilateral and bilateral oocyte retrieval. Subsequently, clinical pregnancy and live birth rates were found to be similar between the groups. The ROC curve analysis revealed (AUC = 0.74, 95% CI 0.63–0.86, p = 0.001) that retrieved COCs ≥ 5 from single ovary had sensitivity of 76.0% and specificity of 64.2% for occurrence of a clinical pregnancy. Conclusion. The patients with unilateral oocyte retrieval have reasonable chance of success with IVF. The retrieval of ≥5 COCs from accessible ovary might result in better treatment outcomes among these patients. PMID:27123444

  20. Does Unilateral Oocyte Retrieval due to Transvaginally Inaccessible Ovaries, Contrary to Common Beliefs, Affect IVF/ICSI Treatment Outcomes That Much?

    Directory of Open Access Journals (Sweden)

    Safak Olgan

    2016-01-01

    Full Text Available Objective. To investigate in vitro fertilization (IVF treatment outcomes of unilateral oocyte retrieval in patients with transvaginally inaccessible ovaries. Study Design. Ninety-two women who underwent unilateral oocyte retrieval were retrospectively matched for age, antral follicle count, and body mass index with 184 women who underwent bilateral oocyte retrieval. Each patient in bilateral oocyte retrieval group had the same number of cumulus oophorus complexes (COCs from single ovary and had comparable number of follicles (±2 on contralateral site where follicular aspiration was performed. Results. The number of COCs, metaphase-2 oocytes, 2-pronuclei, and top-quality embryos was significantly lower in unilateral oocyte retrieval group. However, proportion of patients with an embryo transfer of at least one top-quality embryo was found to be comparable between unilateral and bilateral oocyte retrieval. Subsequently, clinical pregnancy and live birth rates were found to be similar between the groups. The ROC curve analysis revealed (AUC = 0.74, 95% CI 0.63–0.86, p=0.001 that retrieved COCs ≥ 5 from single ovary had sensitivity of 76.0% and specificity of 64.2% for occurrence of a clinical pregnancy. Conclusion. The patients with unilateral oocyte retrieval have reasonable chance of success with IVF. The retrieval of ≥5 COCs from accessible ovary might result in better treatment outcomes among these patients.

  1. Development of an in vitro test battery for assessing chemical effects on bovine germ cells under the ReProTect umbrella

    International Nuclear Information System (INIS)

    Current European legislation for the registration and authorisation of chemicals (REACH) will require a dramatic increase in the use of animals for reproductive toxicity testing. Since one objective of REACH is to use vertebrates only as last resort, the development and validation of alternative methods is urgently needed. For this purpose ReProTect, an integrated research project funded by the European Union, joining together 33 partners with complementary expertise in reproductive toxicology, was designed. The study presented here describes a battery of two tests developed within ReProTect. The objective of these tests is the detection of chemical effects during the processes of oocyte maturation and fertilisation in a bovine model. The corresponding toxicological endpoints are the reaching of metaphase II and the formation of the pronuclei respectively. Fifteen chemicals have been tested (Benzo[a]pyrene, Busulfan, Butylparaben, Cadmium Chloride, Carbendazim, Cycloheximide, Diethylstilbestrol, Genistein, Ionomycin, Ketoconazole, Lindane, Methylacetoacetate, Mifepristone, Nocodazole and DMSO as solvent) demonstrating high intra-laboratory reproducibility of the tests. Furthermore, the responses obtained in both tests, for several substances, had a good correlation with the available in vivo and in vitro data. These tests therefore, could predictably become part of an integrated testing strategy that combines the bovine models with additional in vitro tests, in order to predict chemical hazards on mammalian fertility

  2. Fator de crescimento derivado das plaquetas, retinol e insulina na regulação da maturação nuclear de oócitos bovinos e suas conseqüências no desenvolvimento embrionário Platelet-derived growth factor, retinol and insulin in the regulation of bovine oocyte nuclear maturation and their consequent effect in the embryonic development

    Directory of Open Access Journals (Sweden)

    E.B. Bortolotto

    2001-04-01

    Full Text Available O objetivo deste trabalho foi verificar as ações do fator de crescimento derivado das plaquetas (PDGF; P, da insulina (I, do retinol (R e de suas associações (PI, PIR, IR e PR na maturação nuclear (MN de oócitos bovinos e suas conseqüências no desenvolvimento embrionário (DE. O meio básico para maturação dos oócitos nos diferentes tratamentos foi o TCM-199 modificado acrescido de PVA (controle. No DE, foram utilizados os grupos R, PIR, IR, um controle negativo (PVA e um controle positivo, contendo soro fetal bovino e gonadotrofinas (SFBHOR. Os fatores P, I, R e suas associações não aceleraram a MN em 7h mas sim após 18h (PThe aim of the present study was to determine the effect of platelet-derived growth factor (PDGF; P, insulin (I retinol, (R and their interactions (PI, PIR, IR and PR on oocyte nuclear maturation (NM and, consequent, embryonic development (ED. The basic medium for oocyte maturation in the treatments was the modified TCM-199, supplemented with PVA (control. To study the embryonic development, the oocytes were divided in three treatments, R, PIR e IR, a negative (PVA and a positive control group (containing calf fetal serum and gonadotrophic hormones; FCSHOR. The PDGF, insulin, retinol and their interactions did not change the kinetic of the NM, in seven hours of culture (P=0.4492 but it changed after 18 hours of maturation (P<0.001 except in the treatments R and PR (P<0.001, in which the percentages of metaphase II were, respectively, 4.7% and 8.3%. These results were similar to the control group (0.0%. Considering a significant level of P<0.0001 in comparison to the control group, the higher rates of metaphase II were obtained in the presence of IR (19.0% and PIR (21.3%. The higher rates of MII were observed when the oocytes were matured in the presence of insulin and retinol. In the embryonic development, R (18.3%, PIR (13.9% and IR (10.6% increased the rate of cleavage when compared to PVA group (0.0%; P<0

  3. Antiviral effects of bovine interferons on bovine respiratory tract viruses.

    OpenAIRE

    Fulton, R W; Downing, M M; Cummins, J M

    1984-01-01

    The antiviral effects of bovine interferons on the replication of bovine respiratory tract viruses were studied. Bovine turbinate monolayer cultures were treated with bovine interferons and challenged with several bovine herpesvirus 1 strains, bovine viral diarrhea virus, parainfluenza type 3 virus, goat respiratory syncytial virus, bovine respiratory syncytial virus, bovine adenovirus type 7, or vesicular stomatitis virus. Treatment with bovine interferons reduced viral yield for each of the...

  4. Effect of gibberellic acid on the quality of sperm and in vitro fertilization outcome in adult male rats

    Directory of Open Access Journals (Sweden)

    Mohammadreza Hosseinchi

    2014-12-01

    Full Text Available Gibberellic acid (GA3 is a group of plant hormones identified in various plants. The aim of this study was to determine the effects of GA3 on sperm parameters and in vitro fertilization (IVF. Fifty six adult male rats were divided into seven groups as, control, treatment and sham. Following 15, 30 and 45 days of GA3 and methanol alcohol (MA administration, rats were euthanized and epididymis tail was transferred to human tubular fluid (HTF medium containing 4 mg mL-1 bovine serum albumin (BSA .Total number of sperms, the percentage of live sperms, immature sperms and sperms with damaged chromatin and IVF were examined. The oocytes were obtained from immature rats after the injection of pregnant mare's serum (PMSG and human chorionic gonadotropin (HCG hormones. Human tubular fluid was used as the fertilization medium and zygotes transferred to fresh 1-cell rat embryos culture medium (mR1ECM to reach the blastocyst stage. This study showed that GA3 could decrease the number of total sperms on days 30 and 45 in treated group comparison with the control and sham groups. Additionally, GA3 increased the immature sperms and sperms with damaged chromatin. The percentage of fertilization, two-cell embryos and blastocyst resulting from the treatment group on days 30 and 45 also decreased and showed significant differences with the control and sham groups (p < 0.05. The results obtained from this study indicated that the oral use of GA3 could reduce the fertility in rats by influencing the sperm number and the quality of sperm’s chromatins.

  5. A Role of Lipid Metabolism during Cumulus-Oocyte Complex Maturation: Impact of Lipid Modulators to Improve Embryo Production

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    E. G. Prates

    2014-01-01

    Full Text Available Oocyte intracellular lipids are mainly stored in lipid droplets (LD providing energy for proper growth and development. Lipids are also important signalling molecules involved in the regulatory mechanisms of maturation and hence in oocyte competence acquisition. Recent studies show that LD are highly dynamic organelles. They change their shape, volume, and location within the ooplasm as well as their interaction with other organelles during the maturation process. The droplets high lipid content has been correlated with impaired oocyte developmental competence and low cryosurvival. Yet the underlying mechanisms are not fully understood. In particular, the lipid-rich pig oocyte might be an excellent model to understand the role of lipids and fatty acid metabolism during the mammalian oocyte maturation and their implications on subsequent monospermic fertilization and preimplantation embryo development. The possibility of using chemical molecules to modulate the lipid content of oocytes and embryos to improve cryopreservation as well as its biological effects during development is here described. Furthermore, these principles of lipid content modulation may be applied not only to germ cells and embryo cryopreservation in livestock production but also to biomedical fundamental research.

  6. Ect of Glutathion on resumpsion of Meiosi in-vitro Maturation, and development of immature Mouse oocytes

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    H. Eimani

    2005-01-01

    Full Text Available Background and purpose : In this study the effects of glutathion on resumption of meiosis, in-vitro maturation and development of immature mouse oocytes was evaluated.Materials and methods : Immature oocytes recoverd from NMRI mouse strain (4-6 weeks in four different experimental groups. Group 1 (control group: 345 immature oocytes were matured in MEM-α medium containing FCS 5%, group 1: 338 immature oocytes were cultured in MEM-α medium containing FCS 5% and 1mM glutathione, group 2: 237 immature oocytes curtured in MEM-α medium containing FCS 5%, 7.5 IuhCG, 100mIUrFSH and group 3: 327 immature oocytes were growed in MEM-α medium FCS5%, 7.5 IUhCG, 100mIUrFSH, 1mM glutathione. Fertilization and embryo development were done in T6 medium.Results : There were significant differences between in-vitro maturation rates (group 1: 59.71%, group 2: 79.28%, group 3: 52.74 and group 4: 70.22% and resumpsion of meiosis (group 1: 74.20%, group 2: 92.88%, group 3: 74.26%, group 4: 86.20% in different groups.Conclusion : The results of this study showed that glutathione (1mM enhanced resumpsion of meiosis and in-vitro maturation and development of embryoes.

  7. 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.

  8. Rescue in vitro maturation (IVM) of immature oocytes in stimulated cycles in women with low functional ovarian reserve (LFOR).

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    Lee, Ho-Joon; Barad, David H; Kushnir, Vitaly A; Shohat-Tal, Aya; Lazzaroni-Tealdi, Emanuela; Wu, Yan-Guang; Gleicher, Norbert

    2016-04-01

    Rescue in vitro maturation (IVM) is currently not a routine procedure in association with in vitro fertilization (IVF). We compared in a prospectively cohort study of 10 patients with normal functional ovarian reserve (NFOR) and of 25 with low functional ovarian reserve (LFOR), defined by abnormally high FSH and/or abnormally low AMH levels), IVM dynamics of immature oocytes. Following controlled ovarian hyperstimulation in IVF cycles, only immature oocytes underwent rescue IVM (for up to 48 h). Oocyte maturation dynamics, fertilization rates, embryo development, and pregnancy rates were then compared between NFOR and LFOR patients. Though proportion of MI and GV oocytes reaching MII stages within 48 h and rate of maturation of MI oocytes did not differ, in women with LFOR significantly more GV oocytes reached MII stage within 24 h (30.4 vs. 66.9 %; P = 0.013), while fertilization rates and embryo generation numbers were similar between both groups. Rescue IVM, thus, produced 1.5 additional embryos for transfer in women with LFOR and 1.6 in patients with NFOR, a highly significant difference in relative improvement in available embryo numbers for LFOR (+60.0 %) and NFOR women (+16.5 %). Rescue IVM, thus, not only demonstrates different time dynamics between women with LFOR and NFOR but also disproportionate efficacy in improving available embryo numbers for transfer in favor of LFOR patients. 1/7 patients, who reached embryo transfer with only embryos produced via rescue IVF conceived and delivered, proving that rescue IVF in women with LFOR also improves pregnancy and delivery chances. Because of the small number of embryos LFOR patients produce, every additional embryo is of considerable potential clinical significance for them, suggesting that rescue IVM in women with LFOR should become routine practice. PMID:26419849

  9. An Update on Oxidative Damage to Spermatozoa and Oocytes.

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    Opuwari, Chinyerum S; Henkel, Ralf R

    2016-01-01

    On the one hand, reactive oxygen species (ROS) are mandatory mediators for essential cellular functions including the function of germ cells (oocytes and spermatozoa) and thereby the fertilization process. However, the exposure of these cells to excessive levels of oxidative stress by too high levels of ROS or too low levels of antioxidative protection will render these cells dysfunctional thereby failing the fertilization process and causing couples to be infertile. Numerous causes are responsible for the delicate bodily redox system being out of balance and causing disease and infertility. Many of these causes are modifiable such as lifestyle factors like obesity, poor nutrition, heat stress, smoking, or alcohol abuse. Possible correctable measures include foremost lifestyle changes, but also supplementation with antioxidants to scavenge excessive ROS. However, this should only be done after careful examination of the patient and establishment of the individual bodily antioxidant needs. In addition, other corrective measures include sperm separation for assisted reproductive techniques. However, these techniques have to be carried out very carefully as they, if applied wrongly, bear risks of generating ROS damaging the germ cells and preventing fertilization. PMID:26942204

  10. An Update on Oxidative Damage to Spermatozoa and Oocytes

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    Chinyerum S. Opuwari

    2016-01-01

    Full Text Available On the one hand, reactive oxygen species (ROS are mandatory mediators for essential cellular functions including the function of germ cells (oocytes and spermatozoa and thereby the fertilization process. However, the exposure of these cells to excessive levels of oxidative stress by too high levels of ROS or too low levels of antioxidative protection will render these cells dysfunctional thereby failing the fertilization process and causing couples to be infertile. Numerous causes are responsible for the delicate bodily redox system being out of balance and causing disease and infertility. Many of these causes are modifiable such as lifestyle factors like obesity, poor nutrition, heat stress, smoking, or alcohol abuse. Possible correctable measures include foremost lifestyle changes, but also supplementation with antioxidants to scavenge excessive ROS. However, this should only be done after careful examination of the patient and establishment of the individual bodily antioxidant needs. In addition, other corrective measures include sperm separation for assisted reproductive techniques. However, these techniques have to be carried out very carefully as they, if applied wrongly, bear risks of generating ROS damaging the germ cells and preventing fertilization.

  11. The Effect of Insulin-like Growth Factor-1, Cysteamine and β-Mercaptoethanol on the In Vitro Maturation of Immature Mice Oocytes

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    A Dehghan Manshadi

    2015-11-01

    Full Text Available Background & aim: In vitro maturation of oocytes is a promising technique for reducing the costs and complications of ovarian stimulation by gonadotropins. The aim of this study was to investigate the effects of combination of insulin-like growth factor-1 and antioxidant cysteamine and &beta-Mercaptoethanol on maturation and fertilization of immature oocytes. Methods: in this experimental study, following 48 hrs injection of 7.5 IU PMSG to immature female mice, the germinal vesicle oocytes from ovaries were removed and transferred to TCM199 culture medium containing 50 ng /ml insulin-like growth factor-1 and 100 &mumol Cysteamine and &beta -Mercaptoethanol. After 24 hrs of culture, the oocytes of MII in IVF were fertilized and embryonic development to the two cells was studied under an inverted microscope. Data analysis was performed by using ANOVA and Post hoc Tukey test. Results: The results showed that the rate of maturation, fertilization and 2-cell embryo formation in GV oocytes with cumulus cells in TCM199 medium containing insulin-like growth factor-1, Cysteamine and BME were 92.10, 93.30, 80.60% and in the GV oocytes without Cumulus cells were cultured in the same medium were 65.80, 64.00, 58.60% respectively which were statistically significant (P <0.001. Conclusion: In the present study, the simultaneous combination of insulin-like growth factor-1, &beta-Mercaptoethanol and CYS increased maturation, fertilization and developmental rate to 2-cells stage with cumulus cells more than the oocyte without cumulus cells to a greater extent. This represented the need of adding supplemental growth factors and antioxidants to the medium and is associated with cumulus cells.

  12. Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability.

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    Di Giacomo, Monica; Camaioni, Antonella; Klinger, Francesca G; Bonfiglio, Rita; Salustri, Antonietta

    2016-02-19

    Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability. PMID:26694612

  13. In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model.

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    Barbara Ambruosi

    Full Text Available Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl phthalate (DEHP is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM, CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL and intracellular reactive oxygen species (ROS levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05. This effect was related to increased CC apoptosis (P<0.001 and reduced ROS levels (P<0.0001. At higher doses (12 and 1200 µM, DEHP induced apoptosis (P<0.0001 and ROS increase (P<0.0001 in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity, intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05, possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into

  14. Nomogram to predict the number of oocytes retrieved in controlled ovarian stimulation

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    Moon, Kyoung Yong; Kim, Hoon; Lee, Joong Yeup; Lee, Jung Ryeol; Jee, Byung Chul; Suh, Chang Suk; Kim, Ki Chul; Lee, Won Don; Lim, Jin Ho

    2016-01-01

    Objective Ovarian reserve tests are commonly used to predict ovarian response in infertile patients undergoing ovarian stimulation. Although serum markers such as basal follicle-stimulating hormone (FSH) or random anti-Müllerian hormone (AMH) level and ultrasonographic markers (antral follicle count, AFC) are good predictors, no single test has proven to be the best predictor. In this study, we developed appropriate equations and novel nomograms to predict the number of oocytes that will be retrieved using patients' age, serum levels of basal FSH and AMH, and AFC. Methods We analyzed a database containing clinical and laboratory information of 141 stimulated in vitro fertilization (IVF) cycles performed at a university-based hospital between September 2009 and December 2013. We used generalized linear models for prediction of the number of oocytes. Results Age, basal serum FSH level, serum AMH level, and AFC were significantly related to the number of oocytes retrieved according to the univariate and multivariate analyses. The equations that predicted the number of oocytes retrieved (log scale) were as follows: model (1) 3.21–0.036×(age)+0.089×(AMH), model (2) 3.422–0.03×(age)–0.049×(FSH)+0.08×(AMH), model (3) 2.32–0.017×(age)+0.039×(AMH)+0. 03×(AFC), model (4) 2.584–0.015×(age)–0.035×(FSH)+0.038×(AMH)+0.026×(AFC). model 4 showed the best performance. On the basis of these variables, we developed nomograms to predict the number of oocytes that can be retrieved. Conclusion Our nomograms helped predict the number of oocytes retrieved in stimulated IVF cycles. PMID:27358830

  15. The extracellular matrix of porcine mature oocytes: Origin, composition and presumptive roles

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    Pivko Juraj

    2003-12-01

    Full Text Available Abstract The extracellular matrix (ECM of porcine mature oocytes was revealed by transmission electron microscopy (TEM after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS and on the surface of the zona pellucida (ZP, it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine – precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA bound to proteoglycans – for various times (with or without chase and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI and metaphase II (MII and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a

  16. Effects of IGF-I bioavailability on bovine preantral follicular development in vitro.

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    Thomas, Fiona H; Campbell, Bruce K; Armstrong, David G; Telfer, Evelyn E

    2007-06-01

    The aim of this study was to determine the effect of regulation of IGF-I bioavailability on preantral follicle development in vitro. Bovine preantral follicles were cultured for 6 days in serum-free medium with increasing doses of Long R3 (LR3) IGF-I (an analog with low affinity for IGF-binding proteins (IGFBPs)), or human recombinant IGF-I (hrIGF-I). Follicle diameter and estradiol production were measured every second day. On day 6, ratios of oocyte/follicle diameter and oocyte morphology were assessed by histological examination, and IGFBP-2 and -3 were detected by immunocytochemistry and in situ hybridization respectively. Both types of IGF-I increased follicle diameter in a dose-dependent manner (P LR3 IGF-I and the highest concentration of hrIGF-I (1000 ng/ml) had smaller oocyte/follicle ratios, and increased oocyte degeneration, compared with controls or follicles treated with physiological concentrations of hrIGF-I (P < 0.05). IGFBPs were detected in cultured preantral follicles, indicating a requirement for regulation of IGF bioavailability during the early stages of follicular development. Specifically, IGFBP-3 mRNA was found to be expressed in oocytes, and IGFBP-2 immunoreactivity was detected in oocytes and granulosa cells of cultured follicles. In summary, the regulation of IGF-I bioavailability by IGFBPs is necessary for the co-ordination of oocyte and follicle development in vitro. PMID:17636166

  17. Effect of anti-Mullerian hormone in culture medium on quality of mouse oocytes matured in vitro.

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    Yihui Zhang

    Full Text Available Anti-mullerian hormone (AMH is thought to reflect the growth of follicles and the ovarian function. However, the role of AMH in culture medium during in vitro maturation (IVM on oocyte quality and subsequent development potential is unclear. The objective of this study is to investigate the effect of recombinant human AMH (rh-AMH supplemented into IVM medium on oocyte quality. Cumulus-oocyte complexes (COCs were obtained from ICR mice and cultured in vitro with the different concentrations (0-1,000 ng/ml of rh-AMH. Following 16-18 h of culture, quantitative PCR and ELISA were performed to analyze GDF9 and BMP15 mRNA expression and protein production from the oocytes. Subsequently, in vitro fertilization (IVF and early embryonic development were employed to further evaluate the quality of in vitro matured oocytes. The results showed that AMH was only expressed in cumulus cells but not in the oocytes. However, AMH most specific receptor, AMHR-II, was expressed in both oocytes and cumulus cells. The levels of GDF9 and BMP15 expression and blastocyst formation rate were significantly increased (p<0.05 when the IVM medium was supplemented with 100 ng/ml of rh-AMH. With AdH1-SiRNA/AMH for knocking down of AMH expression during IVM significantly reduced (p<0.05 the levels of GDF9 and BMP15 expression and blastocysts formation rate. These results suggest that AHM improves oocytes quality by up-regulating GDF9 and BMP15 expressions during IVM.

  18. Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

    Science.gov (United States)

    Wu, Linda L; Russell, Darryl L; Wong, Siew L; Chen, Miaoxin; Tsai, Te-Sha; St John, Justin C; Norman, Robert J; Febbraio, Mark A; Carroll, John; Robker, Rebecca L

    2015-02-15

    Over-nutrition in females causes altered fetal growth during pregnancy and permanently programs the metabolism of offspring; however, the temporal and mechanistic origins of these changes, and whether they are reversible, are unknown. We now show that, in obese female mice, cumulus-oocyte complexes exhibit endoplasmic reticulum (ER) stress, high levels of intracellular lipid, spindle abnormalities and reduced PTX3 extracellular matrix protein production. Ovulated oocytes from obese mice contain normal levels of mitochondrial (mt) DNA but have reduced mitochondrial membrane potential and high levels of autophagy compared with oocytes from lean mice. After in vitro fertilization, the oocytes of obese female mice demonstrate reduced developmental potential and form blastocysts with reduced levels of mtDNA. Blastocysts transferred to normal weight surrogates that were then analyzed at E14.5 showed that oocytes from obese mice gave rise to fetuses that were heavier than controls and had reduced liver and kidney mtDNA content per cell, indicating that maternal obesity before conception had altered the transmission of mitochondria to offspring. Treatment of the obese females with the ER stress inhibitor salubrinal or the chaperone inducer BGP-15 before ovulation increased the amount of the mitochondrial replication factors TFAM and DRP1, and mtDNA content in oocytes. Salubrinal and BGP-15 also completely restored oocyte quality, embryo development and the mtDNA content of fetal tissue to levels equivalent to those derived from lean mice. These results demonstrate that obesity before conception imparts a legacy of mitochondrial loss in offspring that is caused by ER stress and is reversible during the final stages of oocyte development and maturation. PMID:25670793

  19. Fertility preservation in young patients with cancer

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    Virender Suhag

    2015-01-01

    Full Text Available Infertility can arise as a consequence of treatment of oncological conditions. The parallel and continued improvement in both the management of oncology and fertility cases in recent times has brought to the forefront the potential for fertility preservation in patients being treated for cancer. Many survivors will maintain their reproductive potential after the successful completion of treatment for cancer. However total body irradiation, radiation to the gonads, and certain high dose chemotherapy regimens can place women at risk for acute ovarian failure or premature menopause and men at risk for temporary or permanent azoospermia. Providing information about risk of infertility and possible interventions to maintain reproductive potential are critical for the adolescent and young adult population at the time of diagnosis. There are established means of preserving fertility before cancer treatment; specifically, sperm cryopreservation for men and in vitro fertilization and embryo cryopreservation for women. Several innovative techniques are being actively investigated, including oocyte and ovarian follicle cryopreservation, ovarian tissue transplantation, and in vitro follicle maturation, which may expand the number of fertility preservation choices for young cancer patients. Fertility preservation may also require some modification of cancer therapy; thus, patients' wishes regarding future fertility and available fertility preservation alternatives should be discussed before initiation of therapy.

  20. Fertility preservation in young cancer patients

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    Ariel Revel

    2010-01-01

    Full Text Available As a result of advances in treatment, almost 80% of children and adolescents who receive a diagnosis of cancer become long-term survivors. The increased survival rate of children and adolescents with cancer has resulted in a major interest in the long-term effects of cancer treatment on the possibility for future fertility. Currently established methods for the preservation of fertility are available only for pubertal males and females. Pubertal male cancer patients should be encouraged to freeze numerous sperm samples even when sperm count and motility are poor. In these cases, intracytoplasmic sperm injection is a powerful technique compared with intrauterine insemination since thawed sperm samples with poor parameters can produce relatively high fertilization rates resulting in normal pregnancies and deliveries. Married pubertal women should be proposed ovulation induction, follicular aspiration, and fertilization with husband sperm. Single women could benefit from vitrification of oocytes. This requires a delay of about 3 weeks in the commencement of chemotherapy to enable follicular growth. Fertility preservation for prepubertal patients is more of a problem. Young girls could be offered cryopreservation of gametes in the gonadal tissue. Cryopreservation of testicular tissue was suggested for fertility preservation for young boys, but this method is totally experimental and not currently offered. Discussing future fertility is part of the consultation of young female and male patients facing potentially gonadotoxic cancer therapy. It is the role of reproductive specialists to create various options in their laboratory to preserve fertility potential of cancer patients.

  1. Ovarian morphometric characterization and in vitro maturation of oocytes obtained from buffalo (Bubalus bubalis ovaries – partial results

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    F.C. Landim-Alvarenga

    2010-02-01

    Full Text Available Buffalo ovaries were collected from a slaughterhouse (Frigol, Brazil and transported to the laboratory in saline solution at 36º C. The ovaries were dissected to realize the evaluations (weight, length, width and height of the ovary; corpus luteum and dominant follicle diameters. The Cumulus-oocyte complexes (COCs were recovered by aspiration of 2-8 mm follicles. Selected COCs were matured in TCM 199 supplemented with 10% fetal bovine serum, sodium pyruvate, LH, FSH, estradiol and gentamicin. In vitro maturation was carried out at 38.5° C for 22-24 h and 34-36 h. For the evaluation of the nuclear maturation the oocytes were placed in TCM 199 medium added with type v hialuronidase where the granulosa cells were extracted. The denuded oocytes were transferred to 10 μl of Hoescht 33342 and the chromosomic configuration was evaluated. The oocytes were classified according to meiosis stage in: Germinal Vesicle, Germinal Vesicle Breakdown, Metaphase I, Metaphase II and Degenerated. The means of weight, length, width and height of the ovary were 3.83 g, 2.27 cm, 1.08 cm and 1.56 cm, respectively. The means of corpus luteum and dominant follicle diameters were 1.40 cm and 7.77 mm. The proportion of oocytes that reached metaphase II stage was: 36.68%.

  2. Bovine endometrial stromal cells display osteogenic properties

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    Cavirani Sandro

    2008-12-01

    Full Text Available Abstract The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.

  3. In vitro fertility rate of 129 strain is improved by Buserelin (GnRH) administration prior to superovulation

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    Vasudevan, K; Sztein, J M

    2012-01-01

    The 129 mice are well recognized for their low fertility and it is speculated that this lack of fertility may be due to oocyte condition. In this study we investigated superovulation regimens for 129S1/SvImJ mouse strain to improve the oocyte quality and fertility rate of in vitro fertilization (IVF). Female mice were divided into four groups based on hormone and timing of injection. Group 1 received pregnant mare serum gonatotropin (PMSG) and 48 hours later human chorionic gonadotropin (hCG); using the same dose, group 2 received hCG 52 hours post PMSG and group 3, 55 hours post PMSG. Group 4 received Buserelin (gonadotropin releasing hormone agonist [GnRH]) followed 24 hours later by PMSG and then hCG 55 hours post PMSG. IVF was performed using 129S1/SvImJ oocytes and sperm; C57BL/6J sperm with 129S1/SvImJ oocytes was used as fertility control. The IVF fertility rate was 1% (Groups 1 & 2), 17% (Group 3) and 55% (Group 4) for 129 oocytes fertilized with 129 sperm. For 129 oocytes fertilized with C57BL/6J sperm, the fertility rate was 5% (Group 1) 10% (Group 2) 40% (Group 3) and 59% (Group 4).-These results suggest that extending the interval time between PMSG and hCG and giving GnRH in addition to the standard PMSG and hCG treatment can improve IVF fertility rate of 129S1/SvImJ strain mice significantly. PMID:23097563

  4. In vitro fertility rate of 129 strain is improved by buserelin (gonadotropin-releasing hormone) administration prior to superovulation.

    Science.gov (United States)

    Vasudevan, K; Sztein, J M

    2012-10-01

    The 129 mice are well recognized for their low fertility and it is speculated that this lack of fertility may be due to the oocyte condition. In this study we investigated superovulation regimens for the 129S1/SvImJ mouse strain to improve the oocyte quality and fertility rate of in vitro fertilization (IVF). Female mice were divided into four groups based on hormone and timing of injection. Group 1 received pregnant mare serum gonadotropin (PMSG) and 48 h later human chorionic gonadotropin (hCG); using the same dose, group 2 received hCG 52 h post-PMSG and group 3, 55 h post-PMSG. Group 4 received buserelin (gonadotropin-releasing hormone agonist [GnRH]) followed 24 h later by PMSG and then hCG 55 h post-PMSG. IVF was performed using 129S1/SvImJ oocytes and sperm; C57BL/6J sperm with 129S1/SvImJ oocytes was used as fertility control. The IVF fertility rate was 1% (Groups 1 and 2), 17% (Group 3) and 55% (Group 4) for 129 oocytes fertilized with 129 sperm. For 129 oocytes fertilized with C57BL/6J sperm, the fertility rate was 5% (Group 1), 10% (Group 2), 40% (Group 3) and 59% (Group 4). These results suggest that extending the interval time between PMSG and hCG and giving GnRH in addition to the standard PMSG and hCG treatments can improve IVF fertility rate of 129S1/SvImJ mouse strains significantly.

  5. Fertility preservation in gynecological cancers.

    Science.gov (United States)

    Chhabra, Shakuntala; Kutchi, Imran

    2013-03-21

    For cancers of reproductive system in women, fertility preservation is complex. Fertility is also affected by therapies, however prevention is possible. Radiotherapy affects gonads, uterus, and subsequent pregnancy outcomes in all ages. However, degree and damage depend on dose, irradiation field, and age at the time of exposure. Ovarian transposition is considered if ovarian involvement is unlikely. Gonadotoxic effects of chemotherapy are related to agent's type, cumulative doses, age, and ovarian reserve. Some agents are highly toxic. Rendering follicular development quiescent by suppression of gonadotropins does reduce the ovarian damage. Simple or radical trachelectomy can be used in early cervical cancer. Fertility saving surgery is possible only in early stage low grade epithelial cancers of the ovary, however, in germ cell tumors even in advanced stages it may be possible to preserve fertility. There are no standard recommendations for endometrial cancer. Embryo, oocyte, and ovarian tissue cryopreservation are possible. The human embryo is very resistant to damage. In view of these possibilities, it is advocated that attention to long term health and quality of life in gonadotoxic therapy must be incorporated into plans as early as possible. PMID:24453519

  6. Induction of E-cadherin+ human amniotic fluid cell differentiation into oocyte-like cells via culture in medium supplemented with follicular fluid.

    Science.gov (United States)

    Liu, Te; Huang, Yongyi; Bu, Yanzhen; Zhao, Yanhui; Zou, Gang; Liu, Zhixue

    2014-07-01

    Pluripotent human amniotic fluid cells (HuAFCs) can differentiate into various types of somatic cell in vitro. However, their differentiation into oocyte-like cells has never been described to the best of our knowledge. In the present study, differentiation of E-cadherin+ and E-cadherin- HuAFC sub-populations into oocyte-like cells was induced via culture in medium containing bovine follicular fluid and β-mercaptoethanol. The E-cadherin+ HuAFCs expressed DAZL highly. Post-induction, cells with an oocyte-like phenotype were found among the E-cadherin+ HuAFCs, expressing markers specific to germ cells and oocytes (VASA, ZP3 and GDF9) and meiosis (DMC1 and SCP3). When specific small interfering RNA (siRNA) was used to suppress E-cadherin in the E-cadherin+ HuAFCs, the levels of DAZL expression were reduced. Post-induction, the morphology of the siRNA‑E‑cadherin HuAFCs was poorer and the expression levels of germ cell-specific markers were lower compared with those of the siRNA-mock HuAFCs. Therefore, E-cadherin+ HuAFCs could be more easily induced to differentiate into oocyte-like cells by bovine follicular fluid and β-mercaptoethanol. In addition, the E-cadherin+ HuAFCs exhibited potential characteristics of DAZL protein expression, and thus it was conjectured that bovine follicular fluid acts on DAZL protein and promotes E-cadherin+ HuAFC differentiation into oocyte-like cells.

  7. 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)

  8. Age-dependent radiosensitivity of mouse oocytes

    International Nuclear Information System (INIS)

    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)

  9. Effect of ionomycin on oocyte activation and embryo development in mouse.

    Science.gov (United States)

    Heytens, Elke; Soleimani, Reza; Lierman, Sylvie; De Meester, Simon; Gerris, Jan; Dhont, Marc; Van der Elst, Josiane; De Sutter, Petra

    2008-12-01

    Artificial oocyte activation using the calcium ionophore ionomycin is applied successfully in assisted reproduction but some concern exists on the clinical use. The aims of the present study were to optimize the oocyte activation scheme and to address embryo toxicity in a mouse model. Efficiency of oocyte activation and subsequent development was evaluated and ionomycin was found to be an efficient activator at 10 micromol/l. An improved effect of a second exposure to 5 micromol/l ionomycin on blastocyst development was observed. Toxicity of ionomycin on embryos was then investigated by evaluating pre- and post-implantation development of in-vivo fertilized oocytes following exposure to ionomycin. Blastocyst development, blastocyst cell numbers in trophectoderm and inner cell mass were not different between treated and non-treated zygotes. Also implantation rates and fetal parameters such as length, weight and morphological parameters were similar between the fetuses originating from zygotes treated with ionomycin and non-treated zygotes. Furthermore, healthy offspring originating from ionomycin-treated zygotes was born. In conclusion, no adverse effects of ionomycin on in-vitro or in-vivo mouse embryo development were noticed, giving arguments in favour of the use of ionomycin, although negative long-term effects of this compound cannot be excluded at present.

  10. Effect of low-frequency electromagnetic field exposure on oocyte differentiation and follicular development

    Directory of Open Access Journals (Sweden)

    L Roshangar

    2014-01-01

    Full Text Available Background: The effect of electromagnetic field (EMF as an environmental factor on different organs including female reproductive system is of critical concern. The aim of the present study is to evaluate the effect of low-frequency (LF-EMF on oocyte differentiation and follicular development. Materials and Methods: The experiment was carried out in animal lab of Faculty of Medicine Tabriz University of Medical Sciences. For this purpose, the BALB/c mice were divided into control and experimental group in animal lab. The pregnant mice in the experimental group were exposed to 3 mT EMF field, 4 h/day during the pregnancy period. The LF-EMF was produced by a system using 50 Hz alternative current, in the control group the pregnant mice were kept in a similar condition without exposure to EMF. The neonatal mice from both groups were sacrificed immediately after birth and their ovary was dissected apart and prepared for light and electron microscopy. Result : Microscopy revealed that in the experimental group, in comparison to control group, oocyte nests were mostly broken and irregularly arranged. The primordial follicles were less developed and nuclei of oocytes with an electron microscope appeared heterochromatic, shrunken and had vacuolated cytoplasm. Conclusion: It is concluded that exposure to EMF during the developmental period could affect both oocyte differentiation and folliculogenesis and may result in reduced fertility, by decreasing ovarian reservoir.

  11. How to Achieve High-Quality Oocytes? The Key Role of Myo-Inositol and Melatonin

    Science.gov (United States)

    Rossetti, Paola; Corrado, Francesco; Rapisarda, Agnese Maria Chiara; Condorelli, Rosita Angela; Valenti, Gaetano; Sapia, Fabrizio; Buscema, Massimo

    2016-01-01

    Assisted reproductive technologies (ART) have experienced growing interest from infertile patients seeking to become pregnant. The quality of oocytes plays a pivotal role in determining ART outcomes. Although many authors have studied how supplementation therapy may affect this important parameter for both in vivo and in vitro models, data are not yet robust enough to support firm conclusions. Regarding this last point, in this review our objective has been to evaluate the state of the art regarding supplementation with melatonin and myo-inositol in order to improve oocyte quality during ART. On the one hand, the antioxidant effect of melatonin is well known as being useful during ovulation and oocyte incubation, two occasions with a high level of oxidative stress. On the other hand, myo-inositol is important in cellular structure and in cellular signaling pathways. Our analysis suggests that the use of these two molecules may significantly improve the quality of oocytes and the quality of embryos: melatonin seems to raise the fertilization rate, and myo-inositol improves the pregnancy rate, although all published studies do not fully agree with these conclusions. However, previous studies have demonstrated that cotreatment improves these results compared with melatonin alone or myo-inositol alone. We recommend that further studies be performed in order to confirm these positive outcomes in routine ART treatment. PMID:27651794

  12. P63 null mutation protects mouse oocytes from radio-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Livera, G.; Petre-Lazar, B.; Guerquin, M.J.; Trautmann, E.; Coffigny, H.; Habert, R. [CEA, DSV/DRR/SEGG/LDRG, Unit Gametogenesis et Genotoxic, F-92265 Fontenay Aux Roses (France); Univ Paris 07 Denis Diderot, UFR Biol, UMR S 566, F-92265 Fontenay Aux Roses (France); INSERM, U566, F-92265 Fontenay Aux Roses (France)

    2008-07-01

    Female fertility in mammals is determined by the pool of primordial follicles and low doses of radiation induce a major loss of primordial follicles in the ovary. We investigated the expression of p53 and its homologues, p63 and p73, in the normal and irradiated neonatal ovary. p63 was the only member of the p53 family detected in oocyte nucleus. No p63 transcripts or protein were detected in the early foetal ovary. p63 production began in late pachytene-stage oocytes and peaked in diplotene oocytes in mice and humans. The production of p63 was correlated with meiotic DNA double-strand break repair. Only trans-activation (TA) isoforms were present in the ovary, with TAp63 alpha by far the most abundant in terms of mRNA and protein levels. Complete p63 null mutation did not affect normal ovary development. Irradiation rapidly triggered p63 phosphorylation. p63 null mutation prevented the cleavage of caspases-9 and -3 and the follicle loss induced by ionizing radiation. Thus, our results evidence that irradiation-induced depletion of the primordial follicle pool results from the activation of p63 in quiescent oocytes. (authors)

  13. How to Achieve High-Quality Oocytes? The Key Role of Myo-Inositol and Melatonin.

    Science.gov (United States)

    Vitale, Salvatore Giovanni; Rossetti, Paola; Corrado, Francesco; Rapisarda, Agnese Maria Chiara; La Vignera, Sandro; Condorelli, Rosita Angela; Valenti, Gaetano; Sapia, Fabrizio; Laganà, Antonio Simone; Buscema, Massimo

    2016-01-01

    Assisted reproductive technologies (ART) have experienced growing interest from infertile patients seeking to become pregnant. The quality of oocytes plays a pivotal role in determining ART outcomes. Although many authors have studied how supplementation therapy may affect this important parameter for both in vivo and in vitro models, data are not yet robust enough to support firm conclusions. Regarding this last point, in this review our objective has been to