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Sample records for bovine oocytes fertilized

  1. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization

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    Graham C. Gilchrist

    2016-03-01

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

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

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

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    MAKITA, Miho; UEDA, Mayuko; MIYANO, Takashi

    2016-01-01

    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. PMID:27151093

  4. HIGH INCIDENCE OF POLYSPERMIC FERTILIZATION IN BOVINE OOCYTES MATURED IN VITRO AFTER CRYOTOP VITRIFICATION.

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    Hwang, In-Sul; Kwon, Dae-Jin; Im, Gi-Sun; Tashima, Kazuya; Hochi, Shinichi; Hwang, Seongsoo

    2016-01-01

    Vitrification with the Cryotop device is the most promising technique for oocyte cryopreservation, but the high post-warming morphological survival of bovine oocytes does not guarantee high developmental competence after in vitro fertilization (IVF). This study was designed to examine achievement of normal fertilization in bovine oocytes vitrified-warmed with the Cryotop device. Oocytes were matured in vitro and vitrified-warmed after complete removal of the cumulus layers. Distribution of cortical granules (CGs) was assessed by Lens culinaris agglutinin (LCA) lectin staining. Ten hours after IVF, presumptive zygotes were analyzed for pronuclear formation. Day-8 blastocysts were harvested and stained with Hoechst-33342 for total cell counting. Both yield and mean cell number of the blastocysts were impaired by Cryotop vitrification. Incidence of polyspermic fertilization was three-times higher in vitrified oocytes compared to fresh oocytes. No difference in CG distribution was found between vitrified and fresh oocytes. Polyspermic fertilization induced in vitrified-warmed bovine oocytes may be one of the possible causes responsible for their low developmental potential.

  5. Oxygen tension and oocyte density during in vitro maturation affect the in vitro fertilization of bovine oocytes

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    Angelo Bertani Giotto

    2015-12-01

    Full Text Available Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20% with different oocyte densities (1:10?l or 1:20?l in the in vitro maturation (IVM of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P>0.05. In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P<0.05. Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P<0.05. In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P<0.05. Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte

  6. Role of cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival, fertilization, and blastocyst development.

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    Ortiz-Escribano, N; Smits, K; Piepers, S; Van den Abbeel, E; Woelders, H; Van Soom, A

    2016-07-15

    This study was designed to determine the role of cumulus cells during vitrification of bovine oocytes. Mature cumulus-oocyte complexes (COCs) with many layers of cumulus cells, corona radiata oocytes (CRs), with a few layers of cumulus cells, and denuded oocytes (DOs) without cumulus cells were vitrified in 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5-M sucrose. Oocytes that survived the vitrification process were fertilized. Denuded oocytes were fertilized with or without supplementation of intact COCs (DOsCOCs). First, survival and embryo development rates were studied. Higher survival rates were obtained for DOs and DOsCOCs (94% and 95%, respectively) compared with COCs (82.7%, P vitrification of mature bovine oocytes. Because cumulus cells are required for fertilization, the use of partially DOs (CRs) or the addition of intact COCs (DOsCOCs) during fertilization can result in higher survival and embryo development after vitrification. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Role of cumulus cells during vitrification and fertilization of mature bovine oocytes

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    Ortiz-Escribano, N.; Smits, K.; Piepers, S.; Abbeel, Van den E.; Woelders, H.; Soom, Van A.

    2016-01-01

    This study was designed to determine the role of cumulus cells during vitrification of bovine oocytes. Mature cumulus-oocyte complexes (COCs) with many layers of cumulus cells, corona radiata oocytes (CRs), with a few layers of cumulus cells, and denuded oocytes (DOs) without cumulus cells were

  8. Sericin accelerates the production of hyaluronan and decreases the incidence of polyspermy fertilization in bovine oocytes during in vitro maturation.

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    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% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.

  9. Microtubule assembly and in vitro development of bovine oocytes with increased intracellular glutathione level prior to vitrification and in vitro fertilization.

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    Hara, H; Yamane, I; Noto, I; Kagawa, N; Kuwayama, M; Hirabayashi, M; Hochi, S

    2014-11-01

    Although vitrification is a useful technique for preservation of bovine oocytes, the yield of blastocysts derived from the vitrified oocytes is still low. We have recently reported a new type of cryoinjury, multiple aster formation, by which pronuclear migration and development of vitrified-warmed and in vitro-fertilized bovine oocytes are impaired. The aim of the present study was to investigate the effect of glutathione (GSH) content of vitrified bovine oocytes on multiple aster formation and subsequent in vitro development. Treatment of bovine cumulus-oocyte complexes with β-mercaptoethanol (βME) and L-cysteine (Cys) during in vitro maturation resulted in 2.5-fold higher GSH content not only in fresh control but also in vitrified-warmed oocytes. The percentage of normally fertilized zygotes exhibiting sperm aster(s) was >95% in all four groups (with or without βME/Cys × fresh control or vitrified). The frequency of multiple aster formation in vitrified oocytes (three-fold higher than that in fresh control oocytes) was not affected by the increased level of intracellular GSH with βME/Cys. Consequently, the migration and development of pronuclei as well as the yield of blastocysts from vitrified-warmed oocytes (17 versus 41%) were not improved. In addition, there was no effect of increased GSH level on the yield of blastocysts in fresh control groups.

  10. In vitro oocyte fertilization and subsequent embryonic development after cryopreservation of bovine ovarian tissue, using an effective approach for oocyte collection.

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    Faheem, Marwa S; Carvalhais, I; Chaveiro, A; Moreira da Silva, F

    2011-05-01

    This study was undertaken to assess dissection/puncture combined technique for collecting large number of oocytes from bovine ovaries and to determine the effect of ovarian tissue cryopreservation on the oocytes capability to undergo in vitro maturation, fertilization and subsequent embryonic development. Ovaries (n=31) of slaughtered cows were cut into small fragments using a scalpel blade and the ovarian tissues were randomly assigned to cryopreserved by slow freezing and vitrification and non cryopreserved (fresh) groups. Oocytes were collected from non-atretic follicles from fresh and post-thawing ovarian tissue by the puncture method. The advantage of this technique appeared through morphologically good quality cumulus-oocyte complex (COC) recovery rate from fresh tissue (31.7±2.0 oocytes/ovary). However, the cryopreservation affected the post thawing total and good quality COC recovery rates from slow freezing (26.6±2.0 and 23.5±2.3 oocytes/ovary, respectively) and vitrification groups (21.7±1.1 and 17.6±1.8 oocyte/ovary, respectively). The maturation rate resulted in significant differences between the fresh tissue (94.1±1.1%) and the two cryopreservation groups. Moreover, this rate was significantly higher in the slow freezing group (80.1±1.3%) than in the vitrification group (73.0±1.9%). No statistical differences were observed in the cleavage and the embryonic developmental rates between fresh tissue group and cryopreservation groups. Furthermore the number of embryos produced per animal was statistically higher for fresh tissues than for slow freezing and the vitrification groups (34.4±1.4, 27.8±3.1 and 22.0±0.7, respectively). In conclusion, dissection method followed by puncture of bovine ovaries greatly maximizes the number of good quality oocytes recovered, as well as the number of embryos obtained per animal. Ovarian tissue can be successfully cryopreserved by slow freezing and vitrification. Copyright © 2011 Elsevier B.V. All rights

  11. Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro.

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    Coy, Pilar; Romar, Raquel; Payton, Rebecca R; McCann, Lisa; Saxton, Arnold M; Edwards, J Lannett

    2005-01-01

    The overall objective was to evaluate the effectiveness of the S-enantiomer of roscovitine (inhibitor of p34cdc2/cyclin B kinase) to maintain bovine cumulus-oocyte complexes at the germinal vesicle (GV) stage for extended times after removal from antral follicles without compromising subsequent maturation, fertilization and embryo development. Oocytes were cultured in 0, 12.5, 25 or 50 micromol/l S-roscovitine for 24 h. Hoechst staining showed that 50 micromol/l S-roscovitine maintained >90% of oocytes at the GV stage and inhibited gonadotropin-induced cumulus expansion. Fewer oocytes underwent nuclear maturation after in vitro maturation (Hoechst staining) when cultured in 50 micromol/l S-roscovitine for 66 versus 21 or 42 h. Zona pellucida (ZP) hardening (pronase resistance), cortical granule types (lens culinaris agglutinin-fluorescein isothiocyanate), nuclear maturation and fertilization with frozen-thawed spermatozoa (Hoechst staining) were assessed after culture of oocytes in 50 micromol/l S-roscovitine for 0, 24 or 48 h. Neither ZP hardening, nor nuclear maturation nor fertilization were altered by roscovitine culture for 48 h. A higher proportion of oocytes had a type III cortical granule pattern (premature translocation to the oolemma) after roscovitine culture for 48 h. However, embryo development was not compromised as cleavage, development to 8-16 cell and blastocyst stages were at least comparable in control and roscovitine-treated oocytes. In conclusion, the studies have shown that S-roscovitine reversibly maintained bovine oocytes at the GV stage for 48 h. However, maintenance of oocytes in static culture for 48 h was not sufficient to improve development above non-treated controls.

  12. Reduced competence of immature and mature oocytes vitrified by Cryotop method: assessment by in vitro fertilization and parthenogenetic activation in a bovine model.

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    Bulgarelli, Daiane L; Vireque, Alessandra A; Pitangui-Molina, Caroline P; Silva-de-Sá, Marcos F; de Sá Rosa-E-Silva, Ana Carolina J

    2017-04-01

    This study aimed to evaluate the embryo development competence, the nuclear maturation and the viability of germinal vesicle (GV) and metaphase II (MII) oocytes vitrified by the Cryotop method. Cumulus-oocyte complexes were derived from bovine ovaries and three experiments were conducted. In Experiment 1, GV oocytes were vitrified and underwent in vitro maturation (IVM) or not and their nuclear maturation was assessed by orcein staining. In Experiment 2, GV oocytes and MII oocytes were vitrified or not and the viability was assessed by calcein/ethidium homodimer-1 staining. In Experiment 3, MII oocytes matured before or after vitrification were submitted to in vitro fertilization (IVF) and parthenogenetic activation (PA) in order to evaluate embryo development. No difference was found for the nuclear maturation rate in the GV group (50%) and the GV control group (67%; P = 0.23) and for viability rate (56%; 77%; P = 0.055, respectively). However, in the MII group (27%) viability was significantly lower than that of the MII control group (84%; P vitrification by the Cryotop method reduced the capacity for embryo development. Vitrification of GV oocytes, however, did not influence the capacity of meiotic nuclear maturation and they exhibited higher viability following vitrification at the MII stage.

  13. Closed system for bovine oocyte vitrification

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    Helena Ševelová

    2012-01-01

    Full Text Available The aim of our study was to develop a vitrification carrier for bovine oocyte cryopreservation. The carrier was to be cheap enough, elementary in its construction and meet contemporary requirements for a safe closed system. In a closed system, a cell is prevented from direct exposure to liquid nitrogen, thus minimizing the risk of cross-contamination. Furthermore, two questions regarding the proper vitrification technique were resolved: if it is necessary to partially denude the oocytes before the vitrification process or whether intact cumulus oocyte complexes should be frozen; and if it is more advantageous to preheat the vitrification solutions to female body temperature (39 °C or to keep them at room temperature. Our results show that it is better to partially denude the oocytes prior to vitrification because cryopreserved intact cumulus oocyte complexes often proved dark, non-homogeneous or fragmented cytoplasm after warming, with many of them having visibly widened perivitelline spaces or fractured zonae pellucidae as a result of extensive damage during vitrification. Consequently, intact cumulus oocyte complexes showed significantly lower numbers of cleavage stage embryos on Day 3 compared to partially denuded oocytes (7.4% and 26%, respectively. On the other hand, the survival rate and following development of fertilized oocytes in preheated vitrification solution were equal to results reached at room temperature conditions. In conclusion, results achieved with the newly developed carrier were comparable to previously published studies and therefore they could be recommended for common use.

  14. 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.

  15. Effect of melatonin on in vitro maturation of bovine oocytes

    African Journals Online (AJOL)

    STORAGESEVER

    2010-04-26

    Apr 26, 2010 ... To investigate the effect of different concentrations of melatonin on bovine oocytes in vitro maturation, varying concentrations of melatonin .... solution (30 - 35°C) containing 100 IU/ml penicillin and 100 g/ml streptomycin (15140-122 .... The adverse effect will compromise fertility potential (Peter and Adashi, ...

  16. Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization, perivitelline, and intracytoplasmic sperm injections.

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    Sessions-Bresnahan, D R; Graham, J K; Carnevale, E M

    2014-07-15

    IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4',6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes

  17. Effect of melatonin on in vitro maturation of bovine oocytes ...

    African Journals Online (AJOL)

    ... Vs 17.67, 15.68, 16.53). In conclusion in this experiment, melatonin cannot improve cumulus cell expansion and nuclear maturation of bovine oocytes. When concentrations is high, melatonin may affect bovine oocytes meiotic maturation at metaphase-1 stage, but it is improbable melatonin be toxic for bovine oocytes.

  18. Assisted oocyte activation following ICSI fertilization failure.

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    Vanden Meerschaut, Frauke; Nikiforaki, Dimitra; Heindryckx, Björn; De Sutter, Petra

    2014-05-01

    The capacity of intracytoplasmic sperm injection (ICSI) to permit almost any type of spermatozoa to fertilize oocytes has made it the most successful treatment for male factor infertility. Despite its high success rates, fertilization failure following ICSI still occurs in 1-3% of couples. Assisted oocyte activation (AOA) is being increasingly applied in human assisted reproduction to restore fertilization and pregnancy rates in couples with a history of ICSI fertilization failure. However, controversy still exists mainly because the artificial activating agents do not mimic precisely the initial physiological processes of mammalian oocyte activation, which has led to safety concerns. This review addresses the mechanism of human oocyte activation and the relatively rare phenomenon of fertilization failure after ICSI. Next, it describes the current diagnostic approaches and focuses on the application, efficiency and safety of AOA in human assisted reproduction. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  19. In vitro blastocyst development of post-thaw vitrified bovine oocytes

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    D. J. Dutta,

    2013-08-01

    Full Text Available Aim: To evaluate the developmental competence of post-thaw vitrified bovine cumulus-oocyte complexes (COCs in vitro.Materials and Methods: A total of 129 COCs were cryopreserved using vitrification solution comprising of 15% ethylene glycol (EG + 15% dimethyl sulfoxide (DMSO + 0.6 M sucrose in medium TCM-199 with 10% FBS. Immediately, within a minute they are plunged into liquid nitrogen using 0.25 ml straws. Thawing was made with a step wise dilution method. Post-thaw normal vitrified and non-vitrified oocytes were subjected to in vitro maturation and in vitro fertilization.Results: Post-thaw survival percentage of vitrified oocytes was 88.37% and maturation performance of vitrified oocytes on the basis of cumulus expansion was 81.58% as compared to non-vitrified control 93.85%. The in vitro fertilization performance of vitrified oocytes was 49.46% as compared to the non-vitrified ones (63.11%. Similarly, blastocyst formation of vitrified oocytes was 21.74% as compared to 32.47% in non-vitrified oocytes.Conclusion: Vitrification of immature bovine oocytes using 7.5% EG + 7.5% DMSO for equilibration and 15% EG +15% DMSO + 0.6 M sucrose as vitrification solution yielded better in vitro fertilization and blastocyst formation rate.

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

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    Divyaswetha Peddinti

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

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

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

    2017-10-15

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

  2. 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.

  3. Age-associated potency decline in bovine oocytes is delayed by blocking extracellular Ca(2+) influx.

    Science.gov (United States)

    Zhao, Shuan; Liu, Zhen-Xing; Bao, Zhong-Jian; Wu, Yi; Wang, Kun; Yu, Guang-Min; Wang, Cui-Mei; Zeng, Shen-Ming

    2015-06-01

    Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca(2+)) concentration ([Ca(2+)]i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca(2+)]ivia blocking the influx of extracellular Ca(2+) or chelating ooplasmic free Ca(2+). After IVM, cumulus-oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca(2+)]i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus-oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca(2+)]i was significantly decreased. When oocytes were cultured in Ca(2+)-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca(2+)]i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca(2+)]i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca(2+) or reducing ooplasmic free Ca(2+). 1-Octanol, BAPTA-AM, or Ca(2+)-free medium could be used to lengthen the fertilization windows of aged bovine oocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Rescue of vitrified-warmed bovine oocytes with rho-associated coiled-coil kinase inhibitor.

    Science.gov (United States)

    Hwang, In-Sul; Hara, Hiromasa; Chung, Hak-Jae; Hirabayashi, Masumi; Hochi, Shinichi

    2013-08-01

    Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 μM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.

  5. Nuclear maturation of immature bovine oocytes after vitrification ...

    African Journals Online (AJOL)

    user

    2011-03-21

    Mar 21, 2011 ... compared for cryopreservation of immature bovine oocytes. After warming, COCs were cultured in vitro for 24 h. The polar body (PB+) and metaphase-II (MII) stage rates differed significantly among treatment groups. Oocytes vitrified using cryotop solution and device showed higher percentages of PB+ (36 ...

  6. Nuclear maturation of immature bovine oocytes after vitrification ...

    African Journals Online (AJOL)

    In the second experiment, effectiveness of both vitrification methods was compared for cryopreservation of immature bovine oocytes. After warming, COCs were cultured in vitro for 24 h. The polar body (PB+) and metaphase-II (MII) stage rates differed significantly among treatment groups. Oocytes vitrified using cryotop ...

  7. PTK2b function during fertilization of the mouse oocyte

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-01

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

  8. In vitro maturation and embryo development of bovine oocytes after meiosis blockage with MPF inhibitors

    Directory of Open Access Journals (Sweden)

    Mariana Groke Marques

    2011-12-01

    Full Text Available This study evaluated the bovine oocyte maturation and embryo development after in vitro fertilization. The maturation of the oocytes was blocked using Butyrolactone I and Roscovitine using pre-maturation medium supplemented with fetal calf serum (FCS. The ocytes were divided in four groups: Control 0 hour, Control (24 hours of maturation, Roscovitine (maturation blockage with 50mM Roscovitine during 24 hours followed by 24 hours of maturation, and Butyrolactone I (maturation blockage with 150mM Butyrolactone I during 24 hours followed by 24 hours of maturation. The oocytes were fixed and stained with aceto orcein to evaluate the nuclear maturation. After the maturation period, the remaining oocytes of the Control group, Roscovitine, and Butyrolactone I were fertilized in vitro. Embryo development was assessed by the cleavage rate (D3 and blastocysts formation (D7. The Butyrolactone I group had similar rates of germinal vesical stage oocytes during blockage, and Metaphase 2 after maturation, comparing to Control group at 0 hour and Control group, respectively. On the other hand, the Roscovitine group had lower rates of vesical stage oocytes during blockage, and Metaphase 2 after maturation comparing to Control groups. After in vitro fertilization, higher rates of cleavage were observed in Control and Butyrolactone I groups. For the blastocyst formation rate, the Control group showed better results than Roscovitine group. In summary, Butyrolactone I group had similar results to the Control group, and for this reason, is suitable for pre-maturation of bovine oocytes using FCS. In contrast, Roscovitine group had lower oocyte maturation and embryo development.

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

    Directory of Open Access Journals (Sweden)

    Robert F. Casper

    2010-01-01

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

  10. Cryotop and development of vitrified immature bovine oocytes

    Directory of Open Access Journals (Sweden)

    H Hajarian

    2011-02-01

    Full Text Available The effectiveness of different cryodevices (open-pulled straw (OPS, electron microscopy grid (EMG, and Cryotop was evaluated for vitrification of immature bovine oocytes. Polar body, metaphase II stage (MII, survivability, and subsequent developmental rates were determined. Only oocytes with four or five layers of cumulus cells were used. Oocytes were equilibrated in two vitrification solutions - 1: 10% DMSO + 10% ethylene glycol (EG for 30-45sec and 2: 20% DMSO + 20% EG +0.5M sucrose for 25sec -, mounted on one of the cryodevices and directly plunged into liquid nitrogen for 10 days. Immature vitrified oocytes using Cryotop showed the highest rates of polar body extrusion (PB and nuclear maturity (MII; 41 and 58% respectively. Vitrified oocytes using OPS and EMG showed 26 and 32%; and 35 and 46% of PB and MII rates, respectively. The highest survivability resulted from Cryotop and EMG groups and no significant difference was found between them. Vitrified oocytes using Cryotop had the highest cleavage and blastocyst rates. All of the mean rates for vitrified immature oocytes were significantly lower than that of control group (P<0.05. The results of this study showed the superiority of Cryotop device for vitrification of immature bovine oocytes

  11. Vitrification of bovine oocytes at different meiotic stages using the Cryotop method: assessment of morphological, molecular and functional patterns.

    Science.gov (United States)

    Sprícigo, J F W; Morais, K; Ferreira, A R; Machado, G M; Gomes, A C M; Rumpf, R; Franco, M M; Dode, M A N

    2014-10-01

    This study aimed to investigate the functional, morphological and molecular patterns of bovine oocytes vitrified at different times during in vitro maturation (IVM). Four groups of oocytes were used: non-vitrified control oocytes (CG), oocytes vitrified at 0 h (V0), oocytes vitrified after 8 h of IVM (V8) and oocytes vitrified after 22 h of IVM (V22). After vitrification, the oocytes were warmed and then returned to the incubator to complete a total of 24h of IVM. To evaluate the effect of vitrification, the nuclear maturation and fertilization rates were assessed by lacmoid staining and ultrastructural electron microscopy. The cleavage and blastocyst rates were evaluated at D2, D7 and D8. The expression levels of CASP3, TP53, HDAC2, SUV39H1 and DNMT1 were investigated by RT-qPCR. The nuclear maturation, oocyte fertilization, cleavage and blastocyst rates were higher (P vitrification (P > 0.05). In conclusion, cytoplasm degeneration seems to be the most severe form of damage caused by vitrification. The use of the Cryotop method for vitrification severely reduces bovine oocyte viability regardless of whether it is performed at GV, GVBD or MII stage. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Comparison of Cryotop and micro volume air cooling methods for cryopreservation of bovine matured oocytes and blastocysts

    Science.gov (United States)

    PUNYAWAI, Kanchana; ANAKKUL, Nitira; SRIRATTANA, Kanokwan; AIKAWA, Yoshio; SANGSRITAVONG, Siwat; NAGAI, Takashi; IMAI, Kei; PARNPAI, Rangsun

    2015-01-01

    This study was designed to compare the efficiency of the Cryotop method and that of two methods that employ a micro volume air cooling (MVAC) device by analyzing the survival and development of bovine oocytes and blastocysts vitrified using each method. In experiment I, in vitro-matured (IVM) oocytes were vitrified using an MVAC device without direct contact with liquid nitrogen (LN2; MVAC group) or directly plunged into LN2 (MVAC in LN2 group). A third group of IVM oocytes was vitrified using a Cryotop device (Cryotop group). After warming, vitrified oocytes were fertilized in vitro. There were no significant differences in cleavage and blastocyst formation rates among the three vitrified groups, with the rates ranging from 53.1% to 56.6% and 20.0% to 25.5%, respectively; however, the rates were significantly lower (P vitrification of bovine IVM oocytes and IVP expanded blastocysts. PMID:26119929

  13. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development.

    Science.gov (United States)

    Morató, Roser; Izquierdo, Dolors; Paramio, Maria Teresa; Mogas, Teresa

    2008-10-01

    Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.

  14. Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming.

    Science.gov (United States)

    Chasombat, Jakkhaphan; Nagai, Takashi; Parnpai, Rangsun; Vongpralub, Thevin

    2015-10-01

    The stabilization of spindle fibersis important for successful vitrification of bovine oocytes because microtubules and other cytoskeleton fibers (CSF) can be damaged during vitrification, resulting in failure of fertilization after thawing. Docetaxel, a stabilizing agent, could potentially reduce CSF damage of bovine oocytes induced during vitrification. However, there have been no reports on the effects of docetaxel on their vitrification. Experiment 1 was conducted to investigate the effects of various doses of docetaxel (0.0, 0.05, 0.5, 5.0 and 50 μM) in preincubation medium of in vitro matured (IVM) bovine oocytes on their developmental ability after in vitro fertilization (IVF). The results show that 0.05 μM docetaxel had no adverse effect on embryo development, while docetaxel at a concentration of ⩾0.5 μM inhibited development. Experiments 2 and 3 were conducted to investigate the effects of preincubation of IVM bovine oocytes with 0.05 μM docetaxel for 30 min prior to vitrification-warming on CSF integrity (Experiment 2), and on oocyte survival and viability after IVF (Experiment 3). When preincubated with 0.05 μM docetaxel for 30 min before vitrification, post-thawed oocytes had less CSF damage and higher survival rates compared with those untreated with docetaxel before vitrification. Surviving oocytes also had higher rates of cleavage and development to the blastocyst stage after IVF. In conclusion, preincubation of IVM bovine oocytes with 0.05 μM docetaxel for 30 min before vitrification was effective at preventing CSF damage during vitrification, and improving oocyte viability after warming and subsequent cleavage and blastocyst formation after IVF. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Birth of healthy calves after intra-follicular transfer (IFOT) of slaughterhouse derived immature bovine oocytes.

    Science.gov (United States)

    Hoelker, M; Kassens, A; Salilew-Wondim, D; Sieme, H; Wrenzycki, C; Tesfaye, D; Neuhoff, C; Schellander, K; Held-Hoelker, E

    2017-07-15

    To circumvent the negative impacts of in vitro culture on bovine embryos, we have recently established a new method, the so called intra-follicular oocyte transfer (IFOT), enabling in vivo fertilization and in vivo development of in vitro matured oocytes up to the blastocyst stage as well as to term. In this study, we raised the question whether immature bovine oocytes could also be transferred into a pre-ovulatory follicle to support in vivo maturation prior to subsequent in vivo fertilization, in vivo development as well as to term. To unravel that question, a total of 791 immature oocytes were transferred in groups of ∼50 into pre-ovulatory follicles of 16 recipient heifers. Consequently, we were able to recollect a total of 306 structures 8 days thereafter (38.5%). All in all, 12 heifers (75%) gave embryos developed to the morula or blastocyst stage in addition to the expected native embryos. Among all recollected structures, 40.1% had developed to the morula and/or blastocyst stage, meaning a total efficiency of 17.3% based on all transferred oocytes. Of impact, IFOT-embryos reached significantly higher developmental rates to the Morula and/or blastocyst stage until day 7 compared to in vitro cultured control embryos, despite being derived from the same charge of slaughterhouse ovaries (40.1 vs. 29.3%). This implicates a beneficial effect of the follicular environment for the intrinsic quality of the fertilized embryos during maturation and for subsequent developmental rates up to the blastocyst stage. Finally, the birth of two healthy calves after transfer of frozen-thawed IFOT-derived blastocysts to final recipients established the first proof of principle that IFOT of immature bovine oocytes generates bovine blastocysts bearing developmental capacity to term. Likewise, to the best of our knowledge, these calves are the first calves derived from full in vivo development of immature slaughterhouse derived oocytes. Thus, the results of the present

  16. Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes.

    Directory of Open Access Journals (Sweden)

    Giulia Rusciano

    Full Text Available Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs. Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our

  17. Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes.

    Science.gov (United States)

    Rusciano, Giulia; De Canditiis, Carolina; Zito, Gianluigi; Rubessa, Marcello; Roca, Maria Serena; Carotenuto, Rosa; Sasso, Antonio; Gasparrini, Bianca

    2017-01-01

    Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental

  18. Bovine cumulus-oocyte disconnection in vitro

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul

    1987-01-01

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

  19. Oocyte surface in four teleost fish species postspawning and fertilization

    Directory of Open Access Journals (Sweden)

    Elizete Rizzo

    1998-06-01

    Full Text Available Cytological and cytochemical studies were carried out to investigate the surface characteristics of oocytes of four teleost species from the São Francisco river. The fishes were submitted to hypophysation at the Três Marias Hybrobiology and Fishculture Station, Minas Gerais, Brazil, in January 1996. Postspawning, oocytes of the curimatãs Prochilodus affinis, Prochilodus marggravii and dourado Salminus brasiliensis were surrounded by a thick, three-layered zona pellucida with radial striae. The surface of spawned oocytes of the surubim, Pseudoplatystoma coruscans, was comprised of mucous coat located externally to a thin, two-layered and striated zona pellucida. Oocyte activation during fertilization, lead to cortical reaction, formation of a perivitelline space, reduction of the thickness of the zona pellucida and increase in the oocyte diameter in the four species. Following fertilization, many spermatozoa were embedded in the mucous coat of the surubim oocytes. During embryogenesis, this later coating became thicker, diffuse and less viscous while the zona pellucida (chorion was thinner in all studied species. Cytochemical analyses indicated species-specific differences in the oocyte surface after spawning. It was suggested that the mucous coat of surubim oocytes play a functional role during fertilization. The knowledge of the morphology of the oocyte surface of teleost is important for our understanding of the interactions between their eggs and surrounding environment and may also contribute significantly to phylogenetic studies.

  20. Effect of vitrification on the mRNA transcriptome of bovine oocytes.

    Science.gov (United States)

    Wang, N; Li, C-Y; Zhu, H-B; Hao, H-S; Wang, H-Y; Yan, C-L; Zhao, S-J; Du, W-H; Wang, D; Liu, Y; Pang, Y-W; Zhao, X-M

    2017-08-01

    Vitrification has been shown to decrease the developmental capacity of mammalian oocytes, and this is closely associated with the abnormal mRNA expressions of vitrified oocytes. However, the effect of vitrification on transcriptional machinery of oocytes examined by RNA sequencing (RNA-seq) has yet to be defined. In the present study, the mRNA transcriptomes of fresh and vitrified bovine oocytes were analysed by Smart-seq2 with the differently expressed genes determined by DEseq2 (an adjusted p-value of .05 and a minimum fold change of 2). The differentially expressed mRNAs were then searched against the Gene Ontology (GO) and Genomes (KEGG) database. Finally, the mRNA expressions of 10 candidate genes were validated using quantitative real-time PCR (qRT-PCR). Approximately 12,000 genes were detected in each sample of fresh or vitrified oocytes. Of these, the expression levels of 102 genes differed significantly in vitrified groups: 12 genes mainly involved in cell cycle, fertilization and glucose metabolism were upregulated, and 90 genes mainly involved in mitochondria, ribosomal protein, cytoskeleton, transmembrane protein, cell cycle and calcium ions were downregulated. GO analysis showed that these genes were mainly enriched in terms of membrane-bounded organelles, macromolecular complex, and intracellular part. The mRNA expression levels of 10 candidate genes selected randomly were in agreement with the results of the RNA-seq. In conclusion, our results showed that vitrification affected the mRNA transcriptome of bovine oocytes by downregulating genes, which contributed to the decreased developmental capacity of vitrified oocytes. Our findings will be useful in determining approaches to improve the efficiency of vitrified oocytes. © 2017 Blackwell Verlag GmbH.

  1. Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells

    Directory of Open Access Journals (Sweden)

    S Modina

    2009-06-01

    Full Text Available The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the coolingand- freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs. The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization.

  2. The effect of vitrification of immature bovine oocytes to the subsequent in vitro development and gene expression.

    Science.gov (United States)

    Faheem, Marwa S; Baron, E; Carvalhais, I; Chaveiro, A; Pavani, K; da Silva, F Moreira

    2015-12-01

    Immature bovine oocytes were vitrified using the cryotop method and their post-warming survivability and capability to undergo in vitro maturation, fertilization and subsequent embryonic development were evaluated. In addition throughout the embryonic 2-cell, 4-cell, morula and blastocyst stages, the expression of four developmentally important genes (Cx43, CDH1, DNMT1 and HSPA14) was analysed using the real-time polymerase chain reaction (PCR). Immature oocytes (n = 550) were randomly assigned to non-vitrified (fresh) or cryotop vitrification groups using ethylene glycol (EG) with 1,2 propanediol (PROH) or dimethylsulphoxide (DMSO). After warming, oocytes survivability, embryo cleavage and embryonic developmental rates were not statistically different between the two cryoprotectants groups. However, the DMSO group had a lower (P vitrification of immature bovine oocytes, both for embryonic developmental competence and at the molecular level. Moreover, PROH showed some advantage over DMSO as a cryoprotectant.

  3. Bovine oocytes in secondary follicles grow in medium containing bovine plasma after vitrification.

    Science.gov (United States)

    Taketsuru, Hiroaki; Takajo, Asuka; Bao, Rong-Mei; Hamawaki, Atsushi; Yoshikawa, Motoichi; Miyano, Takashi

    2011-02-01

    There has been no culture system that supports the growth of bovine oocytes for more than 2 weeks. In the present study, bovine secondary follicles were cultured for 4 weeks, and the effects of supplemented protein components and FSH in the culture medium on the growth of the oocytes were examined. The effect of vitrification of secondary follicles on the subsequent oocyte growth was also examined. Secondary follicles (150 to 200 µm in diameter) containing growing oocytes (approximately 60 µm in diameter) were dissected from ovaries and cultured in a medium supplemented with FSH (0, 25 or 50 ng/ml) and one of the following four kinds of protein components: bovine serum albumin (BSA), bovine plasma (BPL), fetal calf serum (FCS) and bovine follicular fluid (BFF). In BSA- and BPL-supplemented media with 0 or 25 ng/ml FSH, more than 50% of follicles showed no degenerative signs during culture, and oocytes significantly increased in size after 4 weeks (Pbovine secondary follicles for 4 weeks, even after vitrification and warming of the follicles.

  4. Calves born after direct transfer of vitrified bovine in vitro-produced blastocysts derived from vitrified immature oocytes.

    Science.gov (United States)

    Vieira, A D; Forell, F; Feltrin, C; Rodrigues, J L

    2008-06-01

    Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super-cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non-vitrified control group, (ii) vitrified in normal (-196 degrees C) liquid nitrogen (LN(2)) and (iii) vitrified in super-cooled LN(2) (vitrification containers. Immature oocytes were in vitro-matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN(2) state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super-cooled LN(2), resulted in viable blastocysts and live calves following in vitro embryo production.

  5. PTK2b function during fertilization of the mouse oocyte.

    Science.gov (United States)

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

    2014-08-01

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

  6. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. 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.

  8. In Vitro Growth and Maturation of Vitrified-Warmed Bovine Oocytes Collected from Early Antral Follicles

    Science.gov (United States)

    HIRAO, Yuji; SOMFAI, Tamás; NARUSE, Kenji

    2013-01-01

    Abstract. Cryopreservation of growing oocytes enriches the choice of timing and location of artificial embryo production. However, completion of oocyte growth after warming is crucial when using such cryopreserved oocytes. Our research objective was to develop a sequential system that incorporates cryopreservation of growing bovine oocytes and their subsequent in vitro growth. Oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 µm were vitrified-warmed and then cultured for 14 days. The percentage of surviving oocytes following cryopreservation and 14-day culture was approximately 80%. More than half of the surviving oocytes were capable of maturing to metaphase II after in vitro maturation; the rate was comparable to that of control oocytes grown in vitro without cryopreservation. Taken together, the combined protocols for vitrification-warming of growing oocytes and subsequent in vitro growth can produce oocytes capable of undergoing meiotic maturation. PMID:24126072

  9. Characteristics of failed fertilized oocytes in patients with severe obesity

    Directory of Open Access Journals (Sweden)

    E A Pigarova

    2012-12-01

    Full Text Available Реферат по статье: Machtinger R, Combelles CM, Missmer SA, Correia KF, Fox JH, Racowsky C. The association between severe obesity and characteristics of failed fertilized oocytes. Hum Reprod. 2012 Nov;27(11:3198-207.

  10. Dynamic changes of the Golgi apparatus during bovine in vitro oocyte maturation.

    Science.gov (United States)

    Racedo, S E; Rawe, V Y; Niemann, H

    2012-04-01

    For successful fertilization by the male gamete, oocyte cytoplasmic organelles such as the Golgi apparatus have to undergo specific changes: the entire process is known as cytoplasmic maturation. The goal of this study was to unravel the dynamics of the Golgi apparatus in bovine oocytes at critical stages of in vitro maturation, i.e. germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI) and metaphase II, and to investigate the role of various molecules critically involved therein. The cytoplasmic distribution of proteins was assessed by immunocytochemistry and laser confocal microscopy. We applied specific inhibitors, including nocodazole to unravel the functional role of the microtubular elements; sodium orthovanadate, which primarily inhibits cytoplasmic dynein ATPase activity; monastrol which inhibits the kinesin EG5; and roscovitine to inhibit the kinase cyclin-dependent kinase 2A (CDC2A). Prior to GVBD, the Golgi apparatus was translocated from the centre of the cytoplasm to the cortical area in the periphery, where it underwent fragmentation. A second translocation was observed between GVBD and MI stages, when the Golgi apparatus was moved from the cortex to the centre of the cytoplasm. Incubation with the specific inhibitors revealed that microtubules played an active role in the final localization at GVBD, while CDC2A was essential for Golgi fragmentation at GVBD stage. This partitioning was a precondition for the second movement. In conclusion, for the first time we show basic mechanisms critically involved in the regulation of the dynamic changes of Golgi apparatus during meiosis of the bovine oocyte.

  11. Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes.

    Science.gov (United States)

    Suttirojpattana, Tayita; Somfai, Tamas; Matoba, Satoko; Parnpai, Rangsun; Nagai, Takashi; Geshi, Masaya

    2017-02-01

    Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non-stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence. © 2016 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.

  12. 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...... ≥6 mm always gave a higher blastocyst rate than oocytes from follicles...

  13. 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

    The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation...... 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...

  14. Ultrastructure and Development of Vitrified/Warmed Bovine Oocytes Matured with 9-cis Retinoic Acid

    OpenAIRE

    Rodríguez, Aida; Gómez, Enrique; Antolín, Isaac; Duque, Paloma; Hidalgo, C.O. (Carlos); Alonso, Cristina; Tamargo, Carolina; Fernández, Lina; Carbajo, Maite; Facal, Nieves; Caamaño, J.N. (José); Díez, Carmen

    2012-01-01

    In this work we analyze the effects of vitrification on the ultrastructure and developmental ability of bovine oocytes matured in the presence of 9-cis-retinoic acid (RA). Bovine cumulus oocyte complexes (COCs) were matured in a basic medium containing 10% fetal calf serum (FCS), 9-cis-RA or polyvinyl alcohol (PVA). Mature oocytes were vitrified using the Open Pulled Straw method with minor modifications. A group of fresh and vitrified/warmed COCs was fixed for ultrastructural analysis, wh...

  15. Alterations in transcript abundance of bovine oocytes recovered at growth and dominance phases of the first follicular wave

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    Kanitz Wilhelm

    2007-07-01

    Full Text Available Abstract Background Oocyte developmental competence is highly affected by the phase of ovarian follicular wave. Previous studies have shown that oocytes from subordinate follicles recovered at growth phase (day 3 after estrus are developmentally more competent than those recovered at dominance phase (day 7 after estrus. However, the molecular mechanisms associated with these differences are not well elucidated. Therefore, the objective of this study was to investigate transcript abundance of bovine oocytes retrieved from small follicles at growth and dominance phases of the first follicular wave and to identify candidate genes related to oocyte developmental competence using cDNA microarray. Results Comparative gene expression analysis of oocytes from growth and dominance phases and subsequent data analysis using Significant Analysis of Microarray (SAM revealed a total of 51 differentially regulated genes, including 36 with known function, 6 with unknown function and 9 novel transcripts. Real-time PCR has validated 10 transcripts revealed by microarray analysis and quantified 5 genes in cumulus cells derived from oocytes of both phases. The expression profile of 8 (80% transcripts (ANAXA2, FL396, S100A10, RPL24, PP, PTTG1, MSX1 and BMP15 was in agreement with microarray data. Transcript abundance of five candidate genes in relation to oocyte developmental competence was validated using Brilliant Cresyl Blue (BCB staining as an independent model. Furthermore, localization of mRNA and protein product of the candidate gene MSX1 in sections of ovarian follicles at days 0, 1, 3 and 7 of estrous cycle showed a clear fluorescent signal in both oocytes and cumulus cells with higher intensity in the former. Moreover, the protein product was detected in bovine oocytes and early cleavage embryos after fertilization with higher intensity around the nucleus. Conclusion This study has identified distinct sets of differentially regulated transcripts between

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

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    Kenji Ezoe

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

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

    Science.gov (United States)

    Ezoe, Kenji; Yabuuchi, Akiko; Tani, Tetsuya; Mori, Chiemi; Miki, Tetsuya; Takayama, Yuko; Beyhan, Zeki; Kato, Yoko; Okuno, Takashi; Kobayashi, Tamotsu; Kato, Keiichi

    2015-01-01

    Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming. PMID:25965267

  18. Coenzyme Q10 supplementation during in vitro maturation of bovine oocytes (Bos taurus) helps to preserve oocyte integrity after vitrification.

    Science.gov (United States)

    Ruiz-Conca, M; Vendrell, M; Sabés-Alsina, M; Mogas, T; Lopez-Bejar, M

    2017-10-01

    Oocyte vitrification causes less cell stress than slow cooling, but cytoskeletal and spindle alterations may occur affecting the oocyte competence. In vitro maturation (IVM) supplementation with different antioxidant molecules has been performed to attenuate this harmful stress. Coenzyme Q10 (CoQ10 ) supplementation has previously shown to have positive effects in bovine and mouse in vitro embryo development. The aim of this study was to evaluate the effects of CoQ10 during bovine oocyte IVM and vitrification. Cumulus-oocyte complexes (COCs) (n = 311) were cultured under standard maturation conditions with 0 μM (control), 25 μM and 50 μM CoQ10 supplementation. After 22 hr, a cohort of 170 oocytes both from the control and from CoQ10 -supplemented groups were vitrified, warmed and returned to incubation until 24 hr of maturation, while the rest of the oocytes (n = 141) remained fresh. Then, oocyte survival was assessed morphologically by stereomicroscopy. Oocytes from all groups were then fixed and stained for assessing cortical granules (CG) migration and nuclear stage. High rates of oocyte MII progression and appropriate CG migration as a continuous layer beneath the plasma membrane were obtained both in control and in CoQ10 groups. Results showed that although vitrification has great impact in survival of IVM bovine oocytes, 50 μM CoQ10 supplementation significantly improved oocyte survival (p = .045) and reduced the premature CG exocytosis, helping to preserve the CG migration pattern (31.3% control vs. 54.5% in 50 μM CoQ10 ; p = .039), attenuating the negative effects of vitrification. © 2017 Blackwell Verlag GmbH.

  19. Evaluation of different factors affecting the efficiency of oocytes cryopreservation in the bovine model

    OpenAIRE

    De Blasi, Marina

    2011-01-01

    Interest in oocyte cryopreservation has recently increased. Cattle oocytes are sensitive to low temperatures, and despite the efforts of numerous research groups cryopreservation of oocytes remains a difficult task. This problem may be in part due to the large size of bovine oocytes, which consequently have a low surface to volume ratio, making it more difficult for water and cryoprotectants (CP) to move across the cell plasma membranes. Several attempts to improve the survival rate of oocy...

  20. Protein Tyrosine Kinase Signaling During Oocyte Maturation and Fertilization

    Science.gov (United States)

    McGinnis, Lynda K.; Carroll, David J.; Kinsey, William H.

    2011-01-01

    The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans. PMID:21681843

  1. Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone.

    Science.gov (United States)

    Mota, Gustavo Bruno; Oliveira e Silva, Ingrid; de Souza, Danielle Kaiser; Tuany, Flavia; Pereira, Michele Munk; Camargo, Luiz Sergio de Almeida; Rosa e Silva, Alzira Amélia Martins

    2015-08-01

    The aim of this study was to evaluate the dose-response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus-oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus-oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.

  2. Recent cadmium exposure among male partners may affect oocyte fertilization during in vitro fertilization (IVF)

    Science.gov (United States)

    Kim, Keewan; Fujimoto, Victor Y.; Parsons, Patrick J.; Steuerwald, Amy J.; Browne, Richard W.

    2010-01-01

    Purpose We recently reported evidence suggesting associations between urine cadmium concentrations, reflecting long-term exposure, measured in 25 female patients (relative risk = 1.41, P = 0.412) and 15 of their male partners (relative risk = 0.19, P = 0.097) and oocyte fertilization in vitro. Blood cadmium concentrations reflect more recent exposure. Methods We here incorporate those measures into our prior data set and employ multivariable log-binomial regression models to generate hypotheses concerning the relative effects of long-term and recent cadmium exposure on oocyte fertilization in vitro. Results No association is indicated for blood cadmium from women and oocyte fertilization, adjusted for urine cadmium and creatinine, blood lead and mercury, age, race/ethnicity and cigarette smoking (relative risk = 0.88, P = 0.828). However, we suggest an inverse adjusted association between blood cadmium from men and oocyte fertilization (relative risk = 0.66, P = 0.143). Conclusions These results suggest that consideration of long-term and recent exposures are both important for assessing the effect of partner cadmium levels on oocyte fertilization in vitro. PMID:20508982

  3. Cytochalasin B efficiency in the cryopreservation of immature bovine oocytes by Cryotop and solid surface vitrification methods.

    Science.gov (United States)

    Sripunya, Nucharin; Liang, Yuanyuan; Panyawai, Kanchana; Srirattana, Kanokwan; Ngernsoungnern, Apichart; Ngernsoungnern, Piyada; Ketudat-Cairns, Mariena; Parnpai, Rangsun

    2014-12-01

    The present study was undertaken to compare the efficacies of Cryotop (CT), solid surface vitrification (SSV) methods and cytochalasin B (CB) treatment for the cryopreservation of immature bovine oocytes, in terms of survival, nuclear maturation, and in vitro development. Solution exposed oocytes were in vitro maturated and fertilized. No difference was found in the rates of survival, nuclear maturation and blastocyst among solution exposed groups and fresh control group, except blastocysts rates in oocytes exposed to CB, cryoprotectant (CPA) and fluorescein diacetate (FDA) group (CB-CPA-FDA) (23%) significantly lower than that of control group (32%). CB pretreated ((+)CB) or non-pretreated ((-)CB) COCs were vitrified either by SSV or CT. Among four vitrified groups the nuclear maturation rates (CT(-)CB: 58%, CT(+)CB: 57%, SSV(-)CB: 60%, SSV(+)CB: 63%), cleavage (CT(-)CB: 36%, CT(+)CB: 24%, SSV(-)CB: 34%, SSV(+)CB: 26%) and blastocysts rates (CT(-)CB: 6%, CT(+)CB: 7%, SSV(-)CB: 4%, SSV(+)CB: 6%) did not differ, but the rates of the four vitrified groups were significantly lower than those of non-vitrified group (81%, 71% and 26%, respectively). We thus conclude that CT and SSV perform equally in vitrification of bovine immature oocytes, and CB did not increase the viability, nuclear maturation, or in vitro development of vitrified oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification.

    Science.gov (United States)

    Apparicio, M; Ruggeri, E; Luvoni, G C

    2013-04-01

    The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p vitrification compared to 60.9% of those submitted to slow freezing procedure (p bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture. © 2012 Blackwell Verlag GmbH.

  5. Hyperactivation of stallion sperm is required for successful in vitro fertilization of equine oocytes.

    Science.gov (United States)

    McPartlin, L A; Suarez, S S; Czaya, C A; Hinrichs, K; Bedford-Guaus, S J

    2009-07-01

    Capacitation is a complex and not well-understood process that encompasses all the molecular changes sperm must undergo to successfully fertilize an oocyte. In vitro fertilization has remained elusive in the horse, as evidenced by low in vitro fertilization (IVF) rates (0%-33%); moreover, only two foals have ever been produced using IVF. Incubation of stallion sperm in modified Whittens supplemented with bovine serum albumin and sodium bicarbonate yielded significant rates of time-dependent protein tyrosine phosphorylation and induced acrosomal exocytosis, consistent with capacitation. The objective of this study was to characterize stallion sperm hyperactivation and to test whether hyperactivation of capacitated sperm supported equine IVF. Treatment of sperm with procaine, an anesthetic shown to induce hyperactivation in other mammalian species, resulted in the decrease of three motility variables indicative of hyperactivation: straight line velocity (P = 0.029), straightness (P = 0.001), and linearity (P = 0.002). We demonstrated that procaine-induced hyperactivation was not regulated by changes in protein tyrosine phosphorylation and that it did not induce acrosomal exocytosis in capacitated sperm compared with calcium ionophore (P > 0.05), similar to findings in the bovine. Most notably, by coupling our capacitating conditions with the induction of hyperactivation using procaine, we have achieved the novel result of substantial and reproducible percentages of fertilized mare oocytes (60.7%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with procaine did not fertilize (0%). These results support the hypothesis that capacitation and hyperactivation are required for successful IVF in the equine.

  6. 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.

  7. Oocyte pre-IVM with caffeine improves bovine embryo survival after vitrification.

    Science.gov (United States)

    Bernal-Ulloa, Sandra Milena; Lucas-Hahn, Andrea; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Baulain, Ulrich; Niemann, Heiner

    2016-09-15

    Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until Day 8. Expanded blastocysts derived from either standard control or the 10-mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before and after IVM. Matured oocytes, cleavage, and blastocyst rates were reduced in the 30-mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10-mM caffeine group (73.8%) compared with the standard control (59.7%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Structural changes of in vitro matured buffalo and bovine oocytes following cryopreservation

    OpenAIRE

    Marina De Blasi; Evelina Mariotti; Salvatore Velotto; Marcello Rubessa; Serena Di Francesco; Bianca Gasparrini

    2010-01-01

    The aim of this work was to evaluate chromatin and spindle organization of buffalo and bovine in vitro matured oocytes after vitrification/warming by Cryotop and after their exposure to cryoprotectants (CP). In vitro matured oocytes were vitrified/warmed and exposed to the vitrification/warming solutions containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose as CP. Two hours after warming, oocytes were fixed and immunostained for microtubules and nuclei and examined by fluores...

  9. Cryopreservation of bovine oocytes: is cryoloop vitrification the future to preserving the female gamete?

    OpenAIRE

    Mavrides, Andreas; Morroll, David

    2002-01-01

    International audience; The cryoloop is a technique where a thin nylon loop is used to suspend a film of cryoprotectant containing the oocytes and directly immersing them in liquid nitrogen. 508 bovine oocytes were collected, of these 351 were cryopreserved by slow freezing using standard straws or a new vitrification method using our self-constructed cryoloops and the remainder were controls. After thawing, the oocytes were inseminated by ICSI or standard IVF. The cryoloop vitrification meth...

  10. Development of the competence of bovine oocytes to release cortical granules and block polyspermy after meiotic maturation.

    Science.gov (United States)

    Wang, W; Hosoe, M; Li, R; Shioya, Y

    1997-10-01

    Bovine immature oocytes do not have the ability to block polyspermic penetration. The present study was conducted to determine whether this is correlated to cortical granule (CG) distribution and the competence of oocytes to release CG upon sperm penetration, and whether the ability of bovine oocytes to release CG develops during in vitro maturation. Fluorescein isothiocyanate-conjugated Lens culinaris agglutinin was used for detecting CG in immature and mature oocytes before and after sperm penetration and electric stimulation. The labeled oocytes were examined with laser confocal and fluorescent microscopes. The results show that CG exist as clusters in all immature oocytes. The CG were not released from immature oocytes exposed to electric pulse or penetrated by spermatozoa, resulting in 94% of oocytes being polyspermic. When immature oocytes were cultured for 22 h in vitro, 81% extruded the first polar body and reached metaphase II. In mature oocytes, 25% of oocytes showed CG clusters, 42% and 33% of oocytes showed partial and complete CG dispersion, respectively. When mature oocytes were inseminated in vitro, only 15% of oocytes were polyspermic. Cortical granule exocytosis occurred in 97% of oocytes after sperm penetration and 84% of oocytes released all of the CG 18 h after insemination. Electric pulse induced all of the mature oocytes to release CG but only 55% released all of their CG 18 h post stimulation. These results indicate that polyspermy in immature bovine oocytes is the result of the complete failure of the oocyte to release CG after sperm penetration. Bovine oocytes became competent to release CG by sperm penetration and electric stimulation after meiotic maturation. These results provide evidence that CG exocytosis plays an important role(s) in the establishment of the block to polyspermy in bovine oocytes.

  11. Structural changes of in vitro matured buffalo and bovine oocytes following cryopreservation

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    Marina De Blasi

    2010-01-01

    Full Text Available The aim of this work was to evaluate chromatin and spindle organization of buffalo and bovine in vitro matured oocytes after vitrification/warming by Cryotop and after their exposure to cryoprotectants (CP. In vitro matured oocytes were vitrified/warmed and exposed to the vitrification/warming solutions containing ethylene glycol (EG, dimethyl sulfoxide (DMSO and sucrose as CP. Two hours after warming, oocytes were fixed and immunostained for microtubules and nuclei and examined by fluorescence microscopy. Data were analyzed by Chi Square test. A higher percentage of Telophase II stage oocytes was found in the toxicity (26 and 34% in bovine and buffalo and the vitrification groups (13 and 7% in bovine and buffalo compared to the control, indicating occurrence of activation. An increased percentage of oocytes with abnormal spindle and chromosome organization was found in oocytes exposed to CP (24 and 13% in bovine; 32 and 30% in buffalo respectively and in those vitrified (26 and 31% in bovine; 26 and 29% in buffalo respectively compared to the control (0 in bovine and 2.5 % in buffalo.

  12. Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification.

    Science.gov (United States)

    Maziero, R R D; Guaitolini, C R F; Paschoal, D M; Kievitsbosch, T; Guastali, M D; Moraes, C N; Landim-Alvarenga, F C

    2016-04-01

    This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL-I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm), BL-I (50 μm) and association of drugs (ROS 6.25 μm and BL-I 25 μm). Oocytes were cultured for 18 h in an agent-free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL-I and its association with the ROS increased the rates of cleavage and blastocyst formation (p vitrification process, presenting a higher rate of embryonic re-expansion (p < 0.05). In conclusion, block of meiosis using BL-I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL-I resulted in a better resistance to the embryo cryopreservation process. © 2016 Blackwell Verlag GmbH.

  13. Melatonin inhibits apoptosis and improves the developmental potential of vitrified bovine oocytes.

    Science.gov (United States)

    Zhao, Xue-Ming; Hao, Hai-Sheng; Du, Wei-Hua; Zhao, Shan-Jiang; Wang, Hao-Yu; Wang, Na; Wang, Dong; Liu, Yan; Qin, Tong; Zhu, Hua-Bin

    2016-03-01

    Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10(-9) m melatonin. We analyzed the ROS, mitochondrial Ca(2+) (mCa(2+) ) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase-3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa(2+) , Bax mRNA, and caspase-3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa(2+) , Bax mRNA expression, and caspase-3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10(-9) m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes.

    Science.gov (United States)

    Chouzouris, Thomas-Markos; Dovolou, Eleni; Krania, Fotini; Pappas, Ioannis S; Dafopoulos, Konstantinos; Messinis, Ioannis E; Anifandis, George; Amiridis, Georgios S

    2017-04-01

    The purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus-oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml-1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P ghrelin resulted in substantially reduced (P Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

  15. Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein-4 increases oocyte diameter, but impairs subsequent developmental competence.

    Science.gov (United States)

    Yang, Yinghua; Kanno, Chihiro; Sakaguchi, Kenichiro; Yanagawa, Yojiro; Katagiri, Seiji; Nagano, Masashi

    2017-11-01

    Bone morphogenetic protein-4 (BMP-4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo-grown oocytes. We herein investigated whether an extended IVG culture with BMP-4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte-granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP-4 (10 ng/mL), while a 12 day culture with BMP-4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP-4 (83.2%), but was significantly lower without BMP-4 (58.9%) than the control (83.0%). Prolong-cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP-4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP-4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP-4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes. © 2017 Japanese Society of Animal Science.

  16. Characterization of the IGF2 Imprinted Gene Methylation Status in Bovine Oocytes during Folliculogenesis.

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    Anelise dos Santos Mendonça

    Full Text Available DNA methylation reprogramming occurs during mammalian gametogenesis and embryogenesis. Sex-specific DNA methylation patterns at specific CpG islands controlling imprinted genes are acquired during this window of development. Characterization of the DNA methylation dynamics of imprinted genes acquired by oocytes during folliculogenesis is essential for understanding the physiological and genetic aspects of female gametogenesis and to determine the parameters for oocyte competence. This knowledge can be used to improve in vitro embryo production (IVP, specifically because oocyte competence is one of the most important aspects determining the success of IVP. Imprinted genes, such as IGF2, play important roles in embryo development, placentation and fetal growth. The aim of this study was to characterize the DNA methylation profile of the CpG island located in IGF2 exon 10 in oocytes during bovine folliculogenesis. The methylation percentages in oocytes from primordial follicles, final secondary follicles, small antral follicles, large antral follicles, MII oocytes and spermatozoa were 73.74 ± 2.88%, 58.70 ± 7.46%, 56.00 ± 5.58%, 65.77 ± 5.10%, 56.35 ± 7.45% and 96.04 ± 0.78%, respectively. Oocytes from primordial follicles showed fewer hypomethylated alleles (15.5% than MII oocytes (34.6% (p = 0.039; spermatozoa showed only hypermethylated alleles. Moreover, MII oocytes were less methylated than spermatozoa (p<0.001. Our results showed that the methylation pattern of this region behaves differently between mature oocytes and spermatozoa. However, while this region has a classical imprinted pattern in spermatozoa that is fully methylated, it was variable in mature oocytes, showing hypermethylated and hypomethylated alleles. Furthermore, our results suggest that this CpG island may have received precocious reprogramming, considering that the hypermethylated pattern was already found in growing oocytes from primordial follicles. These results

  17. Polyadenylated tail length variation pattern in ultra-rapid vitrified bovine oocytes

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    D. J. Dutta

    2016-10-01

    Full Text Available Aim: The current study aims at investigating the polyadenylated (poly[A] tail length of morphologically high and low competent oocytes at different developmental stages. Furthermore, effect of ultra-rapid vitrification on the poly(A tail length was studied. Materials and Methods: Fresh bovine cumulus oocyte complexes from abattoir originated ovaries were graded based on morphological characters and matured in vitro. Cryopreservation was done by ultra-rapid vitrification method. mRNA was isolated from different categories of oocyte and subjected to ligation-mediated poly(A test followed by polymerase chain reaction for determining the poly(A tail length of β actin, gap junction protein alpha 1 (GJA1, poly(A polymerase alpha (PAPOLA, and heat shock 70 kDa protein (HSP70 transcripts. Results: GJA1, PAPOLA, and HSP70 showed significantly higher poly(A in immature oocytes of higher competence irrespective of vitrification effects as compared to mature oocytes of higher competence. Conclusion: mRNA poly(A tail size increases in developmentally high competent immature bovine oocytes. There was limited effect of ultra-rapid vitrification of bovine oocytes on poly(A.

  18. In vitro fertilization of porcine oocytes is affected by spermatic coincubation time

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    Guilherme Oberlender

    Full Text Available Abstract: The aim was to study the effects of different gamete coincubation times on porcine in vitro fertilization (IVF, and to verify whether efficiency could be improved by reducing oocyte exposure time to spermatozoa during IVF. In groups of 50, a total of 508 immature cumulus-oocyte complexes (COCs were matured in NCSU-37 medium. The COCs were cultured for 44 hours and then inseminated with in natura semen (2,000 spermatozoa/oocyte. The sperm and oocytes were coincubated according to the following treatments (T: T1 = oocytes exposed to spermatozoa for one hour (173 oocytes, T2 = oocytes exposed to spermatozoa for two hours (170 oocytes, and T3 = oocytes exposed to spermatozoa for three hours (165 oocytes. After these coincubation periods, the oocytes were washed in fertilization medium (TALP medium to remove spermatozoa not bound to the zona pellucida and cultured in another similar medium (containing no sperm. Eighteen to twenty hours after fertilization, the putative zygotes were stained in Hoechst-33342 to evaluate the IVF results. The penetration rate was higher (P0.05 between oocytes exposed to spermatozoa for one (T1 and three hours (T3. However, optimum (P=0.048 results were obtained after two hours of coincubation, when the rate of fertilization performance was 50.16±8.52%. The number of penetrated sperm per oocyte, as well as male pronucleus formation, did not differ (P>0.05 between the treatments evaluated. Under these assay conditions, especially in relation to the sperm concentration used, gamete coincubation for a period of two hours appears to be optimal for monospermy and fertilization performance. Thus, it is the optimal time period for obtaining a large number of pig embryos capable of normal development.

  19. Remodeling epigenetic modifications at tumor suppressor gene promoters with bovine oocyte extract.

    Science.gov (United States)

    Wang, Zhenfei; Yue, Yongli; Han, Pengyong; Sa, Rula; Ren, Xiaolv; Wang, Jie; Bai, Haidong; Yu, Haiquan

    2013-09-01

    Epigenetic silencing of tumor suppressor genes by aberrant DNA methylation and histone modifications at their promoter regions plays an important role in the initiation and progression of cancer. The therapeutic effect of the widely used epigenetic drugs, including DNA methyltransferase inhibitors and histone deacetylase inhibitors, remains unsatisfactory. One important underlying factor in the ineffectiveness of these drugs is that their actions lack specificity. To investigate whether oocyte extract can be used for epigenetic re-programming of cancer cells, H460 human lung cancer cells were reversibly permeabilized and incubated with bovine oocyte extract. Bisulfite sequencing showed that bovine oocyte extract induced significant demethylation at hypermethylated promoter CpG islands of the tumor suppressor genes RUNX3 and CDH1; however, the DNA methylation levels of repetitive sequences were not affected. Chromatin immunoprecipitation showed that bovine oocyte extract significantly reduced transcriptionally repressive histone modifications and increased transcriptionally activating histone modifications at the promoter regions of RUNX3 and CDH1. Bovine oocyte extract reactivated the expression of RUNX3 and CDH1 at both the messenger RNA and the protein levels without up-regulating the transcription of pluripotency-associated genes. At the functional level, anchorage-independent proliferation, migration and invasion of H460 cells was strongly inhibited. These results demonstrate that bovine oocyte extract reactivates epigenetically silenced tumor suppressor genes by remodeling the epigenetic modifications at their promoter regions. Bovine oocyte extract may provide a useful tool for investigating epigenetic mechanisms in cancer and a valuable source for developing novel safe therapeutic approaches that target epigenetic alterations. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  20. Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos.

    Science.gov (United States)

    Balasubramanian, S; Rho, Gyu-Jin

    2007-04-01

    In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM-IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM-IVF-IVC methods were chilled to 0 degrees C for 30 min at 2-cell (30-34 h post-insemination, hpi), 8-cell (48-52 hpi) or blastocyst (166-170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 microM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7+/-0.9% versus 7.2+/-0.9%; Pchilling of blastocysts produced by IVM-IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.

  1. In vitro maturation supplements affect developmental competence of bovine cumulus-oocyte complexes and embryo quality after vitrification.

    Science.gov (United States)

    Räty, Mervi; Ketoja, Elise; Pitkänen, Timo; Ahola, Virpi; Kananen, Kirsi; Peippo, Jaana

    2011-12-01

    Oocyte quality affects subsequent embryo development and quality. We examined the impact of bovine oocyte in vitro maturation (IVM) conditions on subsequent embryo yield, quality and cryosurvival. Cumulus-oocyte complexes (COCs) were sampled for cytological and gene expression analysis after IVM in TCM199 supplemented with 10% fetal bovine serum (FBS), 4 mg/ml of fatty-acid-free bovine serum albumin (FAFBSA), 4 mg/ml of polyvinylpyrrolidone (PVP), FAFBSA with epidermal growth factor (EGF, 100 ng/ml) and insulin-like growth factor 1 (IGF-I, 100 ng/ml) (FAFBSAGF), PVP with EGF and IGF-I (PVPGF) or PVP with single strength BME and MEM amino acids (PVPAA). The remaining COCs were fertilized. On day 7 (IVF=day 0) quality 1 blastocysts were vitrified or analyzed for glucose transporter 1 (Glut-1) expression levels. The remaining blastocysts (days 7-9) were evaluated for morphology and total cell counts. After warming, survival and hatching rates were evaluated followed by total cell counts and Glut-1 expression levels. Only PVPGF IVM resulted in embryo production rates comparable to those recorded with FBS IVM. Growth factors with FAFBSA and amino acids with PVP reduced embryo production rates whereas the effect of the growth factors with PVP was negligible. Insulin-like growth factor 2 binding protein 3 (IGF2BP3) and beta cell translocation gene 4 (BTG4) were revealed as potential candidates for oocyte developmental competence, and secreted protein, acidic and rich in cysteine (SPARC) for cumulus cell expansion. There were no differences among treatments in hatching rates of vitrified embryos after warming. However, total cell numbers and Glut-1 expression levels at 72 h were affected. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Osteopontin reduces polyspermy during in vitro fertilization of porcine oocytes.

    Science.gov (United States)

    Hao, Yanhong; Mathialagan, Nagappan; Walters, Eric; Mao, Jiude; Lai, Liangxue; Becker, Donald; Li, Wensheng; Critser, John; Prather, Randall S

    2006-11-01

    This study was designed to determine the role of osteopontin (SPP1) in in vitro fertilization (IVF) in swine. The initial objective was to evaluate the effect of various concentrations of SPP1 (0, 0.001, 0.01, 0.1 and 1 microg/ml) on spermatozoa and oocytes during IVF. The results demonstrate that SPP1 reduced the rate of polyspermy in a dose-dependent manner (P polyspermy was specific to SPP1, a mixture of pregnancy-associated glycoproteins was included in the IVF protocol and shown to have no effect on polyspermy. Furthermore, Western blotting demonstrated that a 50-kDa SPP1 form was present in the oviducts on Days 0, 3, and 5 in pregnant and nonpregnant gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5. The current study represents the first report to demonstrate that SPP1 plays an important role in the regulation of pig polyspermic fertilization; it decreases polyspermy and increases fertilization efficiency during IVF.

  3. Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes.

    Science.gov (United States)

    Arcarons, Núria; Morató, Roser; Vendrell, Meritxell; Yeste, Marc; López-Bejar, Manel; Rajapaksha, Kosala; Anzar, Muhammad; Mogas, Teresa

    2017-01-01

    This study examines whether incorporating cholesterol-loaded methyl-β-cyclodextrin (CLC) in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV) and in vitro matured (MII) oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage.

  4. Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes.

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    Núria Arcarons

    Full Text Available This study examines whether incorporating cholesterol-loaded methyl-β-cyclodextrin (CLC in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV and in vitro matured (MII oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage.

  5. Downsizing cumulus cell layers to improve cryotolerance of germinal vesicle-stage bovine oocytes.

    Science.gov (United States)

    Tashima, Kazuya; Kubo, Yuki; Hirabayashi, Masumi; Hochi, Shinichi

    2017-06-01

    This study was undertaken to investigate whether complete removal or downsizing of the cumulus cell layers in germinal vesicle (GV)-stage bovine cumulus-oocyte complexes (COCs) can improve blastocyst development rate following Cryotop vitrification. Downsized COCs (196 μm in mean diameter) and denuded oocytes (141 μm in mean diameter) were prepared by vortex-mixing of full-sized COCs (330 μm in mean diameter) retrieved from abattoir-derived ovaries. Nuclear maturation rates, assessed by the first polar body extrusion, after vitrification and the subsequent 22-h IVM were comparable (61.9-62.9%). Approximately one-third (30.5-31.2%) of the matured oocytes derived from the downsized COCs could develop into high quality blastocysts after 6-h IVF and 8-d IVC, while 13.4 and 23.7% of the matured oocytes derived from denuded oocytes and full-size COCs reached to the blastocysts, respectively. Cytoplasmic lipid droplets of matured oocytes in vitrification group were more clustered with decreased number and increased size of the droplets, when compared to those in fresh control group. However, individual oocyte culture in well-of-the well system suggested that change of lipid droplet distribution in the matured oocytes had no adverse effect on their subsequent developmental competence up to the blastocyst stage. In conclusion, Cryotop vitrification of downsized GV-stage bovine COCs allowed blastocyst yields as high as >30%. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Oocyte maturation, embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification.

    Science.gov (United States)

    Azari, Mehdi; Kafi, Mojtaba; Ebrahimi, Bita; Fatehi, Roya; Jamalzadeh, Mahboobeh

    2017-03-01

    To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification. Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions. The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P vitrification groups (P vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.

  7. Whole-depth change in bovine zona pellucida biomechanics after fertilization: how relevant in hindering polyspermy?

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    Massimiliano Papi

    Full Text Available Polyspermy is a common problem in bovine in vitro fertilization (IVF and has a still unclear etiology. In this specie, after IVF, despite the lack of a biochemical post-fertilization hardening, the stiffness of the outer ZP layer is significantly increased. Therefore, polyspermy might be related to an incomplete or insufficient stiffening of the ZP. We obtained, by using atomic force spectroscopy in physiological conditions, a complete characterization of the biomechanical changes of the inner and outer ZP layers occurring during oocyte maturation/fertilization and correlated them to the ultrastructural changes observed by transmission electron microscopy using ruthenium red and saponin technique. In both the inner and outer ZP layers, stiffness decreased at maturation while, conversely, increased after fertilization. Contextually, at the nanoscale, during maturation both ZP layers displayed a fine filaments network whose length increased while thickness decreased. After fertilization, filaments partially recovered the immature features, appearing again shorter and thicker. Overall, the observed biomechanical modifications were substantiated by ultrastructural findings in the ZP filament mesh. In fertilized ZP, the calculated force necessary to displace ZP filaments resulted quite similar to that previously reported as generated by bovine sperm flagellum. Therefore, in bovine IVF biomechanical modifications of ZP appear ineffective in hindering sperm transit, highlighting the relevance of additional mechanisms operating in vivo.

  8. Whole-depth change in bovine zona pellucida biomechanics after fertilization: how relevant in hindering polyspermy?

    Science.gov (United States)

    Papi, Massimiliano; Brunelli, Roberto; Familiari, Giuseppe; Frassanito, Maria Cristina; Lamberti, Luciano; Maulucci, Giuseppe; Monaci, Maurizio; Pappalettere, Carmine; Parasassi, Tiziana; Relucenti, Michela; Sylla, Lakamy; Ursini, Fulvio; De Spirito, Marco

    2012-01-01

    Polyspermy is a common problem in bovine in vitro fertilization (IVF) and has a still unclear etiology. In this specie, after IVF, despite the lack of a biochemical post-fertilization hardening, the stiffness of the outer ZP layer is significantly increased. Therefore, polyspermy might be related to an incomplete or insufficient stiffening of the ZP. We obtained, by using atomic force spectroscopy in physiological conditions, a complete characterization of the biomechanical changes of the inner and outer ZP layers occurring during oocyte maturation/fertilization and correlated them to the ultrastructural changes observed by transmission electron microscopy using ruthenium red and saponin technique. In both the inner and outer ZP layers, stiffness decreased at maturation while, conversely, increased after fertilization. Contextually, at the nanoscale, during maturation both ZP layers displayed a fine filaments network whose length increased while thickness decreased. After fertilization, filaments partially recovered the immature features, appearing again shorter and thicker. Overall, the observed biomechanical modifications were substantiated by ultrastructural findings in the ZP filament mesh. In fertilized ZP, the calculated force necessary to displace ZP filaments resulted quite similar to that previously reported as generated by bovine sperm flagellum. Therefore, in bovine IVF biomechanical modifications of ZP appear ineffective in hindering sperm transit, highlighting the relevance of additional mechanisms operating in vivo.

  9. Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine.

    Science.gov (United States)

    Chen, Jun-Yi; Li, Xiao-Xia; Xu, Ya-Kun; Wu, Hua; Zheng, Jun-Jun; Yu, Xue-Li

    2014-12-01

    The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (Pvitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; Pvitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Polyspermy in in vitro fertilization of human oocytes: frequency and possible causes.

    Science.gov (United States)

    van der Ven, H H; Al-Hasani, S; Diedrich, K; Hamerich, U; Lehmann, F; Krebs, D

    1985-01-01

    In an IVF program a total of 585 oocytes (180 patients) were examined for the presence of pronuclei 16 to 20 hours after the addition of spermatozoa. The overall fertilization rate was 71%, and in 58 (10%) of the fertilized oocytes, three or more pronuclei, indicating a failure of the block to polyspermy, could be observed. The frequency of polyspermy was related to the maturity of the oocyte, determined according to morphologic criteria. Immature oocytes showed a higher percentage of polyspermic fertilization (32%) compared to that of mature oocytes (6%). Preincubation of oocytes (for 0.5-1.5, 2-4, and 5-8 hours) prior to the addition of spermatozoa increased the fertilization rate (to 67%, 70%, and 83%, respectively). The polyspermy rate, however, was not significantly different between the various preincubation intervals (13%, 14%, and 19%, respectively). The polyspermy rate was affected by the number of spermatozoa used for in vitro fertilization. Insemination with 0.5-0.8, 1.0, or 1.5-2.0 X 10(6) spermatozoa/oocyte resulted in a polyspermy rate of 6%, 20%, and 32%, respectively. The appearance of polyspermic fertilization was not related to the age of the patient (which ranged from 20 to 45 years) nor to the method of ovarian stimulation (clomiphene, hMG, or clomiphene/hMG). Because of the high incidence of polyspermy under in vitro conditions it seems to be important to routinely examine the oocytes in the pronuclear stage. Reduction of the number of spermatozoa used for in vitro fertilization and the exact timing of insemination according to the maturity of the oocyte might reduce the occurrence of polyspermic fertilization.

  11. Release of sICAM-1 in oocytes and in vitro fertilized human embryos.

    Directory of Open Access Journals (Sweden)

    Monica Borgatti

    Full Text Available During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G by 48-72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection.The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes.The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading, with a possible interesting rebound in assisted reproduction techniques.

  12. Effects of oocyte vitrification on epigenetic status in early bovine embryos.

    Science.gov (United States)

    Chen, Huanhuan; Zhang, Lei; Deng, Tengfei; Zou, Pengda; Wang, Yongsheng; Quan, Fusheng; Zhang, Yong

    2016-08-01

    Oocyte cryopreservation has a great impact on subsequent embryonic development. Currently, several studies have primarily focused on the consequences of vitrification and the development potential of cellular structures. This study determined whether oocyte vitrification caused epigenetic instabilities of bovine embryos. The effects of oocyte vitrification on DNA methylation, histone modifications, and putative imprinted genes' expression in early embryos derived by intracytoplasmic sperm injection were examined. Results showed that oocyte vitrification did not affect zygote cleavage rates (67.0% vs. 73.8% control, P > 0.05) but reduced the blastocyst rate (9.6% vs. 23.0%, P vitrification group during the early cleavage phases. No differences were observed for DNA methylation, H3K9me3, and acH3K9 in the inner cell mass of blastocysts, whereas decreased levels of DNA methylation and acH3K9 (P vitrification. The expression of putative-imprinted genes PEG10, XIST, and KCNQ1O1T was upregulated in blastocysts. These epigenetic abnormalities may be partially explained by altered expression of genes associated with epigenetic regulations. DNA methylation and H3K9 modification suggest that oocyte vitrification may excessively relax the chromosomes of oocytes and early cleavage embryos. In conclusion, these epigenetic indexes could be used as damage markers of oocyte vitrification during early embryonic development, which offers a new insight to assess oocyte vitrification. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2017-08-01

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

  14. 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 (pvitrification groups [enucleated-vitrified-thawed (EVT) group, 13.7%; VT group, 15.0%; pvitrification groups (pvitrification groups than in the non-V group (pvitrification 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.

  15. Retinoic acid effects on nuclear maturation of bovine oocytes in vitro

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-18

    Aug 18, 2009 ... Experimental design and statistical analysis. In the current study different concentrations of t-RA (RA ... In this experiment 735 immature bovine oocytes were selected and randomly allocated to control, vehicle .... been implicated as important regulators of redox signalling pathways (Olson, 1993; Imam et al., ...

  16. Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17 beta-estradiol levels in mural granulosa cell cultures.

    Science.gov (United States)

    Minervini, F; Dell'Aquila, M E; Maritato, F; Minoia, P; Visconti, A

    2001-01-01

    Moulds parasites of livestock foodstuffs alter the quality of grains by synthesizing mycotoxins. Zearalenone (ZEA) and its derivatives (alpha- and beta-zearalenol, zeranol, taleranol and zearalanone) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to fertility disturbances and other reproductive pathologies. Zearalenone, alpha-zearalenol and zearalanone were tested, at levels ranging from 0.3 to 30 microg/ml, in order to evaluate the effect on the in vitro maturation (IVM) rate of bovine oocytes and on the formation of 17 beta-estradiol in supernatants of mural granulosa cells (GC) cultures. These compounds induced dose-dependent oocyte maturation delay and chromatin abnormalities. Maturation of oocytes to metaphase II (M II) was inhibited in oocytes cultured in the presence of 30 microg/ml ZEA, alpha-zearalenol or zearalanone, with a significant increase in chromatin abnormalities occurring in the presence of ZEA (Pzearalenol (Pzearalenol (mean value 1.6 ng/ml) with respect to ZEA and zearalanone (mean estradiol concentrations of 0.06 and 0.5 ng/ml, respectively). These data demonstrate a negative effect of ZEA and its derivatives on meiotic progression of bovine oocytes, possibly attributable to a toxic mechanism not related to the binding affinity of these compounds to estrogen receptor sites, and support previous observations that alpha-zearalenol acts as a stronger estrogenic inducer than the original molecule (ZEA).

  17. Successful vitrification of bovine immature oocyte using liquid helium instead of liquid nitrogen as cryogenic liquid.

    Science.gov (United States)

    Yu, Xue-Li; Xu, Ya-Kun; Wu, Hua; Guo, Xian-Fei; Li, Xiao-Xia; Han, Wen-Xia; Li, Ying-Hua

    2016-04-01

    The objectives of this study were to compare the effectiveness of liquid helium (LHe) and liquid nitrogen (LN2) as cryogenic liquid for vitrification of bovine immature oocytes with open-pulled straw (OPS) system and determine the optimal cryoprotectant concentration of LHe vitrification. Cumulus oocyte complexes were divided into three groups, namely, untreated group (control), LN2 vitrified with OPS group, and LHe vitrified with OPS group. Oocyte survival was assessed by morphology, nuclear maturation, and developmental capability. Results indicated that the rates of normal morphology, maturation, cleavage, and blastocyst (89.3%, 52.8%, 42.7%, and 10.1%, respectively) in the LHe-vitrified group were all higher than those (79.3%, 43.4%, 34.1%, and 4.7%) in the LN2-vitrified group (P vitrification solutions (EDS30, EDS35, EDS40, EDS45, and EDS50) in LHe vitrification for bovine immature oocytes vitrification were examined. No difference was found in the rates of morphologically normal oocytes among the EDS30 (87.9%), EDS35 (90.1%), EDS40 (89.4%), and EDS45 (87.2%) groups (P > 0.05). The maturation rate of the EDS35 group (65.0%) was higher than those of the EDS30 (51.3%), EDS40 (50.1%), EDS45 (52.1%), and EDS50 groups (36.9%; P vitrification of bovine immature oocytes, and it is more efficient than LN2-vitrified oocytes in terms of blastocyst production. EDS35 was the optimal cryoprotectant agent combination for LHe vitrification in this study. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Computational identification of fertility functions of bovine Reprimo ...

    African Journals Online (AJOL)

    The domain structure of bovine RPRM protein was predicted using simple modular architecture research tool (SMART). Protein tertiary structures (3D structures) of bovine RPRM gene and other cattle fertility genes were predicted with Phyre2 software. To have structural and functional similarity, it has been found that protein ...

  19. Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability.

    Science.gov (United States)

    Diez, Carmen; Duque, Paloma; Gómez, Enrique; Hidalgo, Carlos O; Tamargo, Carolina; Rodríguez, Aida; Fernández, Lina; de la Varga, Santiago; Fernández, Alba; Facal, Nieves; Carbajo, Maite

    2005-07-15

    The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro maturation (IVM) was performed in TCM199, 10% FCS, FSH-LH and 17beta-estradiol for 24 h. Oocytes were vitrified using the open pulled straw method (OPS) with minor modifications. Fresh and vitrified/warmed COCs were fixed as immature, after IVM, after meiotic arrest (MA) and after MA + IVM. Vitrification combined with MA followed by IVM produced the highest rates of degeneration, regardless of the vitrification time. As a consequence, lower proportions of embryos cleaved in these groups, although differences were eliminated at the five-eight cell stage. Development rates up to day 8 were similar in all experimental groups, being significantly lower than those in fresh controls. Only oocytes vitrified after IVM were able to give blastociysts. The morphological alterations observed can be responsible for compromised development. More research is needed to explain the low survival rates of the bovine oocyte after vitrification and warming.

  20. 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

  1. D-penicillamine and granulosa cells can effectively extend the fertile life span of bovine frozen-thawed spermatozoa in vitro: effect on fertilization and polyspermy.

    Science.gov (United States)

    Pavlok, A

    2000-03-15

    The effect of D-penicillamine on the fertile life span of frozen-thawed bull spermatozoa was studied in Experiment 1. After thawing, the washed spermatozoa were incubated for 8 h in fertilization medium with 1 mg/mL polyvinyl alcohol (PVA) and 0.5 mg/mL D-penicillamine. The addition of cumulus-free oocytes together with 10% of bovine serum (BOS) + 4 IU/mL heparin to the 8-h incubated spermatozoa resulted in high fertilization and polyspermy rates (97/103, 94.2% and 49/97, 50.5%, respectively). When BOS was substituted with 3 mg/mL of BSA, the fertilization and polyspermy rates decreased to 33/91 (36.3%) and 3/33 (9.1%), respectively. The total absence of fertilization was observed after substitution of proteins with PVA. The 8-h sperm incubation in fertilization medium without D-penicillamine and following fertilization in medium + different capacitation supplements resulted in the total absence of or a very low fertilization rate. In Experiment 2, the first set of cumulus-oocyte complexes (COC) and cumulus-free oocytes were fertilized for 8 h with nonincubated spermatozoa. The second set of COC and cumulus-free oocytes were fertilized in the same wells with spermatozoa after removal of the first set. The fertilization rate for the first set of COC and cumulus-free oocytes was 65/67 (97%) and 73/73 (100%), respectively, with 21/65 (32.3%) and 32/73 (43.8%) polyspermy, respectively. In the second set, the high penetration rate (67/73, 91.8%) was observed only for COC, while that for cumulus-free oocytes (19/76, 25%) was significantly lower (P polyspermy. In medium + BSA + 10 or 100 IU/mL heparin, the fertilization rate of COC was 64/72 (88.9%) and 70/79 (88.6%), respectively, with polyspermy at 2/64 (3.1%) and 7/70 (10%), respectively. In medium + BOS + 10 or 100 IU/mL heparin, the fertilization rate was 79/82 (96.3%) and 79/80 (98.7%), respectively, with a significantly (P polyspermy rate (34/79, 43% and 30/79, 38%, respectively). The vitality and capacitation

  2. 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. © 2015 Japanese Society of Animal Science.

  3. Vitrification by Cryotop and the Maturation, Fertilization, and Developmental Rates of Mouse Oocytes

    Science.gov (United States)

    Abedpour, Neda; Rajaei, Farzad

    2015-01-01

    Background: Oocyte cryopreservation is an important part of modern fertility treatment. The effect of vitrification on the fertilization and developmental rates of embryo is still a matter of debate. Objectives: This study aimed to investigate the effect of vitrification on the success of mouse oocyte maturation, fertilization, and preimplantation development in vitro. Materials and Methods: In this experimental study, a total of 200 germinal vesicle (GV) and 200 metaphase II (MII) oocytes were obtained from ovaries and fallopian tubes of NMRI mice, respectively and divided into two control and experimental (vitrified) groups. Oocytes in the experimental group were vitrified by Cryotop using vitrification medium (Origio, Denmark) and kept in liquid nitrogen for one month. Then, they were cultured in maturation medium for 24 hours. In vitro maturated metaphase 2 (IVM-MII) and ovulated metaphase 2 (OV-MII) oocytes were inseminated and the fertilized embryos assessed until the hatching blastocyst stage. Outcomes were assessed for statistical significance by Chi-square test using SPSS software. Results: Vitrification caused a significant reduction in the maturation rate of oocytes. Of those that matured, the fertilization rate of vitrified IVM-MII (44.1%) and OV-MII oocytes (50%) was not significantly different from each other but both were significantly lower than the control group (P < 0.05). There was no significant difference in developmental rates of both vitrified groups and the control group. Conclusions: The present study showed that vitrification using Cryotop and freezing medium can damage oocytes by reducing the maturation and fertilization rates in both developmental stages. PMID:26568845

  4. OPTIMISATION OF TOTAL RNA EXTRACTION FROM BOVINE OOCYTES AND EMBRYOS FOR GENE EXPRESSION STUDIES AND EFFECTS OF CRYOPROTECTANTS ON TOTAL RNA EXTRACTION.

    Science.gov (United States)

    Pavani, K C; Baron, E E; Faheem, M; Chaveiro, A; Da Silva, F Moreira

    2015-01-01

    Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a standardized reagent protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For such purpose oocytes (n = 795) recovered from about 80 ovaries were divided in three groups: Group 1 modified Trizol (MTP, n = 355); Group 2 Guanidinium thiocyanate protocol (GNTC, n = 140) and Group 3 Commercial Kit protocol (CKP, n = 60). Oocytes belonging to group 1 (n = 100) and 3 (n = 20) were subjected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The 240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization, and 140 oocytes were vitrified using PROH (n = 70) and DMSO (n = 70) as cryoprotectants, being then fertilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA, (GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos at the stage of blastocyst (n = 10). Efficiency of the extraction was further compared. The purity of all samples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA samples (1.7 to 2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration was 127.8 ± 9.3 ng μl(-1) for MTP, against 46.4 ± 9.5 ng μl(-1) from CKP and 476 ± 12.9 ng μl(-1) for GNTC protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concentration of 61.3 ± 3.3 ng μl(-1) and 40.0 μ 12.4 ng μ(-1), respectively for DMSO and PROH. Regarding total RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to

  5. Comparison of the level(s) of DNA damage using Comet assay in bovine oocytes subjected to selected vitrification methods.

    Science.gov (United States)

    Stachowiak, E M; Papis, K; Kruszewski, M; Iwaneńko, T; Bartłomiejczyk, T; Modliński, J A

    2009-08-01

    It was suggested that the cryodamage to oocytes' DNA has been responsible for the compromised developmental competence of cryopreserved oocytes. Vitrification of bovine oocytes affected not only cellular components, but also nuclear material. A significant rate of DNA fragmentation was found in bovine frozen or vitrified oocytes analysed by Comet assay regardless of cryopreservation method. Our method of vitrification using droplet system after gentle pre-equilibration treatment is one of the most effective cryopreservation methods employed for bovine oocytes so far, making it possible to develop 30% blastocyst stage embryos. In this study, the extent of DNA damage in bovine oocytes vitrified using three vitrification methods (droplet system, Open Pulled Straw and traditional vitrification in 0.25 ml insemination straws) was compared using Comet assay. Vitrification in droplet system and Open Pull Straws vitrification did not result in detectable cryoinjuries of DNA of bovine oocytes. On the contrary, DNA fragmentation was found in four of 26 oocytes vitrified in 0.25 ml straws (15.4%, p vitrification methods).

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

    Science.gov (United States)

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

    2016-12-01

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

  7. Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection.

    Science.gov (United States)

    Li, X; Morris, L H; Allen, W R

    2000-07-01

    The effects of four reagents on the activation and subsequent fertilization of equine oocytes, and the development of these after intracytoplasmic sperm injection, were investigated. Cumulus-oocyte complexes collected from equine ovaries obtained from an abattoir were matured in vitro for 40-44 h in TCM199 medium before being injected, when in metaphase II, with an immobilized stallion spermatozoon. The cumulus-oocyte complexes were then subjected to one of five activation treatments: (a) 10 micromol ionomycin l(-1) for 10 min; (b) 7% (v/v) ethanol for 10 min; (c) 100 micromol thimerosal l(-1) for 10 min; (d) 250 micromol inositol 1,4, 5-triphosphate l(-1) injection; and (e) no treatment (control). After 18-20 h further culture, the cumulus-oocyte complexes were assessed for activation by observing whether they had progressed through second anaphase-telophase and had formed a female pronucleus. The proportions of oocytes activated after each treatment were: 16/27 (59%) for ionomycin; 14/25 (56%) for ethanol; 22/28 (79%) for thimerosal; 15/27 (56%) for inositol 1,4,5-triphosphate; and 0/20 (0%) for the untreated controls. Thus, significantly more oocytes (P fertilization was observed in 2/7 (29%), 2/5 (40%) and 7/11 (64%) of the oocytes treated with ionomycin, ethanol and thimerosal, respectively. These results demonstrated that: (a) it is possible to activate equine oocytes with the chemical stimulants, ionomycin, ethanol, thimerosal and inositol 1,4,5-triphosphate; (b) thimerosal is more effective than the other three reagents in facilitating both meiotic activation and normal fertilization of equine oocytes; and (c) chemical activation may also stimulate parthenogenetic cleavage of oocytes without concurrent changes in the head of the spermatozoon.

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

    Directory of Open Access Journals (Sweden)

    George Anifandis

    2014-07-01

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

  9. Effect of macromolecules in solutions for vitrification of mature bovine oocytes.

    Science.gov (United States)

    Checura, C M; Seidel, G E

    2007-03-15

    This study was designed to evaluate vitrification procedures for in vitro matured bovine oocytes for efficient blastocyst production after warming, IVF and culture. A second goal was to replace serum as the macromolecular component of the vitrification solution, without compromising efficacy. The first experiment compared two containers, open pulled straws (OPS) versus cryoloops, and two vitrification protocols: short equilibration (H-TCM-199+10% EG+10% DMSO+20% FCS for 30s, followed by H-TCM-199+20% EG+20% DMSO+20% FCS+0.48M galactose for 20s) versus long equilibration (H-TCM-199+3% EG+20% FCS for 10min, followed by H-TCM-199+31% EG+20% FCS+1M galactose for 20s). Subsequent experiments used only cryoloops and the short equilibration protocol to evaluate the effect of replacing FCS with defined macromolecules (BSA, Ficoll, PVP, and PVA) in vitrification solutions. Cryoloops were superior to OPS for vitrification of oocytes as determined by blastocyst production (Pvitrification protocols gave similar results. The presence of macromolecules in vitrification solutions for bovine oocytes was necessary for acceptable post-warming developmental capacity; 20% FCS, 1% and 2% BSA, 6% and 18% Ficoll, 6% and 20% PVP, 1% PVA, and the combinations of 18% Ficoll+1% BSA, and 6% PVP+1% BSA provided similar protection during vitrification of oocytes; development ranged from 14.8% to 23.0% blastocysts/oocyte, which was not different (P>0.05) from non-vitrified controls (26.9-34.0% blastocysts/oocyte). Too much (6%) and too little (0.3%) BSA, and 0.3% PVA for vitrification resulted in lower blastocyst production (P<0.05) relative to unvitrified oocytes.

  10. PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

    Science.gov (United States)

    Terzaghi, L; Tessaro, I; Raucci, F; Merico, V; Mazzini, G; Garagna, S; Zuccotti, M; Franciosi, F; Lodde, V

    2016-08-02

    Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

  11. Comparison of normal and abnormal fertilization of in vitro-matured human oocyte according to insemination method.

    Science.gov (United States)

    Park, Ju Hee; Jee, Byung Chul; Kim, Seok Hyun

    2016-04-01

    Our purpose was to compare the normal fertilization rate, multi-pronuclei (PN) formation rate, and embryonic development of in vitro-matured oocytes between conventional insemination and intracytoplasmic sperm injection (ICSI). A total of 213 stimulated in vitro fertilization (IVF) cycles were selected, in which at least one immature oocyte was obtained (from 2010 to 2014). Immature oocytes were assigned to germinal vesicle (GV)-stage or metaphase I (MI)-stage oocyte groups. Cycles with obligatory ICSI due to male-factor infertility were excluded. Cycles were divided into two groups according to fertilization method: there were 97 cycles with conventional insemination and 116 cycles with ICSI. After in vitro maturation of 324 GV-stage oocytes and 341 MI-stage oocytes, the fertilization rate, multi-PN formation rate, and embryonic development were compared according to the fertilization method. The normal fertilization rate was similar in the conventional insemination and the ICSI both in GV-derived and MI-derived oocytes. Both fertilization methods resulted in a similar multi-PN formation rate in GV-derived oocytes; however, in MI-derived oocytes, the multi-PN formation rate was zero with ICSI and this was significantly lower than that with conventional insemination (9.6%, P = 0.001). In non-male-factor infertility, ICSI should be considered when MI oocytes are matured. © 2016 Japan Society of Obstetrics and Gynecology.

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

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    Quetglas M.D.

    2001-01-01

    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.

  13. Oocyte surface in four teleost fish species postspawning and fertilization

    OpenAIRE

    Rizzo,Elizete; Moura,Thais F.C.; Sato,Yoshimi; Bazzoli,Nilo

    1998-01-01

    Cytological and cytochemical studies were carried out to investigate the surface characteristics of oocytes of four teleost species from the São Francisco river. The fishes were submitted to hypophysation at the Três Marias Hybrobiology and Fishculture Station, Minas Gerais, Brazil, in January 1996. Postspawning, oocytes of the curimatãs Prochilodus affinis, Prochilodus marggravii and dourado Salminus brasiliensis were surrounded by a thick, three-layered zona pellucida with radial striae. Th...

  14. In vitro maturation of canine oocytes co-cultured with bovine and canine granulosa cell monolayers.

    Science.gov (United States)

    Abdel-Ghani, Mohammed Ali; Shimizu, Takashi; Asano, Tomoyoshi; Suzuki, Hiroshi

    2012-01-15

    The present study investigated the effects of bovine granulosa cell monolayers (BGML) and canine granulosa cell monolayers (CGML) on nuclear maturation of canine oocytes with and without cumulus cells. Cumulus-oocyte complexes (COCs) or cumulus-free oocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM, control group), DMEM with BGML (BGML group), or DMEM with CGML (CGML group) for 72 h at 38.5 °C in 5% CO(2), 5% O(2,) and 90% N(2). All media were supplemented with 10% of FCS, 50 ng/mL of EGF, 2 μg/mL of estradiol-17β, 0.1 IU/mL of hCG, 0.1 IU/mL of FSH, 0.25 mM of pyruvic acid, 100 μM of β-mercaptoethanol, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. In cumulus-enclosed oocytes retrieved from ovaries at estrus and/or diestrus, the highest percentage of M-II oocytes (P 0.05) to proportions achieved with control (3.0%). However, the presence of BGML improved (P < 0.05) the ability of denuded oocytes to develop into M-II (10.2%). The BGML group had the highest overall meiotic resumption (P < 0.05), and least oocyte degeneration (P < 0.05) among experimental groups. In conclusion, BGML had a positive impact on the in vitro maturation system, as well as meiotic resumption of canine oocytes. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Fecundação e clivagem após a ativação da proteína quinase C durante a maturação de oócitos bovinos Fertilization and cleavage after protein kinase C activation during bovine oocyte maturation

    Directory of Open Access Journals (Sweden)

    Rafael Gianella Mondadori

    1999-03-01

    bind on PK-C, phorbol ester control, ECS (10% of estrus cow serum, positive control and in a negative control (basic maturation media for all treatments, except on ECS group where the polyvinyl alcohol was not added. The COCs were kept in presence of PMA for 1, 4 or 7 hours and then transferred to basic maturation media until the 24 hours maturation period was completed. The PK-C stimulation in these periods did not alter the pronucleus formation but caused a decrease in the zygotes cleavage rate. The 4alpha-PDD group results showed that the alterations in PMA group were caused by PK-C activation and not by direct phorbol ester action on the cell. The protein composition analysis showed an additional protein band on ECS group around 74 kilo Daltons (kDa. Altogether, these results with the preliminary Western Blot analysis, indicate an important role of PK-C on bovine oocyte maturation, fertilization and embryo cleavage.

  16. 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

    Directory of Open Access Journals (Sweden)

    Dorota Boruszewska

    2014-01-01

    Full Text Available 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 in oocytes and decreased mRNA abundance of CTSs in cumulus cells. Additionally, oocytes stimulated with LPA had higher transcription levels of BCL2 and lower transcription levels of BAX resulting in the significantly lower BAX/BCL2 ratio. Blastocyst rates on day 7 were similar in the control and the LPA-stimulated COCs. Our study demonstrates for the first time that bovine COCs are a potential source and target of LPA action. We postulate that LPA exerts an autocrine and/or paracrine signaling, through several LPARs, between the oocyte and cumulus cells. LPA supplementation of maturation medium improves COC quality, and although this was not translated into an enhanced in vitro development until the blastocyst stage, improved oocyte competence may be relevant for subsequent in vivo survival.

  17. Loss of Bmal1 decreases oocyte fertilization, early embryo development and implantation potential in female mice.

    Science.gov (United States)

    Xu, Jian; Li, Yan; Wang, Yizi; Xu, Yanwen; Zhou, Canquan

    2016-10-01

    Biological clock genes expressed in reproductive tissues play important roles in maintaining the normal functions of reproductive system. However, disruption of female circadian rhythm on oocyte fertilization, preimplantation embryo development and blastocyst implantation potential is still unclear. In this study, ovulation, in vivo and in vitro oocyte fertilization, embryo development, implantation and intracellular reactive oxygen species (ROS) levels in ovary and oviduct were studied in female Bmal1+/+ and Bmal1-/- mice. The number of naturally ovulated oocyte in Bmal1-/- mice decreased (5.2 ± 0.8 vs 7.8 ± 0.8, P fertilization rate and obtained blastocyst number were observed in Bmal1-/- female mice either mated with wild-type in vivo or fertilized by sperm from wild-type male mice in vitro (all P fertilization rate of oocytes derived from Bmal1-/- increased significantly compared with in vivo study (P fertilization rate, early embryo development and implantation potential in female mice, and these may be possibly caused by excess ROS levels generated in ovary and oviduct.

  18. Retinoic acid effects on nuclear maturation of bovine oocytes in vitro ...

    African Journals Online (AJOL)

    In the present study, the effect of all-trans retinoic acid (t-RA) administration during in vitro maturation (IVM) on bovine oocytes maturation was determined. Concentrations of t-RA (RA; 0, 0.25, 0.5 and 1 μM) and 0.1% ethanol (vehicle) were included in the maturation medium. Ovaries collected from the local abattoir were ...

  19. 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.

  20. IMPACT OF OOCYTE SIZE ON LHRHa INDUCED OVULATION AND FERTILIZED EGG QUALITY IN SADDLED BREAM OBLADA MELANURA (LINNAEUS, 1758

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    Nenad Antolović

    2013-01-01

    Full Text Available The objective of this study was to evaluate the effects of oocyte size and luteinizing hormone releasing hormone analogue (LHRHa on ovulatory success in artificial fertilization. Vitellogenic females with maximum oocyte diameters 400-550 µm were repeatedly injected with LHRHa (20 µg kg-1 per injection. Fish with maximum oocyte diameters 500 µm spawned within 48-54 h. These results demonstrate that injected LHRHa is effective for ovulation of saddled bream with maximum oocyte diameters >500µm.

  1. Artificial oocyte activation with calcium ionophore allowed fertilization and pregnancy in a couple with long-term unexplained infertility where the female partner had diminished EGG reserve and failure to fertilize oocytes despite intracytoplasmic sperm injection.

    Science.gov (United States)

    Check, J H; Summers-Chase, D; Cohen, R; Brasile, D

    2010-01-01

    To determine if fertilization and embryo development and pregnancy was possible following in vitro fertilization (IVF) in a couple with long-term unexplained infertility where the female partner had diminished egg reserve and where fertilization failure occurred despite conventional oocyte insemination and intracyoplasmic sperm injection (ICSI). In vitro fertilization was performed using a low-dose follicle stimulating hormone (FSH) stimulation protocol. Prior to ICSI, artificial oocyte activation with calcium ionophore was used. Only one mature oocyte was retrieved but it fertilized and cleaved to a good quality 8-cell embryo on day 3. A pregnancy with fetal viability was achieved but she subsequently miscarried. A second attempt successful. Fertilization and pregnancy is possible even in women with diminished egg reserve with previous failed fertilization with ICSI by performing artificial oocyte activation with calcium ionophore. It is not clear if the sperm lacked oscillin or if the eggs were not responsive to oscillin.

  2. Bovine oocyte vitrification using the Cryotop method: effect of cumulus cells and vitrification protocol on survival and subsequent development.

    Science.gov (United States)

    Zhou, X L; Al Naib, A; Sun, Da-Wen; Sun, D W; Lonergan, P

    2010-08-01

    The ability to successfully cryopreserve mammalian oocytes has numerous practical, economical and ethical benefits, which may positively impact animal breeding programs and assisted conception in humans. However, oocyte survival and development following vitrification remains poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) bovine oocytes, (2) to compare empirical and theoretical vitrification protocols, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. In Experiment 1, cumulus-enclosed and partially-denuded GV and MII oocytes were vitrified in 15% EG+15% Me(2)SO+0.5M sucrose in two steps. In Experiment 2, GV oocytes were vitrified either as above or using theoretical modeling based on permeability and osmotic tolerance characteristics in 30% EG+11.4% trehalose in three steps or 40% EG+11.4% trehalose in four steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA) 1% X-1000, 1% Z-1000 or both in three steps. In Experiment 1, the survival, cleavage and blastocyst rate of cumulus-enclosed oocytes was significantly higher than those of partially-denuded oocytes when vitrified at the GV stage (93.8% vs. 81.3%, 65.8% vs. 47.3%, 11.3% vs. 4.0%, respectively, P0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes using Cryotop method and conventional IVF procedure. Theoretical analysis of permeability characteristics and tolerance limits could not explain the low developmental competence of vitrified oocytes. (c) 2010 Elsevier Inc. All rights reserved.

  3. 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

  4. Effect of soybean phosphatidylcholine on lipid profile of bovine oocytes matured in vitro.

    Science.gov (United States)

    Pitangui-Molina, Caroline P; Vireque, Alessandra A; Tata, Alessandra; Belaz, Katia Roberta A; Santos, Vanessa G; Ferreira, Christina R; Eberlin, Marcos N; Silva-de-Sá, Marcos Felipe; Ferriani, Rui A; Rosa-E-Silva, Ana Carolina J S

    2017-04-01

    The phospholipid (PL) composition of embryo and oocyte membranes affects thermal phase behavior and several physicochemical properties such as fluidity and permeability. The characterization of PL profiles and the development of suitable in vitro maturation (IVM) protocols, that are able to modify membrane's composition, may result in significant improvements in oocyte developmental potential and cryotolerance. Using soybean phosphatidylcholine (PC) as a model supplement, we evaluated the effect of PL supplementation during IVM on bovine cumulus-oocyte-complex (COC). Substantial changes in the lipid profiles of oocyte membrane were observed and associated with pre-implantation data. The propensity of the PC supplement to become soluble in the maturation medium and/or diffuse into mineral oil was also assessed. Oocytes were matured in TCM without supplementation, i.e. control, (n=922) or supplemented with 50 or 100μM PC (n=994). The maturation media and mineral oil pre- and post- IVM, along with control and PC-treated oocytes were then analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the lipid profiles were compared via principal component analysis (PCA). Soybean PCs are bioavailable and stable in IVM medium; further, PCs did not diffuse to the mineral oil, which also remained unaltered by the metabolism of treated oocytes. PC supplementation at 100μM resulted in substantially greater relative abundances of polyunsatured PL, namely PC (32:1), PC (34:2), PC (36:6), PC (36:4), and PC (38:6), in oocyte membrane. These differences indicated that short-term exposure to the PC supplement could indeed modify the lipid composition of IVM-oocytes in a dose-dependent manner. Membrane incorporation of polyunsaturated molecular species of PC was favored, and does so without compromising the viability of the subsequent embryo in regards to cleavage, blastocyst development and hatching rate. The reported approach will allow for the

  5. Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

    Science.gov (United States)

    Canosa, S; Adriaenssens, T; Coucke, W; Dalmasso, P; Revelli, A; Benedetto, C; Smitz, J

    2017-05-01

    Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P ZP area and thickness in mature oocytes than in immature oocytes (all P ZP retardance was

  6. Selective Regulation of Oocyte Meiotic Events Enhances Progress in Fertility Preservation Methods

    Directory of Open Access Journals (Sweden)

    Onder Celik

    2015-01-01

    Full Text Available Following early embryonic germ cell migration, oocytes are surrounded by somatic cells and remain arrested at diplotene stage until luteinizing hormone (LH surge. Strict regulation of both meiotic arrest and meiotic resumption during dormant stage are critical for future fertility. Intercellular signaling system between the somatic compartment and oocyte regulates these meiotic events and determines the follicle quality. As well as the collected number of eggs, their qualities are also important for in vitro fertilization (IVF outcome. In spontaneous and IVF cycles, germinal vesicle (GV–stage oocytes, premature GV breakdown, and persistence of first meiotic arrest limit the reproductive performance. Likewise, both women with premature ovarian aging and young cancer women are undergoing chemoradiotherapy under the risk of follicle loss because of unregulated meiotic events. Understanding of oocyte meiotic events is therefore critical for the prevention of functional ovarian reserve. High levels of cyclic guanosine monophophate (cGMP, cyclic adenosine monophophate (cAMP and low phosphodiesterase (PDE 3A enzyme activity inside the oocyte are responsible for maintaining of meiotic arrest before the LH surge. cGMP is produced in the somatic compartment, and natriuretic peptide precursor C (Nppc and natriuretic peptide receptor 2 (Npr2 regulate its production. cGMP diffuses into the oocyte and reduces the PDE3A activity, which inhibits the conversion of cAMP to the 5′AMP, and cAMP levels are enhanced. In addition, oocyte itself has the ability to produce cAMP. Taken together, accumulation of cAMP inside the oocyte induces protein kinase activity, which leads to the inhibition of maturation-promoting factor and meiotic arrest also continues. By stimulating the expression of epidermal growth factor, LH inhibits the Nppc/Npr2 system, blocks cGMP synthesis, and initiates meiotic resumption. Oocytes lacking the functional of this pathway may lead to

  7. Reprogramming of fibroblast nuclei after transfer into bovine oocytes.

    Science.gov (United States)

    De Sousa, P A; Winger, Q; Hill, J R; Jones, K; Watson, A J; Westhusin, M E

    1999-01-01

    Recent landmark achievements in animal cloning have demonstrated that the events of cell differentiation can, in principle, be reversed. This reversal necessarily requires large-scale genetic reprogramming, of which little is known. In the present study we characterized the extent to which blastocyst stage-specific mRNA expression would be conserved in bovine embryos produced by nuclear transfer (NT) using fetal fibroblasts as nuclei donors (FF NT). The mRNA pool of FF NT embryos was compared with that of NT embryos reconstructed from embryonic blastomeres (Emb NT), with embryos produced under in vivo or in vitro conditions, and finally with fibroblast cells. Embryo/cell-specific mRNA pools were contrasted using differential display methodology. Random oligonucleotide primer pair combinations were used to subfractionate mRNA populations and represent individual mRNAs as copy DNA (cDNA) bands ranging in size from 100 to 800 base pairs. Regardless of whether bovine blastocysts developed in vivo or in vitro, or were derived after nuclear transplantation with embryonic blastomeres or fetal fibroblasts, their mRNA profile was highly conserved and distinct from that of fetal fibroblast cells. There was approximately 95% conservation in cDNA banding patterns between FF NT, Emb NT, and in vivo derived blastocysts, when compared with in vitro derived blastocysts. In contrast, the cDNA banding in fibroblasts was only 67% conserved with in vitro derived blastocysts (p types to serve as nuclear donors for embryo reconstruction and provide information that can be used to improve the efficiency of cloning animals by nuclear transplantation.

  8. Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification.

    Science.gov (United States)

    Lai, D; Ding, J; Smith, G W; Smith, G D; Takayama, S

    2015-01-01

    Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. The use of more 'steps' of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem-Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less

  9. An efficient method for the sanitary vitrification of bovine oocytes in straws.

    Science.gov (United States)

    Zhou, Yanhua; Fu, Xiangwei; Zhou, Guangbin; Jia, Baoyu; Fang, Yi; Hou, Yunpeng; Zhu, Shien

    2014-01-01

    At present, vitrification has been widely applied to humans, mice and farm animals. To improve the efficiency of vitrification in straw, bovine oocytes were used to test a new two-step vitrification method in this study. When in vitro matured oocytes were exposed to 20% ethylene glycol (EG20) for 5 min and 40% ethylene glycol (EG40) for 30 s, followed by treatment with 30% glycerol (Gly30), Gly40 or Gly50, a volume expansion was observed in Gly30 and Gly40 but not Gly50. This indicates that the intracellular osmotic pressure after a 30 s differs between EG40 and ranged between Gly40 (approximately 5.6 mol/L) and Gly50 (approximately 7.0 mol/L). Since oocytes are in EG40 just for only a short period of time (30 s) and at a lower temperature (4°C), we hypothesize that the main function of this step in to induce dehydration. Based on these results, we omitted the EG40 step, before oocytes were pretreated in EG20 for 5 min, exposed to pre-cooled (4°C) Gly50, for 30 s, and then dipped into liquid nitrogen. After warming, 81.1% of the oocytes survived, and the surviving oocytes developed into cleavage stage embryos (63.5%) or blastocysts (20.0%) after parthenogenetic activation. These results demonstrate that in a two-step vitrification procedure, the permeability effect in the second step is not necessary. It is possible that the second step is only required to provide adequate osmotic pressure to condense the intracellular concentration of CPAs to a level required for successful vitrification.

  10. Characterization of FSH signalling networks in bovine cumulus cells: a perspective on oocyte competence acquisition.

    Science.gov (United States)

    Khan, D R; Guillemette, C; Sirard, M A; Richard, F J

    2015-09-01

    Understanding the mechanisms regulating oocyte developmental competence is essential to enhance the clinical efficiency of assisted reproduction. FSH orchestrates the acquisition of oocyte competence, both in vivo and in vitro. Multiple pathways are implicated in FSH signalling; however, their precise coordination remains unresolved. A robust system to investigate FSH signalling is oocyte in vitro maturation (IVM) and we have previously demonstrated better bovine embryo development after FSH addition for the first 6 h during IVM. Using this model, we investigated FSH signalling in cumulus through transcriptomic and pharmacological tools. We demonstrate modulation of cumulus transcriptome by FSH mainly through protein kinase A (PKA) and epidermal growth factor (EGF) pathways. Differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism and oocyte competence. FSH required rouse-sarcoma oncogene (SRC) for EGF receptor transactivation. PKA and EGF pathway crosstalk was investigated using extracellular signal-regulated kinases (ERK1/2) phosphorylation as the functional end-point. FSH enhanced ERK1/2 activation by the EGF pathway with a simultaneous diminution through PKA. More specifically, FSH increased dual specific phosphatase (DUSP1) transcripts via PKA although DUSP1 protein did not change since EGF was required to prevent degradation. Our findings implicate FSH in PKA and EGF pathway activation, which interact to maintain appropriate levels of ERK1/2 phosphorylation and eventually cumulus expansion, metabolism and steroidogenesis. Moreover, considering the implication of the EGF pathway in GDF9 and BMP15 actions, our findings suggest that FSH may have a role in modulation of the cumulus response to oocyte-secreted factors. This information has implications for improvement of IVM and hence oocyte developmental competence. © The Author 2015. Published by Oxford University Press on behalf of the European Society of

  11. Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

    Science.gov (United States)

    Buschiazzo, Jorgelina; Ríos, Glenda L; Canizo, Jesica R; Antollini, Silvia S; Alberio, Ricardo H

    2017-01-01

    Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol

  12. Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

    Directory of Open Access Journals (Sweden)

    Jorgelina Buschiazzo

    Full Text Available Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane and cholesteryl esters (stored in lipid droplets, revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs among seasons and a dynamic organizational structure

  13. Vitrification of bovine matured oocytes and blastocysts in a paper container.

    Science.gov (United States)

    Paul, Ashit Kumar; Liang, Yuanyuan; Srirattana, Kanokwan; Nagai, Takashi; Parnpai, Rangsun

    2017-10-10

    In the present study, we aimed to determine the applicability of a paper container for the vitrification of in vitro matured (IVM) bovine oocytes. In experiment 1, IVM oocytes were exposed to vitrification solution (20% dimethylsulfoxide (DMSO), 20% ethylene glycol (EG), and 5 mol/L sucrose), using a two-step method, for 30 s; loaded onto either a paper container or Cryotop; and stored in liquid nitrogen. No significant difference (P vitrification was observed between the paper container and Cryotop. In experiment 2, IVM oocytes were exposed to either a two- or three-step vitrification solution. The three-step vitrification solution was not significantly different from the two-step solution in terms of oocyte survival, cleavage and blastocyst rates. In experiment 3, in vitro produced blastocysts were graded according to the manual of the International Embryo Transfer Society (grades 1 and 2) and vitrified using the two- and three-step methods. For grade 2 blastocysts, the three-step method showed significantly higher (P vitrification. © 2017 Japanese Society of Animal Science.

  14. The use of antifreeze protein type III for vitrification of in vitro matured bovine oocytes.

    Science.gov (United States)

    Chaves, Dowglish F; Campelo, Iana S; Silva, Mirelly M A S; Bhat, Maajid H; Teixeira, Darcio I A; Melo, Luciana M; Souza-Fabjan, Joanna M G; Mermillod, Pascal; Freitas, Vicente J F

    2016-12-01

    The aim of this study was to evaluate the use of antifreeze protein type III (AFP III) into vitrification medium on meiotic spindle morphology of in vitro matured bovine oocytes as well as the fertilization and blastocyst rates. Mature cumulus-oocyte complexes (COC) were distributed in four groups: control (untreated), vitrified without supplementation (AFP0) or supplemented with 500 (AFP500) or 1000 ng/mL (AFP1000) into vitrification solutions. Samples from each group were used to analyze the organization of meiotic spindle by confocal microscopy and the remaining COC were submitted to in vitro fertilization and culture for eight days. Control group exhibited only 15% of abnormal spindle. However, the spindle morphology was affected in all vitrified groups regardless to AFP concentration: 75.8%, 76.1% and 69.2% (P > 0.05) for AFP0, AFP500 and AFP1000, respectively. Similar cleavage rate was obtained among the vitrified groups (AFP0 = 17.9%, AFP500 = 16.9% and AFP1000 = 17.8%), but lower (P < 0.05) compared with control group (68.7%). At Day 5 of culture, embryo production rate of AFP500 (30.8%) and AFP1000 (25.0%) were similar to control group (49.4%). However, at Day 8 of culture, AFP0, AFP500 and AFP1000 groups exhibited lower (P < 0.05) blastocyst rates (10.0%, 3.8% and 9.4%, respectively) when compared to control (41.1%). In conclusion, AFP III did not preserve meiotic spindle organization against the cryoinjuries. However, the use of AFP III improved embryo development at Day 5 of culture, although this effect was not maintained up to the blastocyst formation. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules.

    Science.gov (United States)

    Vireque, A A; Camargo, L S A; Serapião, R V; Rosa E Silva, A A M; Watanabe, Y F; Ferreira, E M; Navarro, P A A S; Martins, W P; Ferriani, R A

    2009-03-01

    In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured in TCM plus FSH and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P<0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless, there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. Similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis.

  16. CoQ10 increases mitochondrial mass and polarization, ATP and Oct4 potency levels, and bovine oocyte MII during IVM while decreasing AMPK activity and oocyte death.

    Science.gov (United States)

    Abdulhasan, M K; Li, Q; Dai, J; Abu-Soud, H M; Puscheck, E E; Rappolee, D A

    2017-09-12

    We tested whether mitochondrial electron transport chain electron carrier coenzyme Q10 (CoQ10) increases ATP during bovine IVM and increases %M2 oocytes, mitochondrial polarization/mass, and Oct4, and decreases pAMPK and oocyte death. Bovine oocytes were aspirated from ovaries and cultured in IVM media for 24 h with 0, 20, 40, or 60 μM CoQ10. Oocytes were assayed for ATP by luciferase-based luminescence. Oocyte micrographs were quantitated for Oct4, pAMPK (i.e., activity), polarization by JC1 staining, and mitochondrial mass by MitoTracker Green staining. CoQ10 at 40 μM was optimal. Oocytes at 40 μM enabled 1.9-fold more ATP than 0 μM CoQ10. There was 4.3-fold less oocyte death, 1.7-fold more mitochondrial charge polarization, and 3.1-fold more mitochondrial mass at 40 μM than at 0 μM CoQ10. Increased ATP was associated with 2.2-fold lower AMPK thr172P activation and 2.1-fold higher nuclear Oct4 stemness/potency protein at 40 μM than at 0 μM CoQ10. CoQ10 is hydrophobic, and at all doses, 50% was lost from media into oil by ~ 12 h. Replenishing CoQ10 at 12 h did not significantly diminish dead oocytes. The data suggest that CoQ10 improves mitochondrial function in IVM where unwanted stress, higher AMPK activity, and Oct4 potency loss are induced.

  17. Successful pregnancy after SrCl2 oocyte activation in couples with repeated low fertilization rates following calcium ionophore treatment.

    Science.gov (United States)

    Kim, Jun-Woo; Kim, Sang-Don; Yang, Seong-Ho; Yoon, San-Hyun; Jung, Jae-Hoon; Lim, Jin-Ho

    2014-06-01

    This report describes a successful pregnancy and delivery following oocyte activation with strontium chloride (SrCl2) in couples with repeated complete fertilization failure or low fertilization rates even after calcium ionophore treatment. Eight infertile couples who showed complete fertilization failure or low fertilization rates after conventional intracytoplasmic sperm injection (ICSI) and calcium ionophore treatment. When the results of fertilization were not satisfactory in the cycles, the oocytes were artificially activated by SrCl2 for the next attempts. Oocyte activation with SrCl2 significantly increased the fertilization rates, when compared with conventional ICSI or calcium ionophore treatment (61.7% vs. 20.0% or 25.3%, respectively). There was significant increase in the proportions of good-quality cleaved embryos (50.0% vs. 0% or 12.5%, respectively). The rate of surplus embryos that developed to blastocyst stage increased in SrCl2-treated oocytes, when compared with that in ICSI with or without calcium ionophore treatment (25.7% vs. 0% or 9.1%, respectively). Five successful pregnancies were attained after oocyte activation with SrCl2, of which eight healthy children were born. Physical and mental development of the children were normal from birth to 60 months. These results suggest that SrCl2 in treatment should be considered as an effective method for artificial oocyte activation (AOA) to improve fertilization rates and embryo quality in cases with complete fertilization failure or low fertilization rates after calcium ionophore treatment.

  18. Biochemical heterogeneity, migration, and pre-fertilization release of mouse oocyte cortical granules

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    Calarco Patricia

    2003-11-01

    Full Text Available Abstract Background Oocyte cortical granules are important in the fertilization of numerous species including mammals. Relatively little is known about the composition, migration, and pre-fertilization release of mammalian oocyte cortical granules. Results Results obtained with confocal scanning laser microscopy indicated that mouse oocytes have at least two populations of cortical granules, one that bound both the lectin LCA and the antibody ABL2 and one that bound only LCA. Both types of granules were synthesized at the same time during oocyte maturation suggesting that the ABL2 antigen is targeted to specific granules by a sorting sequence. The distribution of both populations of cortical granules was then studied during the germinal vesicle to metaphase II transition. As the oocytes entered metaphase I, the first cortical granule free domain, which was devoid of both populations of cortical granules, formed over the spindle. During first polar body extrusion, a subpopulation of LCA-binding granules became concentrated in the cleavage furrow and underwent exocytosis prior to fertilization. Granules that bound ABL2 were not exocytosed at this time. Much of the LCA-binding exudate from the release at the cleavage furrow was retained in the perivitelline space near the region of exocytosis and was deduced to contain at least three polypeptides with approximate molecular weights of 90, 62, and 56 kDa. A second cortical granule free domain developed following pre-fertilization exocytosis and subsequently continued to increase in area as both, LCA and LCA/ ABL2-binding granules near the spindle became redistributed toward the equator of the oocyte. The pre-fertilization release of cortical granules did not affect binding of sperm to the overlying zona pellucida. Conclusions Our data show that mouse oocytes contain at least two populations of cortical granules and that a subset of LCA-binding cortical granules is released at a specific time (during

  19. Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro.

    Science.gov (United States)

    Roasa, L M; Choi, Y H; Love, C C; Romo, S; Varner, D D; Hinrichs, K

    2007-09-01

    In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; Pfertilization rates (4% versus 39.6%; Pfertilization rates (range, 0-3%). In Experiment 3, fertilization rates of semen frozen in an extender containing 21.5% egg yolk were lower than fertilization rates of semen from the same ejaculate but frozen with a 3% egg-yolk extender (0% versus 15%, respectively; Pfertilization in this species. These factors may help to explain the great variability in fertilization rates reported with equine IVF, both among and within laboratories.

  20. Effect of age on sperm fertility potential: oocyte donation as a model.

    Science.gov (United States)

    Gallardo, E; Simón, C; Levy, M; Guanes, P P; Remohí, J; Pellicer, A

    1996-08-01

    To determine the effect of age on sperm fecundability using oocyte donation as an in vivo model. Oocyte donation and IVF programs at the Instituto Valenciano de Infertilidad. Retrospective study in which four groups of oocyte donation cycles were established according to age of the male providing the semen sample: group 1 (n = 31) 51 years, the oldest being 64 years. All donated oocytes were obtained from patients < 35 years old. Male age, sperm characteristics (volume, concentration, motility, morphology), fertilization, embryo quality, pregnancy, implantation, and abortion rates among recipients. Similar sperm characteristics in fresh as well as after preparation for IVF were observed among males of different ages. Fertilization, embryo quality, pregnancy, and implantation were similar among the established groups. The mean age of the females included in each group significantly increased from group 1 to group 4. Age (up to 64 years) does not affect sperm characteristics or its ability to fertilize human eggs. Similarly, embryo development in vitro as well as implantation in recipient uteri are not affected by age of the male providing the semen sample.

  1. In vitro fertilization and polyspermy in the pig: factors affecting fertilization rates and cytoskeletal reorganization of the oocyte.

    Science.gov (United States)

    Suzuki, Hiroyuki; Saito, Yosuke; Kagawa, Noriko; Yang, Xiangzhong

    2003-07-01

    Polyspermy is a common phenomenon in the pig. Extensive information has become available from in vitro studies on not only the quality of oocytes but also the quality of spermatozoa. However, little information is available on the relative penetration rates of fresh and frozen spermatozoa from the same ejaculate from boars of different breeds. The present results, based on a total of 15 boars of three different breeds, revealed that the inter-breed variation in fertilization and polyspermic rates is larger than intra-breed variation. It was also shown that the incidence of polyspermy as well as penetration rate was greatly decreased by freezing and thawing, even if a higher number of sperm was coincubated with cumulus-free oocytes for a longer period compared to fresh sperm of the same ejaculate. This study focuses on the cytoskeletal organization of the oocyte with respect to the status of cumulus investment, and monospermic and polyspermic fertilization. The status of cumulus cells correlated with the density of transzonal cumulus-cell processes and with the maturation rate of oocytes and, to some degrees, the incidence of polyspermy. Polyspermic zygotes formed multiple microtubule domains in association with individual male pronuclei (PN), but in a high degree of polyspermy (more than trispermy), the pronuclear apposition did not proceed. The effect of multiple PN of paternal and maternal origin on the cytoskeletal reorganization is also discussed. Copyright 2003 Wiley-Liss, Inc.

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

    Science.gov (United States)

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

    2015-06-01

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

  3. The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes.

    Directory of Open Access Journals (Sweden)

    Gabriella Mamede Andrade

    Full Text Available The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells and cumulus-oocyte complexes (COCs to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.

  4. The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes.

    Science.gov (United States)

    Andrade, Gabriella Mamede; da Silveira, Juliano Coelho; Perrini, Claudia; Del Collado, Maite; Gebremedhn, Samuel; Tesfaye, Dawit; Meirelles, Flávio Vieira; Perecin, Felipe

    2017-01-01

    The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells) and cumulus-oocyte complexes (COCs) to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.

  5. The effect of cumulus cells on domestic cat (Felis catus) oocytes during in vitro maturation and fertilization.

    Science.gov (United States)

    Sowińska, N; Frankowska, K; Filipczyk, A; Adamaszek, A; Nalik, K; Fic, K; Pietsch-Fulbiszewska, A

    2017-04-01

    The aim of this study was to evaluate the effect of co-culture of denuded oocytes with cumulus cells (CC) or cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) and in vitro fertilization (IVF). Immature oocytes were collected from ovaries of domestic cats following a routine ovariectomy. Oocytes were matured in vitro for 24 hr within four groups: (i) denuded oocytes (DO), (ii) DO co-cultured with CC, (iii) DO co-cultured with COC and (iv) COC as a control group. In further experiments, COCs were matured in vitro for 24 hr, and then, oocytes were randomly divided into four groups as previously described and fertilized in vitro. Embryos were cultured for up to 7 days. At the end of each experiment, oocytes/embryos were stained with Hoechst 33342 solution and observed under an inverted fluorescence microscope. The results of oocyte maturation showed that their meiotic competence decreased significantly in all experimental groups, compared to the control group. The maturation rates were approximately 45%, 24%, 43% and 76% in experiment 1, and 21%, 14%, 33% and 50% in experiment 2 in groups (i), (ii), (iii) and (iv), respectively. Examination of in vitro fertilization revealed that embryos developed up to the morula stage in all experimental groups. DO and oocytes cultured with COC during fertilization showed a lower cleavage rate-36% and 25% as opposed to those co-cultured with loose CC and the control group-43% and 42%, respectively. Results of this study indicate that cumulus cells connected with an oocyte into a cumulus-oocyte complex are irreplaceable for the maturation of domestic cat oocyte, but that the addition of loose CC may be beneficial for IVF. © 2016 Blackwell Verlag GmbH.

  6. Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes.

    Science.gov (United States)

    Dietzel, E; Floehr, J; Van de Leur, E; Weiskirchen, R; Jahnen-Dechent, W

    2017-01-01

    Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate? Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts. Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate. We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching. Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm. In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM). Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined. The astacin

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

    Science.gov (United States)

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

    2014-01-01

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

  8. Failed fertilization with conventional oocyte insemination can be overcome with the ability of ICSI according to binding or failing to bind to the zona pellucida.

    Science.gov (United States)

    Check, J H; Bollendorf, A; Wilson, C

    2016-01-01

    To determine the frequency of failed fertilization with conventional oocyte insemination and to determine the ability of intracytoplasmic sperm injection (ICSI) to overcome the failed fertilization according to binding or failing to bind to the zona pellucida. Retrospective review of 12,448 in vitro fertilization (IVF) cycle to identify cycles where failed fertilization occurred following conventional oocyte insemination with seemingly normal sperm. A number of three oocytes retrieved was required. There were only 12 cases of failed fertilization (0.1%). Six were related to failure of any or few sperm attaching to the zona pellucida These six had high fertilization rates with ICSI. Six had normal attachment and five attempted another cycle, this time with ICSI. Only 60% had good fertilization. When there is failed fertilization with normal sperm oocyte binding following conventional oocyte insemination, ICSI may still be effective in 60% of the cases, but it would be probably recommended to combine ICSI with artificial oocyte activation by calcium ionophore.

  9. Oocyte retrieval timing based on spontaneous luteinizing hormone surge during natural cycle in vitro fertilization treatment.

    Science.gov (United States)

    Bodri, Daniel; Kawachiya, Satoshi; Kondo, Masae; Kato, Ryutaro; Matsumoto, Tsunekazu

    2014-04-01

    To determine the efficiency of oocyte retrieval (OR) timing based on the occurrence of spontaneous LH surge during natural cycle IVF (ncIVF) treatment. Retrospective cohort study. The cohort was divided into five subgroups according to the presumed stage of spontaneous LH surge on scheduling day (1A: before onset; 1B: surge start; 2: ascending slope; 3: peak; and 4: descending slope). Private infertility clinic. Three hundred sixty-five infertile patients who underwent 1,138 ncIVF treatment cycles during 2008-2011. Drug-free ncIVF treatment. Rate of successfully retrieved, fertilized oocytes, cleaved embryos, and live births per scheduled oocyte retrieval. In 61% of the cycles OR was scheduled before or just at the start of the LH surge (groups 1A-1B), whereas in the remaining cases it was scheduled after the surge had already started (groups 2-4). The proportion of cycles with successfully recovered (range, 71%-86%), inseminated (range, 61%-78%), fertilized oocytes (range, 47%-68%), cleaved embryos (range, 45%-66%), and live births (range, 4.1%-9.2%) was not significantly different among subgroups. In ncIVF treatment OR timing based on the occurrence of spontaneous LH surge is feasible, yielding acceptable oocyte recovery, fertilization, and embryo cleavage rates. This strategy combined with a rapid and low-risk OR procedure permits the management of a large ncIVF program on a 7-days-per-week basis within working hours. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Ovarian stimulation for oocyte cryopreservation for prevention of age-related fertility loss: one in five is a low responder.

    Science.gov (United States)

    Tsafrir, Avi; Haimov-Kochman, Ronit; Margalioth, Ehud J; Eldar-Geva, Talia; Gal, Michael; Bdolah, Yuval; Imbar, Tal; Hurwitz, Arye; Ben-Chetrit, Avraham; Goldberg, Doron

    2015-10-01

    Oocyte cryopreservation for age-related fertility loss is gaining interest considering the tendency to postpone motherhood in many societies. Little is currently known about the actual efficiency of this approach. We aimed to explore ovarian response of presumably fertile women undergoing in vitro fertilization for this indication. A total of 105 women underwent 151 stimulation cycles at mean age 37.7 ± 2.4. None had known infertility. Mean daily starting FSH dose was 371 ± 110 (225-600). Mean number of mature oocytes cryopreserved at the first completed cycle was 9.7 ± 7.5 (0-43). However, 21% of started cycles were either cancelled before egg retrieval or resulted in 0-3 mature oocytes retrieved. Therefore, women considering oocyte cryopreservation for prevention of age-related fertility decline should be encouraged to perform this procedure at younger age than, preferably before 35.

  11. The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle.

    Science.gov (United States)

    Montag, Markus; Köster, Maria; van der Ven, Katrin; Bohlen, Ulrike; van der Ven, Hans

    2012-05-01

    Following intracytoplasmic sperm injection (ICSI), some patients present low or zero fertilization rates. Artificial oocyte activation has been proposed as a suitable means to overcome this problem. This study applied artificial oocyte activation in patient cohorts with a history of no fertilization (0%, group 1), fertilization between 1 and 29% (group 2) or fertilization between 30 and 50% (group 3) in initial ICSI cycles. In the following treatment cycles, oocytes were activated after ICSI using calcium ionophore. Fertilization, pregnancy and take-home baby rates were compared with the previous cycle without activation. In group 1, fertilization rate was 41.6%, embryos for transfer were available in 82.1% of cycles, giving a clinical pregnancy rate of 18.8% and take-home baby rate of 12.8%. In group 2, despite a lower transfer rate (87.9% versus 100%, Pfertilization and clinical pregnancy rates (44.4% versus 19.3% and 31.4% versus 12.8%, respectively, Pfertilization rates differed (56.1% versus 36.8%; PArtificial oocyte activation has great potential especially in patients showing compromised fertilization rates below 30% after standard ICSI. Following intracytoplasmic sperm injection (ICSI), some patients present very low or even zero fertilization rates after ICSI. Artificial oocyte activation has been proposed as a suitable means to overcome this problem. We applied artificial oocyte activation in patients which presented a history either no fertilization, fertilization between 0 and 30% or fertilization between 30 and 50% in initial ICSI cycles. In the following treatment cycles, oocytes were activated after ICSI using a calcium ionophore. Fertilization, pregnancy and take-home baby rates were compared to the previous cycle without activation. For the groups with previously 0% or 1-29% fertilization, we noted higher fertilization rates and clinical pregnancy rates per embryo transfer. For the group with moderate fertilization, only fertilization rates

  12. 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

    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...... 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...... 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...

  13. Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization.

    Science.gov (United States)

    Wang, W H; Abeydeera, L R; Prather, R S; Day, B N

    1998-03-01

    This study was conducted to evaluate morphologic differences in pig oocytes matured in vivo and in vitro, with particular reference to the potential relationship between oocyte morphology and the occurrence of polyspermy after in vitro fertilization (IVF). In vivo-matured oocytes were surgically recovered from the oviducts of gilts with ovulated follicles on day 2 of estrus, and in vitro-matured oocytes were obtained by culturing follicular oocytes in a oocyte maturation system that has resulted previously in production of live offspring following IVF. Comparisons were made of the cytoplasm density, the diameter of oocytes with or without zona pellucida (ZP), the thickness of the ZP, the size of the perivitelline space (PVS), ZP dissolution time, and cortical granule (CG) distribution before IVF, and CG exocytosis and polyspermic penetration after IVF. Oviductal oocytes have clear areas in the cytoplasm cortex, while in vitro-matured oocytes have very dense cortex. The diameter of ovulated oocytes with ZPs was significantly (P Polyspermy rate was significantly (P polyspermy in pig oocytes.

  14. Aneuploidy involving chromosome 1 in failed-fertilized human oocytes is unrelated to maternal age

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Jingly Fung; Weier, Heinz-Ulrich G.; Nureddin, Aida.; Pedersen, Roger A.; Racowsky, Catherine

    2004-12-04

    Purpose: To study whether maternal meiotic errors in failed-fertilized oocytes involving chromosome 1 occur at frequencies similar to those involving other autosomes, and whether their frequency is affected by maternal age. Methods: Using fluorescence in situ hybridization (FISH), frequencies of aneusomy and chromatid pre-division involving chromosomes 1, 16, 18, and 21 were determined for 273 failed-fertilized oocytes. Results: The aneuploidy rate for chromosome 1 was 15.8 percent, and was neither age-dependent nor significantly different from that for chromosomes 16,18 or 21. Only chromosome 16 exhibited an age-dependent increase in aneusomy rates. The frequency of chromatid pre-division was lower for chromosome 1 than for chromosome 18 (11.9 percent vs. 25.4 percent; P=0.01), but not different from that for chromosomes 16 or 21. Conclusion: Aneuploidy involving chromosome 1 in failed-fertilized oocytes is unrelated to maternal age and occurs at a frequency similar to that for chromosomes 16, 18 and 21.

  15. Okra yield fertilized with bovine manure and biofertilizer

    Directory of Open Access Journals (Sweden)

    Ademar Pereira de Oliveira

    2013-12-01

    Full Text Available The use of bovine manure becomes an useful and economic practice for the small and medium producers of vegetables, and the okra plant normally demands high doses of organic fertilizers. This study was carried out, from January to July 2011, at the Federal University of Paraíba, in Areia city - PB, aiming to evaluate the effect of bovine manure and biofertilizer on the productive behavior of the okra plant. The experimental design used was randomized blocks, with four repetitions in factorial scheme 6 x 2, with the doses factors of bovine manure (0, 10, 20, 30, 40 and 50 t ha-1 with and without biofertilizer. The average mass of commercial fruit of okra, with and without biofertilizer was 18 and 16.5 g, respectively, in the doses of 27.5 and 60 t ha-1 of manure. The number of fruit plant-1 without biofertilizer was 30 fruits plant-1 of okra in the dose of 60 t ha-1 and with biofertilizer, the number of fruits plant-1 was 33 fruits in the dose of 28 t ha-1 of bovine manure. The productivity of commercial fruits of okra without biofertilizer was 20.4 t ha-1 and 22 t ha-1 with biofertilizer, respectively, in the doses of 60 and 31 t ha-1 of bovine manure.

  16. A mother's gift of life: exploring the concerns and ethical aspects of fertility preservation for mother-to-daughter oocyte donation

    NARCIS (Netherlands)

    Balkenende, E. M. E.; Dondorp, W.; Ploem, M. C.; Lambalk, C. B.; Goddijn, M.; Mol, F.

    2017-01-01

    With the introduction of oocyte vitrification, a special form of intergenerational intrafamilial medically assisted reproduction (IMAR) has now become feasible: fertility preservation for mother-to-daughter oocyte donation (FPMDD). For girls diagnosed with premature ovarian insufficiency (POI),

  17. Effect of urea during in vitro maturation on nuclear maturation and embryo development of bovine cumulus-oocyte-complexes

    NARCIS (Netherlands)

    Wit, de A.A.C.; Cesar, M.L.F.; Kruip, T.A.M.

    2001-01-01

    High concentrations of urea in reproductive tract fluids are detrimental to bovine reproduction. Therefore, in experiment 1, the effect of 6 mM urea on nuclear maturation of cumulus-oocyte-complexes (COC) collected from abattoir ovaries was studied. After 4, 8, 12, 16, 20, and 24 h of in vitro

  18. Validating reference microRNAs for normalizing qRT-PCR data in bovine oocytes and preimplantation embryos.

    Science.gov (United States)

    Mahdipour, Mahdi; van Tol, Helena T A; Stout, Tom A E; Roelen, Bernard A J

    2015-06-12

    MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene targets. Accurate quantification of miRNA expression using validated internal controls should aid in the understanding of their role in epigenetic modification of genome function. To date, most studies that have examined miRNA expression levels have used the global mean expression of all expressed genes or the expression of reference mRNAs or nuclear RNAs for normalization. We analyzed the suitability of a number of miRNAs as potential expression normalizers in bovine oocytes and early embryos, and porcine oocytes. The stages examined were bovine oocytes at the germinal vesicle (GV) and metaphase II stages, bovine zygotes, 2, 4 and 8 cell embryos, morulae and blastocysts, as well as porcine cumulus oocyte complexes, GV, metaphase I and II oocytes. qRT-PCR was performed to quantify expression of miR-93, miR-103, miR-26a, miR-191, miR-23b, Let-7a and U6 for bovine samples and miR-21, miR-26a, miR-93, miR-103, miR-148a, miR-182 and miR-191 for porcine oocytes. The average starting material for each sample was determined using specific standard curves for each primer set. Subsequently, geNorm and BestKeeper software were used to identify a set of stably expressed miRNAs. Stepwise removal to determine the optimum number of reference miRNAs identified miR-93 and miR-103 as the most stably expressed in bovine samples and miR-26a, miR-191 and miR-93 in porcine samples. The combination of miR-93 and miR-103 is optimal for normalizing miRNA expression for qPCR experiments on bovine oocytes and preimplantation embryos; the preferred combination for porcine oocytes is miR-26a, miR-191 and miR-93.

  19. 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.

  20. Influence of cysteamine on in vitro maturation, in vitro and in vivo fertilization of equine oocytes.

    Science.gov (United States)

    Deleuze, S; Dubois, C S; Caillaud, M; Bruneau, B; Goudet, G; Duchamp, G

    2010-02-01

    Contents The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, IVF or in vivo embryonic development under our conditions. Ovum pick up yields (52%) and maturation rates (control group: 47% and cysteamine group: 55%) were similar to those previously reported. From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and five in the cysteamine group (9%). Those two percentages were not statistically different (p > 0.05). No effect of IVF technique was seen on the success rate (6%) in each group.

  1. Holding immature bovine oocytes in a commercial embryo holding medium: High developmental competence for up to 10 h at room temperature.

    Science.gov (United States)

    Pascottini, Osvaldo Bogado; Catteeuw, Maaike; Van Soom, Ann; Opsomer, Geert

    2017-11-04

    matured, fertilized and cultured per 8 zygotes, day 8 blastocyst rate was not affected (19.8 ± 3.5% VS 20.6 ± 3.6% and 18.8 ± 3.6% VS 18.3 ± 3.4%). In conclusion, immature bovine oocytes can be successfully conserved in EHM at RT for up to 10 h without compromising their embryonic developmental competence nor quality. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. MicroRNA-130b is involved in bovine granulosa and cumulus cells function, oocyte maturation and blastocyst formation.

    Science.gov (United States)

    Sinha, Pritam Bala; Tesfaye, Dawit; Rings, Franca; Hossien, Munir; Hoelker, Michael; Held, Eva; Neuhoff, Christaine; Tholen, Ernst; Schellander, Karl; Salilew-Wondim, Dessie

    2017-06-19

    Oocyte maturation and preimplantation embryo development are controlled by array of genes that are post-transcriptionally regulated by microRNAs. With respect to this, previously, we identified altered expression of microRNA-130b (miR-130b) during oocyte maturation. Here, we aimed to investigate the role of miR-130b in bovine granulosa and cumulus cell function, oocyte maturation and preimplantation embryo development using gain- and loss-of- function approach. For this study, the granulosa cells, cumulus cells and the oocytes were collected from ovaries obtained from slaughterhouse. The genes targeted by miR-130b were identified using dual-luciferase reporter assay. The role of miR-130b in granulosa and cumulus cell function was investigated by increasing and inhibiting its expression in in vitro cultured cells using miR-130b precursor and inhibitor, respectively while the role of miR-130b on oocyte development, immature oocytes were microinjected with miR-130b precursor and inhibitor and the polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial were determined in each oocyte group 22 h after microinjection. Moreover, to investigate the role of miR-130b during preimplantation embryo development, zygote stage embryos were microinjected with miR-130b precursor or inhibitor and the cleavage rate, morula and blastocyst formation was analyzed in embryos derived from each zygote group after in vitro culture. The luciferase assay showed that SMAD5 and MSK1 genes were identified as the direct targets of miR-130b. Overexpression of miR-130b increased the granulosa and cumulus cell proliferation, while inhibition showed the opposite phenotype. Apart from these, modulation of miR-130b altered the lactate production and cholesterol biosynthesis in cumulus cells. Furthermore, inhibition of miR-130b expression during oocyte in vitro maturation reduced the first polar body extrusion, the proportion of oocytes reaching to metaphase

  3. Exposure to bisphenol A in young adult mice does not alter ovulation but does alter the fertilization ability of oocytes.

    Science.gov (United States)

    Moore-Ambriz, Teresita Rocio; Acuña-Hernández, Deyanira Guadalupe; Ramos-Robles, Brenda; Sánchez-Gutiérrez, Manuel; Santacruz-Márquez, Ramsés; Sierra-Santoyo, Adolfo; Piña-Guzmán, Belem; Shibayama, Mineko; Hernández-Ochoa, Isabel

    2015-12-15

    Follicle growth culminates in ovulation, which allows for the expulsion of fertilizable oocytes and the formation of corpora lutea. Bisphenol A (BPA) is present in many consumer products, and it has been suggested that BPA impairs ovulation; however, the underlying mechanisms are unknown. Therefore, this study first evaluated whether BPA alters ovulation by affecting folliculogenesis, the number of corpora lutea or eggs shed to the oviduct, ovarian gonadotropin responsiveness, hormone levels, and estrous cyclicity. Because it has been suggested (but not directly confirmed) that BPA exerts toxic effects on the fertilization ability of oocytes, a second aim was to evaluate whether BPA impacts the oocyte fertilization rate using an in vitro fertilization assay and mating. The possible effects on early zygote development were also examined. Young adult female C57BL/6J mice (39 days old) were orally dosed with corn oil (vehicle) or 50 μg/kgbw/day BPA for a period encompassing the first three reproductive cycles (12-15 days). BPA exposure did not alter any parameters related to ovulation. Moreover, BPA exposure reduced the percentage of fertilized oocytes after either in vitro fertilization or mating, but it did not alter the zygotic stages. The data indicate that exposure to the reference dose of BPA does not impact ovulation but that it does influence the oocyte quality in terms of its fertilization ability. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Cryotolerance of porcine blastocysts is improved by treating in vitro matured oocytes with L-carnitine prior to fertilization

    Science.gov (United States)

    LOWE, Jenna L.; BARTOLAC, Louise K.; BATHGATE, Roslyn; GRUPEN, Christopher G.

    2017-01-01

    Sufficient generation of adenosine triphosphate (ATP) by oocytes is critical for fertilization and embryo development. The objective of this study was to determine the effects of supplementing media with L-carnitine, a co-factor required for the metabolism of fatty acids, during the peri-fertilization period on embryo development and energy generation. Firstly, in vitro matured (IVM) porcine oocytes were co-incubated with sperm in IVF medium supplemented with 0‒24 mM L-carnitine. The blastocyst formation rate of the control group was greater than those of the L-carnitine groups (P L-carnitine group. Subsequently, oocytes and/or sperm were treated without or with 3 mM L-carnitine for either the 1 h pre-IVF oocyte incubation; the pre-IVF sperm preparation; the first 30 min of IVF; or the entire 5.5 h of IVF. Despite similar fertilization rates among the groups, the cleavage rate of the pre-IVF oocyte group was significantly greater than those of the other groups, except for the pre-IVF sperm group. Additionally, the oocyte ATP content and the cryotolerance of the resulting blastocysts were examined following the pre-IVF oocyte treatment. Oocyte ATP content was also similar among the groups (P > 0.05). Following vitrification, the post-warming survival rate of blastocysts derived from L-carnitine-treated oocytes was greater than that of blastocysts derived from untreated oocytes (42.4% vs. 24.9%; P L-carnitine immediately prior to insemination enhanced cleavage and improved the cryotolerance of resulting blastocysts. While the findings are suggestive of a lipolytic action, further studies are required to clarify the contributions of lipid metabolism and oxidative mechanisms to the observed effects of the L-carnitine treatment. PMID:28302936

  5. Phospholipase C-zeta deficiency as a cause for repetitive oocyte fertilization failure during ovarian stimulation for in vitro fertilization with ICSI: a case report.

    Science.gov (United States)

    Chithiwala, Zahabiya H; Lee, Hoi Chang; Hill, David L; Jellerette-Nolan, Teru; Fissore, Rafael; Grow, Daniel; Dumesic, Daniel A

    2015-09-01

    The purpose of this study is to describe impaired oocyte fertilization from phospholipase C-zeta (PLC-ζ) deficiency in normal-appearing sperm that was successfully treated using calcium (Ca(2+)) ionophore with intracytoplasmic sperm injection (ICSI) of oocytes matured in vitro. An infertile couple undergoing in vitro fertilization (IVF) experienced failed oocyte fertilization following ICSI with normal-appearing sperm. A semen sample collected from the patient was used to assess the expression of sperm PLC- ζ protein by Western blot analysis and immunofluorescence and PLC-ζ bioactivity by an in vitro model of Ca(2+) release. A second IVF cycle was performed using Ca(2+) ionophore with ICSI to enhance Ca(2+)-induced oocyte activation of oocytes matured in vitro. Sperm PLC-ζ protein deficiency was demonstrated by Western blot analysis and immunofluorescence and confirmed by reduced PLC-ζ bioactivity using an in vitro model of Ca(2+) release. Nevertheless, with this sperm and supplementation of Ca(2+) ionophore following ICSI, fertilization of four of six oocytes matured in vitro was obtained. In addition, four embryos underwent cleavage and two of them reached the blastocyst stage. Transfer of these blastocysts into the uterus led to a single pregnancy and live birth. Deficiency of PLC-ζ in normal-appearing human sperm is associated with impaired Ca(2+)-dependent oocyte activation during ICSI. Under this condition, use of Ca(2+) ionophore following ICSI of oocytes matured in vitro improves embryo developmental competence, possibly through the activation of Ca(2+)-dependent mechanisms governing fertilization and preimplantation embryogenesis.

  6. Oocyte donation in low responders to conventional ovarian stimulation for in vitro fertilization.

    Science.gov (United States)

    Remohi, J; Vidal, A; Pellicer, A

    1993-06-01

    To analyze endometrial response (endometrial dating and implantation) to exogenous administration of E2-valerate and P in women with low response to gonadotropins undergoing oocyte donation. Prospective study. A cycle in which endometrial specimens were obtained and subsequent cycles with ET were evaluated. The control group was made up of patients with premature ovarian failure (POF) undergoing the same procedure. In Vitro Fertilization program at the Instituto Valenciano de Infertilidad. A total of 37 women with low response to gonadotropins in previous cycles and 33 women with POF. First artificial cycle with E2-valerate and P in the absence of previous pituitary suppression to determine endometrial adequacy. Successive artificial cycles in which ET was performed on cycle day 17. Oocytes donated from infertile patients undergoing IVF. Serum steroid levels were measured during the artificial cycle. Histologic dating of the endometrium on cycle days 15 and 26. Ultrasonographically documented IVF-ET pregnancies. Postovulatory changes on cycle day 15 were observed in 36.4% of low responders treated with E2-valerate and P in the absence of simultaneous pituitary suppression. Pregnancy rates were higher in women with previous sufficiently (77.8%) or insufficiently (80%) estrogen-primed endometrium than in the cases showing postovulatory changes (37.5%). Pregnancy rates (PRs) per transfer were significantly higher in low responders (63.8%) than in patients with POF (37.2%). Patients with endometriosis had a 71.4% PR per transfer. Embryos derived from oocytes from polycystic ovaries had a 48.3% PR. Oocyte donation is a reliable alternative for women with low response to gonadotropins, including those with severe endometriosis. The efficacy of the steroid replacement regimen in controlling ovarian function may influence outcome. Thus, women with functional ovaries despite exogenous steroid replacement might be differently treated. Women with polycystic ovaries are an

  7. PYK2: A Calcium-sensitive Protein Tyrosine Kinase Activated in Response to Fertilization of the Zebrafish Oocyte

    Science.gov (United States)

    Sharma, Dipika; Kinsey, William H.

    2012-01-01

    Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone. PMID:23084926

  8. In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium, duration of maturation, and sperm calcium ionophore treatment, and comparison with rates of fertilization in vivo after oviductal transfer.

    Science.gov (United States)

    Hinrichs, K; Love, C C; Brinsko, S P; Choi, Y H; Varner, D D

    2002-07-01

    Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.

  9. Effect of Dipeptides on In vitro Maturation, Fertilization and Subsequent Embryonic Development of Porcine Oocytes

    Science.gov (United States)

    Tareq, K. M. A.; Akter, Quzi Sharmin; Tsujii, Hirotada; Khandoker, M. A. M. Yahia; Choi, Inho

    2013-01-01

    The effects of amino acids and dipeptides on in vitro production of porcine embryos and accumulation of ammonia in culture medium during developmental stages were examined in this study. The maturation, fertilization and development of embryonic cultures were performed in modified Tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, modified Tyrode’s albumin lactate pyruvate (mTALP) medium, and modified North Carolina State University (mNCSU)-23 medium, respectively. In addition, amino acids and dipeptides of different concentrations and combinations were used to treat the embryos. The addition of L-alanyl-L-glutamine (AlnGln)+L-glycyl-L-glutamine (GlyGln) significantly (p<0.05) improved oocyte maturation, fertilization and the incorporation and oxidation of 14C(U)-glucose when compared to the control group and other treatment groups. Additionally, 2–4 cell, 8–16 cell, morula and blastocyst development increased significantly (p<0.05) following treatment with AlnGln+GlyGln when compared to the control group and other treatment groups, while this treatment reduced the accumulation of ammonia. Taken together, these findings suggest that treatment with AlnGln+GlyGln may play an important role in increasing the rate of porcine oocyte maturation, fertilization and embryonic development by reducing the level of accumulated ammonia measured in the culture media. PMID:25049815

  10. Effect of Dipeptides on Maturation, Fertilization and Subsequent Embryonic Development of Porcine Oocytes

    Directory of Open Access Journals (Sweden)

    K. M. A. Tareq

    2013-04-01

    Full Text Available The effects of amino acids and dipeptides on in vitro production of porcine embryos and accumulation of ammonia in culture medium during developmental stages were examined in this study. The maturation, fertilization and development of embryonic cultures were performed in modified Tissue culture medium (mTCM-199 supplemented with 10% (v/v porcine follicular fluid, modified Tyrode’s albumin lactate pyruvate (mTALP medium, and modified North Carolina State University (mNCSU-23 medium, respectively. In addition, amino acids and dipeptides of different concentrations and combinations were used to treat the embryos. The addition of L-alanyl-L-glutamine (AlnGln+L-glycyl-L-glutamine (GlyGln significantly (p<0.05 improved oocyte maturation, fertilization and the incorporation and oxidation of 14C(U-glucose when compared to the control group and other treatment groups. Additionally, 2–4 cell, 8–16 cell, morula and blastocyst development increased significantly (p<0.05 following treatment with AlnGln+GlyGln when compared to the control group and other treatment groups, while this treatment reduced the accumulation of ammonia. Taken together, these findings suggest that treatment with AlnGln+GlyGln may play an important role in increasing the rate of porcine oocyte maturation, fertilization and embryonic development by reducing the level of accumulated ammonia measured in the culture media.

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

    Science.gov (United States)

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

    2013-01-01

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

  12. Prophase I mouse oocytes are deficient in the ability to respond to fertilization by decreasing membrane receptivity to sperm and establishing a membrane block to polyspermy.

    Science.gov (United States)

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

    2013-08-01

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

  13. Drug-Induced Thrombocytopenia following a Transvaginal Oocyte Retrieval for In Vitro Fertilization

    Directory of Open Access Journals (Sweden)

    Ioanna A. Comstock

    2015-01-01

    Full Text Available Drug-induced immune thrombocytopenia has been associated with hundreds of medications and can lead to devastating consequences for the patient. We present a case of a healthy 33-year-old female undergoing in vitro fertilization who developed a severe drug-induced thrombocytopenia, petechiae, and a large hemoperitoneum after receiving Cefazolin antibiotic prophylaxis for a transvaginal oocyte retrieval. The patient was admitted to the intensive care unit for resuscitation with blood products. The presence of drug-dependent platelet antibodies to Cefazolin was confirmed serologically.

  14. Drug-Induced Thrombocytopenia following a Transvaginal Oocyte Retrieval for In Vitro Fertilization.

    Science.gov (United States)

    Comstock, Ioanna A; Longmire, Michelle; Aster, Richard H; Milki, Amin A

    2015-01-01

    Drug-induced immune thrombocytopenia has been associated with hundreds of medications and can lead to devastating consequences for the patient. We present a case of a healthy 33-year-old female undergoing in vitro fertilization who developed a severe drug-induced thrombocytopenia, petechiae, and a large hemoperitoneum after receiving Cefazolin antibiotic prophylaxis for a transvaginal oocyte retrieval. The patient was admitted to the intensive care unit for resuscitation with blood products. The presence of drug-dependent platelet antibodies to Cefazolin was confirmed serologically.

  15. In vitro oocyte maturation, fertilization and culture after ovum pick-up in an endangered gazelle (Gazella dama mhorr).

    Science.gov (United States)

    Berlinguer, F; González, R; Succu, S; del Olmo, A; Garde, J J; Espeso, G; Gomendio, M; Ledda, S; Roldan, E R S

    2008-02-01

    The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks of endangered species. Studies exist on sperm cryopreservation of endangered Mohor gazelle (Gazella dama mhorr), but no work has been carried out yet on oocyte collection, fertilization and culture in this or related species. The purpose of this study was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC in the Mohor gazelle using frozen-thawed spermatozoa. Ovum pick-up was performed after ovarian stimulation with a total dose of 5.28 mg of ovine FSH. A total of 35 oocytes were recovered from 56 punctured follicles (62%) (N=6 females). Out of 29 cumulus-oocyte complexes matured in vitro, 3% were found at germinal vesicle stage, 7% at metaphase I, 21% were degenerated, and 69% advanced to metaphase II. Fertilization and cleavage rates of matured oocytes were 40 and 30%, respectively. Embryos cleaved in vitro up to the 6-8 cell stage but none progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more studies are needed to improve hormonal stimulation and oocyte harvesting, as well as IVMFC conditions, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by ovum pick-up from FSH-stimulated endangered gazelles.

  16. Cytoskeleton and chromatin reorganization in horse oocytes following intracytoplasmic sperm injection: patterns associated with normal and defective fertilization.

    Science.gov (United States)

    Tremoleda, Jordi L; Van Haeften, Theo; Stout, Tom A E; Colenbrander, Ben; Bevers, Mart M

    2003-07-01

    Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and examined by confocal laser scanning microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle and the first polar body. After sperm injection, an aster of microtubules formed adjacent to the sperm head and subsequently enlarged such that at the time of pronucleus migration and apposition it filled the entire cytoplasm. During syngamy, the microtubule matrix reorganized to form a mitotic spindle on which the chromatin of both parents aligned. Finally, after nuclear and cellular cleavage were complete, the microtubule asters dispersed into the interphase daughter cells. Sham injection induced parthenogenetic activation of 76% of oocytes, marked by the formation of multiple cytoplasmic microtubular foci that later developed into a dense microtubule network surrounding the female pronucleus. The finding that a parthenote alone can produce a microtubule aster, whereas the aster invariably forms at the base of the sperm head during normal fertilization, indicates that both gametes contribute to the formation of the zygotic centrosome in the horse. Finally, 25% of sperm-injected oocytes failed to complete fertilization, mostly due to absence of oocyte activation (65%), which was often accompanied by failure of sperm decondensation. In conclusion, this study demonstrated that union of the parental genomes in horse zygotes is

  17. EVALUATION OF IN VITRO FERTILITY OF SEMEN FROM YOUNG LOCAL BULLS IN OOCYTES FROM OVARIES OF SLAUGHTERED ANIMALS

    OpenAIRE

    Cabrera V., Próspero; Departamento de Producción Animal, Facultad de Zootecnia, Universidad Nacional Agraria La Molina (UNALM), Lima; Yoong K., Washington; Departamento de Producción Animal, Universidad Nacional Agraria La Molina, Lima; Gamarra L., Gicell; Investigador del Centro de Investigación y Enseñanza en Transferencia de Embriones (CIETE), Lima

    2012-01-01

    This study was carried out in the Center of Research and Instruction in Embryo Transfer (CIETE), sponsored by a partnership between the National Agrarian University of La Molina (UNALM) and the Ministry of Agriculture (MINAG), located at the UNALM campus. The objective was to evaluate the fertility and in vitro production of embryos of four bulls from the National Semen Bank. This study used 1031 oocytes of quality A and B obtained from ovaries of the local slaughterhouse. The oocytes were ma...

  18. Anti-hyaluronidase oligosaccharide derived from chondroitin sulfate a effectively reduces polyspermy during in vitro fertilization of porcine oocytes.

    Science.gov (United States)

    Tatemoto, Hideki; Muto, Norio; Yim, Sun-Deok; Nakada, Tadashi

    2005-01-01

    The present study was conducted to examine the effects of chondroitin sulfate A-derived oligosaccharide (ChSAO) on hyaluronidase activity and in vitro fertilization (IVF) parameters. The activity of hyaluronidase extracted from preincubated boar sperm was completely blocked by ChSAO at concentrations of 10 microg/ml or higher. After in vitro maturation of porcine cumulus-oocyte complexes, some oocytes were freed from their cumulus cells, and cumulus-intact or cumulus-free oocytes were inseminated with sperm in IVF medium containing various concentrations of ChSAO (0.1-100 microg/ml). In cumulus-intact oocytes, the penetration and the polyspermy rates (39% and 28%, respectively) were significantly decreased by treatment with 100 microg/ml ChSAO compared with those of oocytes treated without ChSAO (63% and 52%, respectively). On the contrary, in cumulus-free oocytes, the addition of 10-100 microg/ml ChSAO significantly reduced the polyspermy rate compared with the control (25-30% versus 53%, respectively), whereas ChSAO had no effect on sperm penetration. Interestingly, ChSAO added to IVF medium significantly decreased the number of sperm bound to the zona pellucida (ZP) of cumulus-free oocytes in a concentration-dependent manner between 0.1 and 100 microg/ml. However, ChSAO had no effect on the time course change in ZP modification after oocyte activation by electrostimulation and the incidence of the acrosome-reacted sperm. Treatment with 100 microg/ml ChSAO during IVF of cumulus-free oocytes significantly increased the proportion of development to the blastocyst stage after in vitro insemination. Therefore, the present findings indicate that hyaluronidase-inhibitory ChSAO is an efficient probe for promoting normal fertilization process in terms of an effective decrease in the incidence of polyspermy during IVF of porcine oocytes.

  19. Heat stress during in vitro fertilization decreases fertilization success by disrupting anti-polyspermy systems of the oocytes.

    Science.gov (United States)

    Sakatani, Miki; Yamanaka, Kenichi; Balboula, Ahmed Z; Takenouchi, Naoki; Takahashi, Masashi

    2015-01-01

    Low pregnancy rates during the summer are due, in part, to reduced fertilization. Given that elevated temperature is associated with this season, we investigated the effect of heat stress during fertilization using an in vitro model. Three experiments were performed to determine the mechanism by which exposure to elevated temperature disrupts fertilization. Oocytes were fertilized for 6 hr at 38.5°C or 41.0°C or 40.0°C with non-pre-incubated sperm, or for 6 hr at 38.5°C with sperm that had been pre-incubated at 38.5°C or 41.0°C for 4 hr. In each experiment, zygotes were cultured at 38.5°C in 5% CO(2) and 5% O(2). Rates of cleavage and blasocyst formation were reduced when fertilization occurs at elevated temperatures. The percent of sperm classified as alive, using fluorescein diacetate labeling, was decreased by pre-incubation and fertilization at 40.0°C. Although no difference was observed in sperm penetration rate, polyspermy tended to be increased by heat stress during fertilization. The zona pellucidae of zygotes formed following fertilization at 40.0°C for 6 hr were more sensitive to digestion with pronase. Furthermore, these zygotes exhibited higher hydrogen peroxide levels, measured by 2,7-dihydrodichlorofluorescein diacetate staining, and showed increased transcript abundance for HSPA1A, a gene involved in the heat-shock response, but decreased transcript abundance for UCHL1, a gene involved in preventing polyspermy. Results indicate that heat stress during fertilization is lethal to sperm, and causes oxidative stress, altered transcript abundance, and a defective block to polyspermy in the zygote. Thus, an increase in polyspermy is likely one cause of the reduced competency of zygotes fertilized under elevated temperatures to develop to the blastocyst stage. © 2014 Wiley Periodicals, Inc.

  20. 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

    2016-01-01

    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

  1. LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse

    Science.gov (United States)

    Rossi, Valerio; Lispi, Monica; Longobardi, Salvatore; Mattei, Maurizio; Rella, Francesca Di; Salustri, Antonietta; De Felici, Massimo; Klinger, Francesca G

    2017-01-01

    Premature ovarian failure and female infertility are frequent side effects of anticancer therapies, owing to the extreme sensitivity of the ovarian reserve oocytes to the damaging effects of irradiation and chemotherapy on DNA. We report here a robust protective effect of luteinizing hormone (LH) on the primordial follicle pool of prepubertal ovaries against the cisplatin (Cs)-induced apoptosis. In vitro LH treatment of prepubertal ovarian fragments generated anti-apoptotic signals by a subset of ovarian somatic cells expressing LH receptor (LHR) through cAMP/PKA and Akt pathways. Such signals, reducing the oocyte level of pro-apoptotic TAp63 protein and favoring the repair of the Cs-damaged DNA in the oocytes, prevented their apoptosis. Noteworthy, in vivo administration to prepubertal female mice of a single dose of LH together with Cs inhibited the depletion of the primordial follicle reserve caused by the drug and preserved their fertility in reproductive age, preventing significant alteration in the number of pregnancy and of delivered pups. In conclusion, these findings establish a novel ovoprotective role for LH and further support the very attracting prospective to use physiological 'fertoprotective' approaches for preventing premature infertility and risks linked to precocious menopause in young patients who survived cancer after chemotherapy. PMID:27689876

  2. 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

  3. 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.

  4. Toxic trace metals and human oocytes during in vitro fertilization (IVF)

    Science.gov (United States)

    Bloom, Michael S.; Parsons, Patrick J.; Steuerwald, Amy J.; Schisterman, Enrique F.; Browne, Richard W.; Kim, Keewan; Coccaro, Gregory A.; Conti, Giulia C.; Narayan, Natasha; Fujimoto, Victor Y.

    2010-01-01

    Trace exposures to the toxic metals mercury (Hg), cadmium (Cd) and lead (Pb) may threaten human reproductive health. The aim of this study is to generate biologically-plausible hypotheses concerning associations between Hg, Cd, and Pb and in vitro fertilization (IVF) endpoints. For 15 female IVF patients, a multivariable log-binomial model suggests a 75% reduction in the probability for a retrieved oocyte to be in metaphase-II arrest for each μg/dL increase in blood Pb concentration (relative risk (RR) = 0.25, 95% confidence interval (CI) 0.03–2.50, P = 0.240). For 15 male IVF partners, each μg/L increase in urine Cd concentration is associated with an 81% decrease in the probability for oocyte fertilization (RR = 0.19, 95% CI 0.03–1.35, P = 0.097). Because of the magnitude of the effects, these results warrant a comprehensive study with sufficient statistical power to further evaluate these hypotheses. PMID:20096775

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

    Science.gov (United States)

    Bilodeau-Goeseels, Sylvie

    2011-01-01

    Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures. Copyright © 2011 Wiley Periodicals, Inc.

  6. 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.

  7. Centrosome changes during meiosis in horse oocytes and first embryonic cell cycle organization following parthenogenesis, fertilization and nuclear transfer.

    Science.gov (United States)

    Li, Xihe; Qin, Y; Wilsher, Sandra; Allen, W R

    2006-04-01

    Various types of cell cycle organization occur in mammals. In this study, centrosome changes during meiosis in horse oocytes, and first cell cycle organization following fertilization, parthenogenesis and nuclear transfer, were monitored. Cumulus oocyte complexes harvested from horse ovaries obtained from slaughtered mares were cultured in vitro. Meiotic oocytes of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and II (MI and MII) stages were selected at various set times during in vitro maturation. Embryos at the first cell cycle stage were generated by subjecting MII stage oocytes to fertilization by intracytoplasmic sperm injection (ICSI), parthenogenetic treatment or nuclear transfer. Centrosome changes during meiosis and the first cell cycle organization were detected by indirect immunofluorescent staining, using a mouse anti-alpha-tubulin antibody for microtubules and a rabbit anti-gamma-tubulin antibody for centrosomes. These examinations showed that the centrosomes of the horse oocyte reorganize themselves from the beginning of GV stage to leave only PCM of gamma-tubulin surrounding both poles of the MI and MII stage spindles. These MII oocytes can organize the separation of metaphase chromosomes during the first embryonic cell cycle by parthenogenetic treatment. When the MII oocytes were subjected to ICSI or nuclear transfer, one or two red-stained centrosomes of gamma-tubulin were introduced by the fertilising spermatozoon or the donor cell which associated with the sperm chromatin in the fertilized embryos and with the donor cell chromatin and microtubules in the cloned embryos. This finding suggests that centrosomes are not an essential component in the formation of the metaphase spindle during meiotic maturation of horse oocytes, but they can be introduced from the spermatozoon or donor cell and are necessary for the organization of normal embryonic development.

  8. Expression and distribution of cell adhesion-related proteins in bovine parthenogenetic embryos: The effects of oocyte vitrification.

    Science.gov (United States)

    Zeng, Yan; Fu, Xiangwei; Zhou, Guangbin; Yue, Mingxing; Zhou, Yanhua; Zhu, Shien

    2013-07-01

    The objective was to investigate expression of cell adhesion-related proteins (E-cadherin, β-catenin, and the cytoskeletal protein F-actin) in bovine parthenogenetic embryos derived from vitrified-warmed oocytes. Bovine oocytes at metaphase II were randomly allocated into three groups: (1) untreated (control); (2) exposed to vitrification solution without freezing (toxicity); and (3) vitrified and warmed by the open-pulled straw method (vitrification). After parthenogenetic activation, in the vitrification group compared with the control, the timing of compaction was delayed in (108-120 vs. 96-108 hours, respectively), and the percentage of blastocysts that developed from eight-cell embryos was lower (32.08% vs. 61.03%; P vitrification delayed embryo compaction by affecting adhesion junction formation and function, immunostaining and quantitative reverse transcription polymerase chain reaction were done to characterize distribution patterns (E-cadherin, β-catenin, and the cytoskeletal protein F-actin) and expression levels of cell adhesion-related proteins (β-catenin). Distribution of β-catenin in eight-cell embryos from the vitrification group changed dramatically compared with the control and toxicity groups. Relative expression of β-catenin at the mRNA and protein levels was lower (P bovine parthenogenetic eight-cell embryos derived from vitrified-warmed oocytes were associated with embryo compaction and reduced competence for subsequent embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. 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.

  10. Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation.

    Science.gov (United States)

    Ferraz, Marcia A M M; Henning, Heiko H W; Costa, Pedro F; Malda, Jos; Melchels, Ferry P; Wubbolts, R; Stout, Tom A E; Vos, Peter L A M; Gadella, Bart M

    2017-02-28

    The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to develop a more physiological in vitro oviduct culture system capable of supporting fertilization. U-shaped chambers were produced using stereo-lithography and mounted with polycarbonate membranes, which were used as culture inserts for primary BOECs. Cells were grown to confluence and cultured at an air-liquid interface for 4 to 6 weeks and subsequently either fixed for immune staining, incubated with sperm cells for live-cell imaging, or used in an oocyte penetration study. Confluent BOEC cultures maintained polarization and differentiation status for at least 6 weeks. When sperm and oocytes were introduced into the system, the BOECs supported oocyte penetration in the absence of artificial sperm capacitation factors while also preventing polyspermy and parthenogenic activation, both of which occur in classical in vitro fertilization systems. Moreover, this "oviduct-on-a-chip" allowed live imaging of sperm-oviduct epithelium binding and release. Taken together, we describe for the first time the use of 3D-printing as a step further on bio-mimicking the oviduct, with polarized and differentiated BOECs in a tubular shape that can be perfused or manipulated, which is suitable for live imaging and supports in vitro fertilization.

  11. Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability

    OpenAIRE

    Díez, Carmen; Duque, Paloma; Gómez, Enrique; Hidalgo, C.O. (Carlos); Tamargo, Carolina; Rodríguez, Aida; Fernández, Lina; Varga, Santiago; Fernández, Alba; Facal, Nieves; Carbajo, Maite

    2011-01-01

    The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24 h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro...

  12. 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

    The final steps of oocyte capacitation and maturation are critical for embryonic development but detailed information is scarce on how the oocyte is affected during this period. In this study, 2033 oocytes were collected from 106 superovulated cattle at four different time points before ovulation...

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

    Science.gov (United States)

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

    2011-06-01

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

  14. 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...... girls and women (0-38 years of age) who each had one ovary cryopreserved for fertility preservation. METHODS: Ovaries were obtained directly from the local hospital or from collaborating hospitals (two to five hours' transport on ice). Immature oocytes were aspirated from large antral follicles visible...... 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...

  15. Random Start Ovarian Stimulation for Oocyte or Embryo Cryopreservation in Women Desiring Fertility Preservation Prior to Gonadotoxic Cancer Therapy.

    Science.gov (United States)

    Danis, Rachel B; Pereira, Nigel; Elias, Rony T

    2017-11-10

    Women of reproductive age diagnosed with cancer are often interested in preserving gametes or reproductive tissue that would allow for future genetic parenthood. Preservation of fertility is often accomplished in young cancer patients via ovarian stimulation followed by oocyte or embryo cryopreservation. Conventional stimulation protocols, however, require 2-4 weeks to complete ovarian stimulation, oocyte retrieval and possible fertilization. Such a strategy may not be feasible in patients requiring urgent cancer treatment. Recent studies have highlighted that random start ovarian stimulation can be initiated irrespective of the phase of the menstrual cycle and is an attractive alternative to conventional ovarian stimulation. The primary aim of the current review is to discuss the feasibility and success of random start ovarian stimulation for oocyte or embryo cryopreservation in women desiring fertility preservation prior to gonadotoxic cancer therapy. We performed a systematic review of medical literature published between January 2000 to June 2017 reporting the utility of random start ovarian stimulation for fertility preservation. Search terms included "fertility preservation," "cancer," "ovarian stimulation," "random-start ovarian stimulation," "embryo cryopreservation, and" "oocyte cryopreservation." Publications were included in this review only if patients underwent random start ovarian stimulation prior to cancer therapy. Nineteen publications were identified and perused by the authors. Most publications described the utility of random start ovarian stimulation in the setting of breast cancer. Radom-start stimulation was associated with a reduced time interval between ovarian stimulation initiation and oocyte or embryo cryopreservation. The yield of mature oocytes and their developmental potential into embryos was comparable between conventional and random-start protocols, albeit with higher gonadotropin doses in the latter. The current review suggests

  16. 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 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.

  17. 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.

  18. A modified swim-up method reduces polyspermy during in vitro fertilization of porcine oocytes.

    Science.gov (United States)

    Park, Chi-Hun; Lee, Sang-Goo; Choi, Don-Ho; Lee, Chang-Kyu

    2009-10-01

    The general method of porcine in vitro fertilization (IVF), involving the co-culture of both gametes in a medium drop, is thought to be the main reason for the high incidence of polyspermy. The aim of this study was to reduce the polyspermic fertilization of porcine embryos during IVF by the modified swim-up method, based on general sperm swim-up technique. Within this design, a 70 microm pore sized cell strainer was used to separate the sperm pellet placed at the bottom of a tube from the mature oocytes placed within the upper region. The separation of gametes using this permeable barrier was to ensure that only motile sperm gained access to the oocytes. It was found that the rate of polyspermy was significantly lowered for the sperm preparations from three boar breeds in modified swim-up method when compared with that of the general microdrop method (p<0.05). However, the penetration rates were found to be similar in both methods for two boar breeds. The average occurrence of blastocysts with more total cell number was higher in the modified swim-up method, while no significant difference in blastocyst rates between the two IVF methods was observed. The frequency of normal diploid embryos was also significantly higher in the modified swim-up method and polyploidy was more frequently observed in microdrop method (p<0.05). Our results demonstrated that the modified swim-up IVF method could reduce polyspermic penetration, and consequently produce better quality and karyotypically normal embryos in porcine IVF.

  19. Power Doppler assessment of follicle vascularity at the time of oocyte retrieval in in vitro fertilization cycles.

    Science.gov (United States)

    Robson, Stephen J; Barry, Michael; Norman, Robert J

    2008-12-01

    To assess the practicality of using power Doppler (PD) to assess follicular vascularity at the time of oocyte retrieval. Prospective study. University-affiliated IVF unit. Twenty-six women undergoing IVF treatment. Evaluation of follicular vascularity by using PD during oocyte retrieval. Subjective assessment of the impact of PD estimation of follicle vascularity during oocyte retrieval; reproducibility of grading of follicle vascularity. Assessment of follicle vascularity by using PD during oocyte retrieval was found to be reproducible and to add minimally to the workload of the clinician and embryologist. The grade of follicle vascularity did not correlate with the yield of oocytes, fertilization rate, or concentration of hormones in follicular fluid. Although the study group was small, there was a statistically significant trend toward higher clinical pregnancy rates when the embryo transfer cohort contained at least one embryo from a highly vascular follicle (50% vs. 15.4%). Assessment of follicle vascularity by using PD at the time of oocyte retrieval was found to be a practical alternative to other methods.

  20. Effect of jasplakinolide on the in vitro maturation of bovine oocytes ...

    African Journals Online (AJOL)

    The results showed that (1) JAS affected oocyte maturation and haploid composition of matured oocytes in a dose-dependent manner. The maturation rates were 70.3, 53.1, 39.7 and 20.6% after culturing oocytes in JAS at 100, 200, 300 and 400 nM for 24 h, respectively, which were significantly lower than the control group ...

  1. Effect of sperm cryopreservation and treatment with calcium ionophore or heparin on in vitro fertilization of horse oocytes.

    Science.gov (United States)

    Alm, H; Torner, H; Blottner, S; Nürnberg, G; Kanitz, W

    2001-09-15

    Little information is available on methods of sperm capacitation for IVF in the horse. In this study, we summarized results of several independent trials that compared acrosome reaction, hyperactivation and chromatin integrity of fresh or cryopreserved stallion spermatozoa after treatment with heparin or with calcium ionophore. We also examined the influence of spermatozoa storage (fresh vs. cryopreserved), capacitation treatment, oocyte maturation time and cumulus morphology on the penetration rate and fertilization rate. We recovered cumulus-oocyte-complexes (COCs) from ovaries by ultrasound guided follicle aspiration or by scraping of follicles from ovaries obtained at a slaughterhouse. Upon recovery, we evaluated the cumulus morphology, and the COCs were matured in vitro for 18 to 24 or 26 to 40 h. Fresh semen and cryopreserved semen were treated either with heparin (200 microg/mL) or calcium ionophore (7.14 microM). Overall, 28.4% (99/349) of the oocytes were penetrated, and 12.9% (45/349) were fertilized. Fresh spermatozoa treated with calcium ionophore showed a higher penetration rate than cryopreserved spermatozoa (36.0 vs. 0%). Fresh and heparin-treated spermatozoa showed a penetration rate of 29.1%, and the same treatment for cryopreserved spermatozoa showed a penetration rate of 33.7%; none of these differences was significant (P>0.05). Fertilization rates after the calcium and heparin treatment followed the same trend and also showed no significant differences. Prolonged maturation period resulted in higher penetration (Pfertilization rates in compact (26 to 40 h: 37.7 and 13.1% vs. 18 to 24 h: 13.1 and 2.8%) and in tendency in expanded COCs (26 to 40 h: 40.0 and 30.3% vs. 18 to 24 h: 29.4 and 13.5%). In oocytes with only a few cumulus cells, the rates tended to be higher after the shorter incubation (18 to 24 h: 33.5 and 18.8% vs. 26 to 40 h: 17.2 and 6.5%). We observed hyperactivation more frequently in fresh than in cryopreserved semen after

  2. 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...

  3. 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...

  4. 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

    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...... oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules...... 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...

  5. 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.

  6. 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.

  7. Microtubule stabilisers docetaxel and paclitaxel reduce spindle damage and maintain the developmental competence of in vitro-mature bovine oocytes during vitrification.

    Science.gov (United States)

    Pitchayapipatkul, Jakkhaphan; Somfai, Tamás; Matoba, Satoko; Parnpai, Rangsan; Nagai, Takashi; Geshi, Masaya; Vongpralub, Thevin

    2017-09-01

    This study compared the efficacy of docetaxel (DT) and paclitaxel (PT) in reducing spindle damage during vitrification and maintaining the developmental competence of in vitro-matured (IVM) bovine oocytes after vitrification and warming. Pretreatment of IVM oocytes with 0.05µM DT for 30min before vitrification resulted in significantly higher (Pvitrification or those vitrified without pretreatment. When nuclear status and spindle morphology of vitrified oocytes were assess after warming by immunostaining, DT pretreatment before vitrification resulted in a significantly higher (Pbovine oocytes with 0.05µM DT or 1.0µM PT for 30min before vitrification reduces spindle damage to the same extent, without side effects on fertilisation and development. Pretreatment with 0.05µM DT improved the developmental competence of vitrified-warmed oocytes to a greater degree than 1.0µM PT pretreatment.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    PURPOSE: The aim of the study was to investigate the maturation rate of immature oocytes collected from ovarian medulla tissue normally discarded during preparation of ovarian cortical tissue for fertility preservation. Further we evaluated survival of derived MII oocytes following vitrification ...

  9. Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility

    Directory of Open Access Journals (Sweden)

    Kaya Abdullah

    2008-02-01

    Full Text Available Abstract Background Male infertility is a major problem for mammalian reproduction. However, molecular details including the underlying mechanisms of male fertility are still not known. A thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder. Results Using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT and identified 125 putative biomarkers of fertility. We next used quantitative Systems Biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. Compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. Our data also suggests a hypothesis that low fertility sperm DNA integrity may be compromised because cell cycle: G2/M DNA damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa. Conclusion This is the first comprehensive description of the bovine spermatozoa proteome. Comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype.

  10. [Comparison of selective oxidative stress parameters in the follicular fluid of infertile women and healthy fertile oocyte donors].

    Science.gov (United States)

    Babuška, V; Cedíková, M; Rajdl, D; Racek, J; Zech, N H; Trefil, L; Mocková, A; Ulčová-Gallová, Z; Novotný, Z; Králíčková, M

    2012-12-01

    Follicular fluid (FF) affects oocyte development and disruption of its homeostasis has a crucial effect on egg developmental potential. The aim of this study was to compare the levels of selected oxidative stress markers in the FF of women with impaired fertility and healthy fertile oocytes donors. A retrospective comparative study. Faculty of Medicine in Pilsen, Charles University in Prague; Institute of Reproductive Medicine and Endocrinology, IVF Center Prof. Zech, Pilsen. Levels of homocysteine (Hcy), malondialdehyde (MDA), glutathione peroxidase (GPx), total antioxidant capacity (AOK) and total protein (CB) were analyzed in the FF. We have analysed FF of 146 women - 74 infertile patients (mean age 31 years, SD = 4.65) and 72 healthy fertile oocyte donors (mean age 26 years, SD = 4.44). Only blood free samples were studied after pooling of all FF samples each patient. The study showed a statistically significantly higher Hcy levels (p healthy fertile women compared with impaired fertility group both - comparing the two groups regardless the age and in groups of the same age range (for the age group between 20 to 29 years isp = 0.0002, for the age group between 30 to 39 years is p < 0.0001). When divided into above age ranges we found statistically significantly higher levels of MDA in the control group aged 20 to 29 years compared to same age infertile patients (p = 0.0374) and statistically significantly higher AOK in infertile women between 30 to 39 years of age compared to same age control group (p = 0.0458). The presence or on the contrary the absence of prooxidant parameters in the FF has an important role in the ability of conception and subsequent embryo development.

  11. Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse.

    Science.gov (United States)

    Bedford, Sylvia J; Kurokawa, Manabu; Hinrichs, Katrin; Fissore, Rafael A

    2004-04-01

    In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.

  12. MicroRNA Expression in Bovine Cumulus Cells in Relation to Oocyte Quality

    Directory of Open Access Journals (Sweden)

    Karen Uhde

    2017-03-01

    Full Text Available Cumulus cells play an essential role during oocyte maturation and the acquisition of fertilizability and developmental competence. Micro(miRNAs can post-transcriptionally regulate mRNA expression, and we hypothesized that miRNA profiles in cumulus cells could serve as an indicator of oocyte quality. Cumulus cell biopsies from cumulus−oocyte−complexes that either yielded a blastocyst or failed to cleave after exposure to sperm cells were analyzed for miRNA expression. On average, 332 miRNA species with more than 10 reads and 240 miRNA species with more than 50 reads were identified in cumulus cells; this included nine previously undescribed microRNAs. The most highly expressed miRNAs in cumulus cells were miR-21, members of the let-7 family and miR-155. However, no repeatable differences in miRNA expression between the cumulus cells from oocytes that became blastocysts versus those from non-cleaved oocytes were identified. Further examination of individual cumulus cell samples showed a wide variability in miRNA expression level. We therefore conclude that miRNA expression in cumulus cells cannot be used as an oocyte quality marker.

  13. Vitrification of immature bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo development.

    Science.gov (United States)

    Prentice-Biensch, Jennifer R; Singh, Jaswant; Mapletoft, Reuben J; Anzar, Muhammad

    2012-09-06

    The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage) bovine cumulus-oocyte complexes (COCs). Two experiments were conducted. In Experiment 1, COCs (n = 420) were randomly assigned to four groups: 1) CONTROL GROUP: no treatment; 2) VS1 group: COCs were exposed to vitrification solution 1 (VS1) containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3) VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS) at 37 C for 45-60 sec; and 4) Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581) were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture. Cleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution.However, both cleavage and blastocyst rates were lower (P bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.

  14. Dataset on lipid profile of bovine oocytes exposed to Lα-phosphatidylcholine during in vitro maturation investigated by MALDI mass spectrometry and gas chromatography-flame ionization detection

    Directory of Open Access Journals (Sweden)

    Alessandra A. Vireque

    2017-08-01

    Full Text Available Data presented in this article are related with the research article entitled “Effect of soybean phosphatidylcholine on lipid profile of bovine oocytes matured in vitro” [1]. This article describes the differences in the relative abundance of the lipid ions detected by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS in control and Lα-phosphatidylcholine-treated oocytes. In addition, the fatty acids (FA content in pure Lα-phosphatidylcholine supplement and oocytes was analyzed by gas chromatography-flame ionization detection (GC-FID. The dataset provides information and inputs for further studies aiming to optimize in vitro maturation conditions and cryotolerance of mammalian oocytes.

  15. A new rolling culture-based in vitro fertilization system capable of reducing polyspermy in porcine oocytes.

    Science.gov (United States)

    Kitaji, Hideki; Ookutsu, Shoji; Sato, Masahiro; Miyoshi, Kazuchika

    2015-05-01

    The high incidence of polyspermy is one of the major obstacles during in vitro fertilization (IVF) in pigs. To overcome this, we developed a novel IVF method, which involves constant rotation. Oocytes matured in vitro were mixed with spermatozoa (0.2 × 10(5) sperm/mL) in an IVF medium (200 μL) using a 200 μL PCR tube. This tube was then rotated at 1 rpm for 6 h at 38.5°C in a rotation mixer (experimental group). A second PCR tube was simultaneously cultured without rotation (control group). The rate of polyspermy was evaluated 12 h after insemination and was significantly (P < 0.05; 21.0% vs. 48.3%) lower in the experimental group than in the control group. Sperm penetration rate was similar in oocytes from the experimental and control groups (75.2% vs. 83.1%). However, monospermic fertilization rate of the oocytes was significantly (P < 0.05; 44.8% vs. 21.2%) higher in the experimental group than in the control group. Furthermore, the rate of blastocyst formation (30.1% vs. 20.8%) increased in the experimental group, as compared to the control group. This present system will contribute to increase the efficacy of blastocyst production through reduction of polyspermic penetration. © 2014 Japanese Society of Animal Science.

  16. Inhibition of boar sperm hyaluronidase activity by tannic acid reduces polyspermy during in vitro fertilization of porcine oocytes.

    Science.gov (United States)

    Tatemoto, Hideki; Tokeshi, Isao; Nakamura, Satoshi; Muto, Norio; Nakada, Tadashi

    2006-11-01

    The present study was conducted to examine the effects of three polyphenols (tannic acid, apigenin and quercetin) on hyaluronidase activity and in vitro fertilization (IVF) parameters. Among them, tannic acid showed by far the strongest potency for blocking hyaluronidase activity extracted from preincubated boar sperm, causing a dose-dependent inhibition over the range of 2-10 microg/ml. When cumulus-intact and cumulus-free oocytes were inseminated in IVF medium containing tannic acid, the penetration and the polyspermy rates were significantly decreased in the presence of 10 microg/ml tannic acid compared with those in the absence of tannic acid, and the addition of 5 microg/ml tannic acid significantly reduced the polyspermy rate (p polyspermy. Interestingly, the incidence of polyspermy was significantly reduced in oocytes inseminated with sperm pretreated with 5 microg/ml tannic acid (p polyspermy after insemination with untreated sperm. Treatment with tannic acid caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization, nor a reduction of the proteolytic activity of acrosomal contents or the number of zona-bound spermatozoa. These data suggest that an appropriate concentration of tannic acid prevents polyspermy through the inhibition of sperm hyaluronidase activity during IVF of porcine oocytes.

  17. Cryopreservation/transplantation of ovarian tissue and in vitro maturation of follicles and oocytes: Challenges for fertility preservation

    Directory of Open Access Journals (Sweden)

    Agarwal Ashok

    2008-10-01

    Full Text Available Abstract Cryopreservation of ovarian tissue and in vitro follicle maturation are two emerging techniques for fertility preservation, especially in cancer patients. These treatment regimes are opening up more options and allow for more suitable choices to preserve fertility according to the patient's specific circumstances. If these technologies are to become widely accepted, they need to be safe, easy to perform and must obtain favorable results. The generation of healthy eggs with the normal genetic complement and the ability to develop into viable and healthy embryos requires tight regulation of oocyte development and maturation. Novel freezing techniques such as vitrification, along with whole ovary cryopreservation and three-dimensional follicle cultures, have shown favorable outcomes. The scope of this article is to take a comprehensively look at the challenges still faced in order for these novel technologies to be routinely employed with the aim of successful fertility preservation.

  18. Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes

    OpenAIRE

    Carbone Maria; Tatone Carla

    2006-01-01

    Abstract Background Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that a...

  19. The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

    Science.gov (United States)

    Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2015-11-01

    Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. © 2015 Wiley Periodicals, Inc.

  20. Sensitivity of eastern oyster (Crassostrea virginica) spermatozoa and oocytes to dispersed oil: Cellular responses and impacts on fertilization and embryogenesis.

    Science.gov (United States)

    Vignier, J; Volety, A K; Rolton, A; Le Goïc, N; Chu, F-L E; Robert, R; Soudant, P

    2017-06-01

    The 2010 Deepwater Horizon (DWH) oil spill released millions of barrels of oil and dispersant into the Gulf of Mexico. The timing of the spill coincided with the spawning season of Crassostrea virginica. Consequently, gametes released in the water were likely exposed to oil and dispersant. This study aimed to (i) evaluate the cellular effects of acute exposure of spermatozoa and oocytes to surface slick oil, dispersed mechanically (HEWAF) and chemically (CEWAF), using flow-cytometric (FCM) analyses, and (ii) determine whether the observed cellular effects relate to impairments of fertilization and embryogenesis of gametes exposed to the same concentrations of CEWAF and HEWAF. Following a 30-min exposure, the number of spermatozoa and their viability were reduced due to a physical action of oil droplets (HEWAF) and a toxic action of CEWAF respectively. Additionally, reactive oxygen species (ROS) production in exposed oocytes tended to increase with increasing oil concentrations suggesting that exposure to dispersed oil resulted in an oxidative stress. The decrease in fertilization success (1-h), larval survival (24-h) and increase in abnormalities (6-h and 24-h) may be partly related to altered cellular characteristics. FCM assays are a good predictor of sublethal effects especially on fertilization success. These data suggest that oil/dispersant are cytotoxic to gametes, which may affect negatively the reproduction success and early development of oysters. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Socio-demographic and fertility-related characteristics and motivations of oocyte donors in eleven European countries.

    Science.gov (United States)

    Pennings, G; de Mouzon, J; Shenfield, F; Ferraretti, A P; Mardesic, T; Ruiz, A; Goossens, V

    2014-05-01

    Do the socio-demographic and fertility-related characteristics and motivations of oocyte donors differ in European countries? The socio-demographic and fertility-related characteristics and motivations of oocyte donors differ considerably across countries. There have been no other international studies comparing the characteristics of oocyte donors. Regarding their motivations, most studies indicate mixed motives. The proposed study was a transversal epidemiological study. Data were collected from 63 voluntarily participating assisted reproduction technology centres practising oocyte donation in 11 European countries (Belgium, Czech Republic, Finland, France, Greece, Poland, Portugal, Russia, Spain, UK and Ukraine). The survey was conducted between September 2011 and June 2012 and ran for 1-6 calendar months depending on the number of cycles of oocyte donation performed at the centre. The sample size was computed in order to allow an estimate of the percentage of a relatively rare characteristic (∼2%) with a precision (95% confidence interval) of 1%. The calculation gave 1118 donors. In total, 1423 forms were obtained from oocyte donors. All consecutive donors in these centres filled out an anonymous questionnaire when they started their hormonal stimulation, asking for their socio-demographic and fertility-related characteristics, their motivations and compensation. Population characteristics were described and compared by country of donation. Motives for donation and mean amount of money were compared between countries and according to the donors characteristics. The socio-demographic and fertility-related characteristics and motivations of oocyte donors varied enormously across European countries. The number of received forms corresponded with a participation rate of 61.9% of the cycles performed by the participating centres. Mean age was 27.4 years. About 49% of donors were fully employed, 16% unemployed and 15% student. The motivation in the total group of

  2. Effect of liquid helium vitrification on the ultrastructure and related gene expression of mature bovine oocytes after vitrifying at immature stage.

    Science.gov (United States)

    Wu, Hua; Yu, Xue-Li; Guo, Xian-Fei; Zhang, Fan; Pei, Xu-Zhe; Li, Xiao-Xia; Han, Wen-Xia; Li, Ying-Hua

    2017-01-01

    This study aimed to investigate the developmental potential of and the ultrastructural changes and gene expression differences resulting from liquid helium (LHe; -269 °C) vitrification in immature bovine oocytes. Immature oocytes were randomly divided into three groups: fresh oocytes (control, negative control), oocytes vitrified in liquid nitrogen (LN group, positive control), and oocytes vitrified in LHe (LHe group). In experiment 1, the rates of normal morphology, maturation, cleavage, and blastocyst in the LHe group were higher than those in the LN group (87.1% vs. 80.5%, 51% vs. 48%, 41.7% vs. 36.8%, and 13% vs. 8.5%, respectively; P vitrification, but more severe degeneration was observed in the LN group, such as formation of several lipid droplets, swelling of mitochondria, and absence of cortical granules. Compared with the LN group, fewer lipid droplets, relatively intact mitochondria, and clustered cortical granules were distributed in the cytoplasm of oocytes in the LHe group. In experiment 3, the mRNA expression levels of p53, CDC20, Eg5, and Npm2 were investigated by real-time quantitative polymerase chain reaction. Expression levels of the kinesin Eg5 and the apoptotic gene p53 expression levels were higher in the LN group compared with the control and LHe groups (P  0.05), the CDC20 expression in the LN and LHe groups were lower than control group (P 0.05). In conclusion, LHe vitrification decreased the negative effect of cryoinjury on the ultrastructure of some organelles and the expression of some related genes, thereby improving the viability of immature bovine oocytes compared to LN vitrification. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. 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.

  4. 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.

  5. 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.

  6. Efficacy of Kisspeptin-54 to Trigger Oocyte Maturation in Women at High Risk of Ovarian Hyperstimulation Syndrome (OHSS) During In Vitro Fertilization (IVF) Therapy.

    Science.gov (United States)

    Abbara, Ali; Jayasena, Channa N; Christopoulos, Georgios; Narayanaswamy, Shakunthala; Izzi-Engbeaya, Chioma; Nijher, Gurjinder M K; Comninos, Alexander N; Peters, Deborah; Buckley, Adam; Ratnasabapathy, Risheka; Prague, Julia K; Salim, Rehan; Lavery, Stuart A; Bloom, Stephen R; Szigeti, Matyas; Ashby, Deborah A; Trew, Geoffrey H; Dhillo, Waljit S

    2015-09-01

    In vitro fertilization (IVF) treatment is an effective therapy for infertility, but can result in the potentially life-threatening complication, ovarian hyperstimulation syndrome (OHSS). This study aimed to investigate whether kisspeptin-54 can be used to effectively and safely trigger oocyte maturation in women undergoing IVF treatment at high risk of developing OHSS. This was a phase 2, multi-dose, open-label, randomized clinical trial of 60 women at high risk of developing OHSS carried out during 2013-2014 at Hammersmith Hospital IVF unit, London, United Kingdom. Following a standard recombinant FSH/GnRH antagonist protocol, patients were randomly assigned to receive a single injection of kisspeptin-54 to trigger oocyte maturation using an adaptive design for dose allocation (3.2 nmol/kg, n = 5; 6.4 nmol/kg, n = 20; 9.6 nmol/kg, n = 15; 12.8 nmol/kg, n = 20). Oocytes were retrieved 36 h after kisspeptin-54 administration, assessed for maturation, and fertilized by intracytoplasmic sperm injection with subsequent transfer of one or two embryos. Women were routinely screened for the development of OHSS. Oocyte maturation was measured by oocyte yield (percentage of mature oocytes retrieved from follicles ≥ 14 mm on ultrasound). Secondary outcomes include rates of OHSS and pregnancy. Oocyte maturation occurred in 95% of women. Highest oocyte yield (121%) was observed following 12.8 nmol/kg kisspeptin-54, which was +69% (confidence interval, -16-153%) greater than following 3.2 nmol/kg. At all doses of kisspeptin-54, biochemical pregnancy, clinical pregnancy, and live birth rates per transfer (n = 51) were 63, 53, and 45%, respectively. Highest pregnancy rates were observed following 9.6 nmol/kg kisspeptin-54 (85, 77, and 62%, respectively). No woman developed moderate, severe, or critical OHSS. Kisspeptin-54 is a promising approach to effectively and safely trigger oocyte maturation in women undergoing IVF treatment at high risk of developing OHSS.

  7. Reduced fertility in high-yielding dairy cows: are the oocyte and embryo in danger? Part II. Mechanisms linking nutrition and reduced oocyte and embryo quality in high-yielding dairy cows.

    Science.gov (United States)

    Leroy, J L M R; Van Soom, A; Opsomer, G; Goovaerts, I G F; Bols, P E J

    2008-10-01

    Dairy cow fertility has been declining during since the mid-80s and this has given rise to numerous scientific studies in which important parts of the pathogenesis are elucidated. Reduced oocyte and embryo quality are acknowledged as major factors in the widely described low conception rates and in the high prevalence of early embryonic mortality. Apart from the importance of the negative energy balance (NEB) and the associated endocrine and metabolic consequences, there is a growing attention towards the effect of the milk yield promoting diets which are rich in energy and protein. Starch-rich diets can improve the energy status and thus the ovarian activity in the early postpartum period but the oocyte and embryo quality can suffer from such insulinogenic diets. Supplementation of dietary fat has a similar dual effect with a beneficial stimulation of the ovarian steroid production while the oocyte and the embryo display an altered energy metabolism and excessive lipid accumulation. High-protein diets can elevate the ammonia and urea concentrations in the blood, leading to changed intrafollicular, oviductal and uterine environments. Oocytes and embryos are highly sensitive to such changes in their microenvironment, possibly leading to a disturbed maturation, fertilization or early cleavage. Several nutrition-linked mechanisms, through which oocyte and/or embryo quality can be affected in modern dairy cows, well after the period of NEB, are proposed and comprehensively reviewed in the present report.

  8. 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%, Pstorage temperature of 15 °C for a 3-4h period had a significant beneficial effect on the quality 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. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Vitrification of immature bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo development

    Directory of Open Access Journals (Sweden)

    Prentice-Biensch Jennifer R

    2012-09-01

    Full Text Available Abstract Background The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage bovine cumulus-oocyte complexes (COCs. Methods Two experiments were conducted. In Experiment 1, COCs (n = 420 were randomly assigned to four groups: 1 Control group: no treatment; 2 VS1 group: COCs were exposed to vitrification solution 1 (VS1 containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3 VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS at 37 C for 45–60 sec; and 4 Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581 were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture. Results Cleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution. However, both cleavage and blastocyst rates were lower (P  Conclusions The permeating cryoprotectants (EG and DMSO present in VS1 and VS2 solutions and the time in the warming solution containing sucrose had no adverse effects on cleavage and blastocyst rates of immature bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.

  10. [Ca2+]i rise at in vitro maturation in bovine cumulus-oocyte complexes.

    Science.gov (United States)

    Silvestre, Francesco; Fissore, Rafael A; Tosti, Elisabetta; Boni, Raffaele

    2012-06-01

    An intracellular calcium ([Ca(2+)]i) rise has been described in cumulus-oocyte complexes (COCs) following luteinizing hormone (LH) exposure. Together with cAMP, Ca(2+) is a candidate signal for resumption of meiosis. Here, we analyzed if the most common hormones involved in oocyte maturation can induce the same Ca(2+) signal. In addition, we characterized the source of this signal. Immature, in vitro-matured, and roscovitine-meiotically arrested COCs were loaded with Fluo-4 AM, stimulated with hormones/growth factors, and tested for [Ca(2+)](i) variations in cumulus cells. Reagents known to inhibit or stimulate [Ca(2+)](i) rises were used to characterize these [Ca(2+)](i) dynamics. Finally, expression of LH receptors (LHRs) in COCs was analyzed by immunofluorescence. In immature COCs, follicle-stimulating hormone (FSH) elicited a single [Ca(2+)](i) rise that was higher than those induced by LH and growth hormone (GH), whereas epithelial growth factor failed to induce any changes in [Ca(2+)](i). The [Ca(2+)](i) rise induced by FSH was higher in immature COCs; was reduced in roscovitine-arrested, immature COCs; and was negligible in gonadotropin-induced, in vitro-matured COCs. In the case of spontaneous- and GH-matured COCs, however, FSH stimulation caused a lower [Ca(2+)](i) rise. The hormone-induced [Ca(2+)](i) rise was due to: (i) external Ca(2+) entry; (ii) intercellular communication; and (iii) intracellular Ca(2+) stores. Immunofluorescence revealed that LHRs were expressed throughout the cumulus cells. The above results show that: (i) gonadotropins and GH cause a [Ca(2+)](i) rise in cumulus cells; (ii) this [Ca(2+)](i) rise results from extra-, inter-, and intra-cellular cumulative Ca(2+) fluxes; and (iii) LHRs are distributed on either outer or inner cumulus cells. Copyright © 2012 Wiley Periodicals, Inc.

  11. 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

  12. Cryopreservation of in vitro matured oocytes in addition to ovarian tissue freezing for fertility preservation in paediatric female cancer patients before and after cancer therapy.

    Science.gov (United States)

    Abir, R; Ben-Aharon, I; Garor, R; Yaniv, I; Ash, S; Stemmer, S M; Ben-Haroush, A; Freud, E; Kravarusic, D; Sapir, O; Fisch, B

    2016-04-01

    Is a protocol that combines in vitro maturation of germinal vesicle-stage oocytes and their vitrification with freezing of cortical ovarian tissue feasible for use in fertility preservation for both chemotherapy-naive paediatric patients as well as patients after initiation of cancer therapy? Follicle-containing ovarian tissue as well as oocytes that can undergo maturation in vitro can be obtained from paediatric patients (including prepubertal girls) both before and after cancer therapy. Anticancer therapy reduces the number of follicles/oocytes but this effect is less severe in young patients, particularly the paediatric age group. Autotransplantation of ovarian tissue has yielded to date 60 live births, including one from tissue that was cryostored in adolescence. However, it is assumed that autografting cryopreserved-thawed ovarian cortical tissue poses a risk of reseeding the malignancy. Immature oocytes can be collected from very young girls without hormonal stimulation and then matured in vitro and vitrified. We have previously shown that there is no difference in the number of ovarian cortical follicles between paediatric patients before and after chemotherapy. A prospective study was conducted in a cohort of 42 paediatric females with cancer (before and after therapy initiation) who underwent fertility preservation procedures in 2007-2014 at a single tertiary medical centre. The study group included girls and adolescent females with cancer: 22 before and 20 after chemotherapy. Following partial or complete oophorectomy, immature oocytes were either aspirated manually ex vivo from visible small antral follicles or filtered from spent media. Oocytes were incubated in oocyte maturation medium, and those that matured at 24 or 48 h were vitrified. Ovarian cortical tissue was cut and prepared for slow-gradual cryopreservation. Anti-Mullerian hormone (AMH) levels were measured in serum before and after oophorectomy. Ovarian tissue was successfully collected from

  13. The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology, fertilization and embryo development

    NARCIS (Netherlands)

    Kidson, A.; Schoevers, E.; Langendijk, P.; Verheijden, J.; Colenbrander, B.; Bevers, M.

    2003-01-01

    In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal

  14. Improving fertilization rate in ICSI cycles by adding myoinositol to the semen preparation procedures: a prospective, bicentric, randomized trial on sibling oocytes.

    Science.gov (United States)

    Rubino, Patrizia; Palini, Simone; Chigioni, Sara; Carlomagno, Gianfranco; Quagliariello, Antonella; De Stefani, Silvia; Baglioni, Andrea; Bulletti, Carlo

    2015-03-01

    To evaluate whether the in vitro incubation of spermatozoa with myoinositol may improve the fertilization rate in ICSI cycles. This is a prospective, bicentric, randomized study on 500 MII sibling oocytes injected in 78 ICSI cycles performed between March and October 2013. Randomization of the oocytes into two groups was performed at the time of the denudation. Fertilization rates (per oocyte injected with spermatozoa treated with myoinositol versus per oocyte injected with spermatozoa treated with placebo) were measured as primary outcome and embryo morphology as secondary outcome. Clinical outcomes were also documented. Fertilization rate (78.9 ± 28.6% vs 63.2 ± 36.7, P = 0.002) and percentage of grade A embryos on day 3 (59.8 ± 35.6% vs 43.5 ± 41.5, P = 0.019) were significantly higher when spermatozoa were treated in vitro with myoinositol versus placebo. No differences were found for the expanded blastocyst formation rate. In vitro treatment of spermatozoa with myoinositol may optimize ICSI outcomes by improving the fertilization rate and embryo quality on day 3. The improvement of the number and the quality of embryos available in an ICSI cycle may have clinical utility if these findings can be confirmed.

  15. The use of mineral oil during in vitro maturation, fertilization, and embryo culture does not impair the developmental competence of pig oocytes.

    Science.gov (United States)

    Martinez, Cristina A; Nohalez, Alicia; Cuello, Cristina; Vazquez, Juan M; Roca, Jordi; Martinez, Emilio A; Gil, Maria A

    2015-03-01

    This study evaluated the effects of mineral oil (MO) overlay during maturation, fertilization, and embryo culture on the timing of nuclear maturation, the progesterone concentrations in the maturation medium, and the subsequent developmental competence of the oocyte. The results from experiment 1 showed that under the typical humidity of laboratory incubators (95%-97%), the culture media osmolality increased in the absence of oil overlay. For this reason, in experiment 2, maturation, fertilization, and embryo culture media were incubated with either an oil cover (MO group) or a microenvironment system for maximum humidity (HM group). Under these conditions, the media osmolality was maintained below 300 mOsm/kg. A portion of oocytes (n = 1414; four replicates) was removed from the maturation medium at 4- to 6-hour intervals to evaluate the nuclear maturation stage. The corresponding medium was used for progesterone measurement. The remaining oocytes were inseminated with frozen-thawed ejaculated sperm and cultured for 12 hours (n = 305) or 7 days (n = 619) to assess fertilization and embryo development parameters, respectively. The progesterone concentration of the maturation medium of the MO group was lower than 1.5 ng/mL at each time point evaluated. The values obtained at 12 hours of maturation and at the end of maturation were 20 and 55 times lower than those of the HM group, respectively. However, compared with the HM group, oil overlay did not delay oocyte progression to metaphase I and II and did not influence normal fertilization, cleavage, blastocyst formation, and total cell number in blastocysts. In conclusion, despite its pronounced impact on progesterone concentration, the use of MO did not affect the time course of oocyte maturation or oocyte developmental competence. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence.

    Directory of Open Access Journals (Sweden)

    Md Mahmodul Hasan Sohel

    Full Text Available Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down in exosomes and 30 miRNAs differentially expressed (21 up and 9 down in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.

  17. Synchrotron X-ray diffraction to detect glass or ice formation in the vitrified bovine cumulus-oocyte complexes and morulae.

    Science.gov (United States)

    Anzar, Muhammad; Grochulski, Pawel; Bonnet, Brennan

    2014-01-01

    Vitrification of bovine cumulus-oocyte complexes (COCs) is not as successful as bovine embryos, due to oocyte's complex structure and chilling sensitivity. Synchrotron X-ray diffraction (SXRD), a powerful method to study crystal structure and phase changes, was used to detect the glass or ice formation in water, tissue culture medium (TCM)-199, vitrification solution 2 (VS2), and vitrified bovine COCs and morulae. Data revealed Debye's rings and peaks associated with the hexagonal ice crystals at 3.897, 3.635, 3.427, 2.610, 2.241, 1.912 and 1.878 Å in both water and TCM-199, whereas VS2 showed amorphous (glassy) appearance, at 102K (-171°C). An additional peak of sodium phosphate monobasic hydrate (NaH2PO4.H2O) crystals was observed at 2.064 Å in TCM-199 only. All ice and NaH2PO4.H2O peaks were detected in the non-vitrified (control) and vitrified COCs, except two ice peaks (3.145 and 2.655 Å) were absent in the vitrified COCs. The intensities of majority of ice peaks did not differ between the non-vitrified and vitrified COCs. The non-vitrified bovine morulae in TCM-199 demonstrated all ice- and NaH2PO4.H2O-associated Debye's rings and peaks, found in TCM-199 alone. There was no Debye's ring present in the vitrified morulae. In conclusion, SXRD is a powerful method to confirm the vitrifiability of a solution and to detect the glass or ice formation in vitrified cells and tissues. The vitrified bovine COCs exhibited the hexagonal ice crystals instead of glass formation whereas the bovine morulae underwent a typical vitrification.

  18. Synchrotron X-ray diffraction to detect glass or ice formation in the vitrified bovine cumulus-oocyte complexes and morulae.

    Directory of Open Access Journals (Sweden)

    Muhammad Anzar

    Full Text Available Vitrification of bovine cumulus-oocyte complexes (COCs is not as successful as bovine embryos, due to oocyte's complex structure and chilling sensitivity. Synchrotron X-ray diffraction (SXRD, a powerful method to study crystal structure and phase changes, was used to detect the glass or ice formation in water, tissue culture medium (TCM-199, vitrification solution 2 (VS2, and vitrified bovine COCs and morulae. Data revealed Debye's rings and peaks associated with the hexagonal ice crystals at 3.897, 3.635, 3.427, 2.610, 2.241, 1.912 and 1.878 Å in both water and TCM-199, whereas VS2 showed amorphous (glassy appearance, at 102K (-171°C. An additional peak of sodium phosphate monobasic hydrate (NaH2PO4.H2O crystals was observed at 2.064 Å in TCM-199 only. All ice and NaH2PO4.H2O peaks were detected in the non-vitrified (control and vitrified COCs, except two ice peaks (3.145 and 2.655 Å were absent in the vitrified COCs. The intensities of majority of ice peaks did not differ between the non-vitrified and vitrified COCs. The non-vitrified bovine morulae in TCM-199 demonstrated all ice- and NaH2PO4.H2O-associated Debye's rings and peaks, found in TCM-199 alone. There was no Debye's ring present in the vitrified morulae. In conclusion, SXRD is a powerful method to confirm the vitrifiability of a solution and to detect the glass or ice formation in vitrified cells and tissues. The vitrified bovine COCs exhibited the hexagonal ice crystals instead of glass formation whereas the bovine morulae underwent a typical vitrification.

  19. Xenotransplantation of cryopreserved human ovarian tissue--a systematic review of MII oocyte maturation and discussion of it as a realistic option for restoring fertility after cancer treatment.

    Science.gov (United States)

    Dittrich, Ralf; Lotz, Laura; Fehm, Tanja; Krüssel, Jan; von Wolff, Michael; Toth, Bettina; van der Ven, Hans; Schüring, Andreas N; Würfel, Wolfgang; Hoffmann, Inge; Beckmann, Matthias W

    2015-06-01

    To systematically review the reporting of MII (MII) oocyte development after xenotransplantation of human ovarian tissue. Systematic review in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). Not applicable. Not applicable. Formation of MII oocytes after xenotransplantation of human ovarian tissue. Any outcome reported in Pubmed. Six publications were identified that report on formation of MII oocytes after xenotransplantation of human ovarian tissue. Xenografting of human ovarian tissue has proved to be a useful model for examining ovarian function and follicle development in vivo. With human follicles that have matured through xenografting, the possibility of cancer transmission and relapse can also be eliminated, because cancer cells are not able to penetrate the zona pellucida. The reported studies have demonstrated that xenografted ovarian tissue from a range of species, including humans, can produce antral follicles that contain mature (MII) oocytes, and it has been shown that mice oocytes have the potential to give rise to live young. Although some ethical questions remain unresolved, xenotransplantation may be a promising method for restoring fertility. This review furthermore describes the value of xenotransplantation as a tool in reproductive biology and discusses the ethical and potential safety issues regarding ovarian tissue xenotransplantation as a means of recovering fertility. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  20. Dynamics of intracellular phospholipid membrane organization during oocyte maturation and successful vitrification of immature oocytes retrieved by ovum pick-up in cattle.

    Science.gov (United States)

    Aono, Akira; Nagatomo, Hiroaki; Takuma, Tetsuya; Nonaka, Rika; Ono, Yoshitaka; Wada, Yasuhiko; Abe, Yasuyuki; Takahashi, Masashi; Watanabe, Tomomasa; Kawahara, Manabu

    2013-05-01

    The objective was to determine if immature bovine oocytes with cumulus cells at the germinal vesicle (GV) stage could be vitrified by aluminum sheets (AS; pieces of sheet-like aluminum foil). Cleavage rates in fertilized oocytes previously vitrified by the AS procedure were higher than those vitrified by a nylon-mesh holder (NM) procedure (89.3 ± 2.1% vs. 65.0 ± 3.7%). Cleaved embryos derived from the AS but not from the NM procedures developed to blastocysts. Furthermore, to investigate the effects of vitrifying GV oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes, the intracellular phospholipid membrane (IM) was stained with the lipophilic fluorescent dye, 3,3'-dioctadecyloxa-carbocyanine perchlorate. After vitrification by AS, the IM remained intact relative to that of oocytes vitrified by NM. During in vitro maturation, reorganization of the IM was also undamaged in oocytes vitrified by AS before oocyte maturation, and the IM within oocytes vitrified by the NM procedure was evidently impaired. Finally, vitrification (AS) was used for GV oocytes collected using the ovum pick-up method. A bull calf was born after in vitro production and subsequent embryo transfer. The vitrification techniques described herein should facilitate generation of viable in vitro production bovine blastocysts using oocytes recovered using the ovum pick-up method. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. [The hormonal micromilieu and oocyte status in the stimulated in vitro fertilization cycle].

    Science.gov (United States)

    Sudik, R; Fliess, F R; Kunkel, S; Blödow, G

    1989-01-01

    FSH, LH, PRL, estradiol-17 beta and progesterone were determined in 651 follicular fluids of 173 patients treated by ovarian stimulation for IVF. The stimulation was performed according to 5 different schemes: clomiphene/HCG, HMG (Pergonal)/HCG, HPG (Anthrogon)/HCG, clomiphene/HPG/HCG, Folistiman (heterologeous pituitary gonadotropin)/HCG. The mean levels of hormones of all follicles of each stimulation scheme were determined and differences between the groups were estimated by Student's t-test and the x-square-test. Additionally, in a hierarchy of follicles made depending on the follicular fluid volume the hormonal levels were compared between different rank numbers of one stimulation group and between different groups of stimulation. The maturation of oocytes judged by a maturation index and their ability for cleavage in culture after insemination was investigated in relation to the hormonal content of the follicular fluid. Stimulation by gonadotropins (Pergonal, Anthrogon, Folistiman) led to an decreased mean level of follicular steroids. This was related to an increased part of follicles poor in steroids after stimulation by gonadotropins. Within the follicular population follicles stimulated by clomiphene/HCG had a reduction of the levels of estradiol in higher rank numbers, but there were no clear evidences for such a reduction in the other stimulated groups. In all stimulation groups a significant reduction of progesterone levels was observed in higher rank numbers of follicles. Oocytes with a high maturation index mainly derived from follicles rich in progesterone. After insemination, development of oocytes in culture was compatible even with very high or low levels of hormones. There was no relation of the levels in FSH and LH to cumulus expansion and levels of estradiol of the follicular fluid. There was also no clear correlation between the levels of prolactin in follicular fluid and the cleavage rate.

  2. 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.

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

    Science.gov (United States)

    Sprícigo, José F W; Diógenes, Mateus N; Leme, Ligiane O; Guimarães, Ana L; Muterlle, Carolle V; Silva, Bianca Damiani Marques; Solà-Oriol, David; Pivato, Ivo; Silva, Luciano Paulino; Dode, Margot A N

    2015-01-01

    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 (Pvitrification because the blastocyst rate at D7 was similar (P>0.05) 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 (Pbovine oocytes to vitrification.

  4. Relationship between bovine fertility and the number of spermatozoa penetrating the cervical mucus within straws.

    Science.gov (United States)

    Taş, Muzaffer; Bacinoglu, Suleyman; Cirit, Umüt; Ozdaş, Ozen Banu; Ak, Kemal

    2007-09-01

    In this study, by using a recently developed test technique, the relationship between the total spermatozoa number penetrating determined sites of bovine cervical mucus in straws and potential fertility of bulls, and other spermatological characteristics were investigated. Furthermore, we aimed to determine the effect on the test results, of two different incubation temperatures (37 and 41 degrees C) and two sperm penetration distance ranges (PDRs). Frozen semen samples of six Holstein bulls were used in the study. The bulls were divided into two fertility groups (high and low fertility) according to the "non-return rates" (NRR). For the penetration test, cervical mucus was drawn into transparent plastic straws and incubated with semen at 37 and 41 degrees C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at -20 degrees C. On the evaluation day, concentrations of spermatozoa penetrated to the PDRs, each of which was 2.5 mm, between 32.5 and 35 mm (first penetration distance range, PDR1), and 50 and 52.5 mm (second penetration distance range, PDR2) distance in the straws from the open end, were measured. When compared with the low fertility group, bulls from the high fertility group showed a higher number of spermatozoa at the determined PDRs, and a significant positive correlation was found between the total number of spermatozoa at the penetration distances and the NRR scores of the bulls.

  5. Efficacy of Kisspeptin-54 to Trigger Oocyte Maturation in Women at High Risk of Ovarian Hyperstimulation Syndrome (OHSS) During In Vitro Fertilization (IVF) Therapy

    Science.gov (United States)

    Abbara, Ali; Jayasena, Channa N.; Christopoulos, Georgios; Narayanaswamy, Shakunthala; Izzi-Engbeaya, Chioma; Nijher, Gurjinder M. K.; Comninos, Alexander N.; Peters, Deborah; Buckley, Adam; Ratnasabapathy, Risheka; Prague, Julia K.; Salim, Rehan; Lavery, Stuart A.; Bloom, Stephen R.; Szigeti, Matyas; Ashby, Deborah A.; Trew, Geoffrey H.

    2015-01-01

    Context: In vitro fertilization (IVF) treatment is an effective therapy for infertility, but can result in the potentially life-threatening complication, ovarian hyperstimulation syndrome (OHSS). Objective: This study aimed to investigate whether kisspeptin-54 can be used to effectively and safely trigger oocyte maturation in women undergoing IVF treatment at high risk of developing OHSS. Setting and Design: This was a phase 2, multi-dose, open-label, randomized clinical trial of 60 women at high risk of developing OHSS carried out during 2013–2014 at Hammersmith Hospital IVF unit, London, United Kingdom. Intervention: Following a standard recombinant FSH/GnRH antagonist protocol, patients were randomly assigned to receive a single injection of kisspeptin-54 to trigger oocyte maturation using an adaptive design for dose allocation (3.2 nmol/kg, n = 5; 6.4 nmol/kg, n = 20; 9.6 nmol/kg, n = 15; 12.8 nmol/kg, n = 20). Oocytes were retrieved 36 h after kisspeptin-54 administration, assessed for maturation, and fertilized by intracytoplasmic sperm injection with subsequent transfer of one or two embryos. Women were routinely screened for the development of OHSS. Main Outcome Measure: Oocyte maturation was measured by oocyte yield (percentage of mature oocytes retrieved from follicles ≥ 14 mm on ultrasound). Secondary outcomes include rates of OHSS and pregnancy. Results: Oocyte maturation occurred in 95% of women. Highest oocyte yield (121%) was observed following 12.8 nmol/kg kisspeptin-54, which was +69% (confidence interval, −16–153%) greater than following 3.2 nmol/kg. At all doses of kisspeptin-54, biochemical pregnancy, clinical pregnancy, and live birth rates per transfer (n = 51) were 63, 53, and 45%, respectively. Highest pregnancy rates were observed following 9.6 nmol/kg kisspeptin-54 (85, 77, and 62%, respectively). No woman developed moderate, severe, or critical OHSS. Conclusion: Kisspeptin-54 is a promising approach to effectively and safely

  6. Sperm dosage and site of insemination in relation to fertility in bovines

    Directory of Open Access Journals (Sweden)

    Tushar Kumar Mohanty

    2018-01-01

    Full Text Available Low sperm numbers in artificial insemination (AI-doses are being used widely to make the best use of high genetic value bulls as well as sex-sorted semen. Sperm concentration needed for AI to obtain reasonable fertility, taking genetic value of bull and numerous others components into consideration is one of the essential constituents for successful AI breeding program. However, low sperm concentrations in AI-doses lead to reducing post-thaw viability. The reduction in viability of low sperm doses may be affected by fresh semen volume, sperm number and seminal plasma level at final dilution. Reduction in quality and fertility of low sperm doses is one of the limitations for their use in successful AI programme. Sperm number per AI required to achieve optimum fertility is one of the main crucial things to AI industry, and numerous efforts have been made in this regard. Due to great variability among bulls, sperm number per AI could be a limiting factor in achieving acceptable fertility values. Fertility of low sperm doses may vary among bulls, and non-return rates (NRRs with low sperm doses may be determined by fertility level of bull. On the basis of individual bulls, sperm numbers in AI doses needed to be adjusted to reduce the variations in NRRs among bulls. Utilizing high fertile bulls for low sperm doses with acceptable non-return rates (NRRs may be a way to cover a large number of bovines under AI in countries like India. Deposition site within the uterine horn may alter non return rates following inseminations with low sperm doses. Following deep-uterine inseminations, acceptable pregnancies may be achieved with low sperm doses and even if ovulation side is unknown.

  7. Low-Dose Urinary Human Chorionic Gonadotropin Is Effective for Oocyte Maturation in In Vitro Fertilization/ Intracytoplasmic Sperm Injection Cycles Independent of Body Mass Index.

    Science.gov (United States)

    R Hoyos, Luis; Khan, Sana; Dai, Jing; Singh, Manvinder; P Diamond, Michael; E Puscheck, Elizabeth; O Awonuga, Awoniyi

    2017-01-01

    Currently, there is no agreement on the optimal urinary derived human chorionic gonadotropin (u-hCG) dose requirement for initiating final oocyte maturation prior to oocyte collection in in vitro fertilization (IVF), but doses that range from 2500- 15000 IU have been used. We intended to determine whether low dose u-hCG was effective for oocyte maturation in IVF/intracytoplasmic sperm injection (ICSI) cycles independent of body mass index (BMI). We retrospectively evaluated a cohort of 295 women who underwent their first IVF/ICSI cycles between January 2003 and December 2010 at the Division of Reproductive Endocrinology and Infertility, Wayne State University, Detroit, MI, USA. Treatment cycles were divided into 3 groups based on BMI (kg/ m(2)): <25 (n=136), 25- <30 (n=84), and ≥30 (n=75) women. Patients received 5000, 10000 or 15000 IU u-hCG for final maturation prior to oocyte collection. The primary outcome was clinical pregnancy rates (CPRs) and secondary outcome was live birth rates (LBRs). Only maternal age negatively impacted (P<0.001) CPR [odds ratio (OR=0.85, confidence interval (CI: 0.79-0.91)] and LBR (OR=0.84, CI: 0.78-0.90). Administration of lower dose u-hCG was effective for oocyte maturation in IVF and did not affect the CPRs and LBRs irrespective of BMI. Women's BMI need not be taken into consideration in choosing the appropriate dose of u-hCG for final oocyte maturation prior to oocyte collection in IVF. Only maternal age at the time of IVF negatively influenced CPRs and LBRs in this study.

  8. Effects of caffeine, cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes.

    Science.gov (United States)

    Maalouf, W E; Lee, J-H; Campbell, K H S

    2009-04-15

    The objectives of these studies were to determine the effects of cumulus cell removal and caffeine treatment on the development of in vitro matured ovine oocytes aged in vitro until until fertilization. Oocytes were denuded (DO) at 24h post-onset of maturation (hpm), control cumulus oocyte complexes (COC's) and DO groups were fertilized at 24 hpm or returned to culture in the presence or absence of 10mM caffeine and fertilized at 30 hpm. Removal of cumulus cells and aging both increased polyspermy, caffeine reduced this increase, however, with the exception of DO's (30 hpm) vs. COC's (24 hpm) the differences were not statistically significant. Aging significantly decreased cleavage between COC groups at 24 hpm and 30 hpm and caffeine did not affect this (68.4%, 73.4%, 74.0% respectively). In contrast, the frequency of cleavage was significantly reduced in the DO (24 hpm) group as compared to COC controls (45.6% vs. 68.4% (P0.05)). In summary caffeine treatment of aged COC's had no significant effect on the frequency of development, however, in aged DO's caffeine treatment statistically increased development to blastocyst and lowered the frequency of polyspermy.

  9. Influence of Insulin-Like Growth Factor-I on Maturation and Fertilization Rate of Immature Oocyte and Embryo Development in NMRI Mouse with TCM199 and α-MEM Medium.

    Science.gov (United States)

    Toori, Mehdi Akbartabar; Mosavi, Esmaeil; Nikseresht, Mohsen; Barmak, Mehrzad Jafari; Mahmoudi, Reza

    2014-12-01

    In vitro maturation (IVM) of oocytes and subsequent, in vitro fertilization (IVF) for the generation of embryos in the laboratory has important values. Growth factors are a component of a complex system of autocrine and paracrine factors that have a regulatory role in ovarian function and affect oocyte maturation. Therefore, the aim of this study is to evaluate the effect of IGF-I on IVM and IVF of mice oocytes during culture with α-MEM and TCM199 medium. Cumulus oocyte complexes (COCs) and denuded oocyte were obtained from 4-6 week old NMRI mice and underwent in vitro maturation and in vitro fertilization in presence or absence of IGF-I with α-MEM and TCM199. Maturation rate (79.6%), fertilization rate (87.2%), two cells development rate (79.5%) and blastocyst rate(43.2%) was higher in COCs cultured in α-MEM with IGF-I, while lower maturation rate (50.6%) fertilization rate (61%), two cells development rate (48.8%) and blastocyst rate(14.6%) were seen in cultured denuded oocytes (DOs) in TCM199 without growth factor. As well as, maturation fertilization, two cells development and blastocyst rates in COCs were higher than DOs. Our findings have shown that IGF-I is involved in the oocyte biology and improve the oocyte maturation, fertilization and embryo development to blastocyst competence in vitro. In addition, it has also shown that cumulus cells are vital for oocyte development when IGF-1 added to the mediums.

  10. XY Sox9 embryonic loss-of-function mouse mutants show complete sex reversal and produce partially fertile XY oocytes.

    Science.gov (United States)

    Lavery, Rowena; Lardenois, Aurélie; Ranc-Jianmotamedi, Fariba; Pauper, Eva; Gregoire, Elodie P; Vigier, Caroline; Moreilhon, Chimene; Primig, Michael; Chaboissier, Marie-Christine

    2011-06-01

    Gonadal differentiation is the first step of mammalian sex determination. The expression of the Y chromosomal testis determining factor Sry leads to up-regulation of the transcription factor Sox9 which promotes testis differentiation. Previous studies showed that Sox9 deficiency induces expression of ovarian markers in XY mutant fetal gonads before they die. To better understand the genome-wide transcriptional profile underlying this process we compared samples from XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads in which Sox9 is ablated in Sertoli-precursor cells during early stages of gonad development to XX Sox9(flox/flox) ovaries and XY Sox9(flox/flox) testes at E13.5. We found a complex mRNA signature that indicates wide-spread transcriptional de-regulation and revealed for XY mutants at E13.5 an intermediate transcript profile between male and female gonads. However, XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads develop as ovaries containing XY developing follicles at P0 but less frequently so than in XX control ovaries. Furthermore, we studied the extent to which developing XY mutant ovaries are able to mediate adult fertility and observed that XY oocytes from XY mutant ovaries are competent for fertilization; however, two thirds of them fail to develop beyond two-cell stage embryos. Taken together, we found that XY Sf1:Cre(Tg/+); Sox9(flox/flox) females are capable of producing viable offspring albeit at a reduced level. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. 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

    cells and then transferred to the oviducts of a recipient ewe, 30 h post oestrus, for a 6.5 day period of in vivo culture. Similar rates of fertilization ( 54-58%) and blastocyst development from cleaving zygotes ( 48-69%) were achieved in both experi- ments in vitro with no evident benefit of including...... oviductal cells. In fact, fewer (P= 0 .02) blastocysts developed from cleaved embryos when co-cultured for the 96 h period (Group 4). Whilst the blastocyst development rates obtained in vitro ( 45-58%) were similar to, or higher than (P=0.03 ), those obtained in vivo ( 43%), more than 50% of in vitro cul...

  12. Effect of Female Body Mass Index on Oocyte Quantity in Fertility Treatments (IVF: Treatment Cycle Number Is a Possible Effect Modifier. A Register-Based Cohort Study.

    Directory of Open Access Journals (Sweden)

    Mette Wulf Christensen

    Full Text Available Overweight and obese women may require higher doses of gonadotrophin when undergoing In Vitro Fertilization Treatment (IVF. Consequently, one may expect a sub-optimal oocyte retrieval in the first treatment cycle and thus a larger compensation in gonadotrophin-dose in the following treatment-cycles and a more favorable outcome. The main objective was to explore if treatment cycle number modifies the outcome when investigating the effect of female Body Mass Index (BMI on oocyte quantity in IVF.A historical cohort study was conducted on 5,342 treatment-cycles during the period 1999-2009. Exclusion criteria were missing information on BMI or treatment type. Further, women were excluded if they had ovulated before oocyte retrieval. According to baseline BMI, women were divided into four categories following the World Health Organization standards. Multiple linear regressions analyses were performed accounting for the non-independence of ≥2 cycles in a woman.Stratification according to cycle number revealed a more suboptimal outcome in the first treatment- cycles than in the following cycles, suggesting a possible interaction or effect modification from cycle number or a factor related to cycle number. The median dose of total follicular stimulating hormone given to the four BMI groups could not straight forwardly explain the less optimal oocyte outcome observed in first treatment cycles. No statistically significant differences were observed in oocyte yield for underweight, overweight and obesity compared to normal weight women when analyzing all treatment-cycles. Overweight women had significantly fewer mature (MII oocytes (p = 0.009 than normal weight women, whereas no differences was observed for underweight and obese women.Our study suggests a possible interaction or effect modification related to treatment cycle number. Investigating the effects of BMI on IVF-results in first treatment-cycles alone should be carried out cautiously.

  13. Gonadotropin-releasing hormone agonist trigger increases the number of oocytes and embryos available for cryopreservation in cancer patients undergoing ovarian stimulation for fertility preservation.

    Science.gov (United States)

    Pereira, Nigel; Kelly, Amelia G; Stone, Logan D; Witzke, Justine D; Lekovich, Jovana P; Elias, Rony T; Schattman, Glenn L; Rosenwaks, Zev

    2017-09-01

    To compare the oocyte and embryo yield associated with GnRH-agonist triggers vs. hCG triggers in cancer patients undergoing controlled ovarian stimulation (COS) for fertilization preservation. Retrospective cohort study. Academic center. Cancer patients undergoing COS with letrozole and gonadotropins or gonadotropin-only protocols for oocyte or embryo cryopreservation. Gonadotropin-releasing hormone agonist or hCG trigger. Number of metaphase II (MII) oocytes or two-pronuclei (2PN) embryos available for cryopreservation were primary outcomes. Separate multivariate linear regression models were used to assess the effect of trigger type on the primary outcomes, after controlling for confounders of interest. A total of 341 patients were included, 99 (29.0%) in the GnRH-agonist group and 242 (71%) in the hCG group. There was no difference in the baseline demographics of patients receiving GnRH-agonist or hCG triggers. Within the letrozole and gonadotropins group (n = 269), the number (mean ± SD, 11.8 ± 5.8 vs. 9.9 ± 6.0) and percentage of MII oocytes (89.6% vs. 73.0%) available for cryopreservation was higher with GnRH-agonist triggers compared with hCG triggers. Similar results were noted with GnRH-agonist triggers in the gonadotropin-only group (n = 72) (i.e., a higher number [13.3 ± 7.9 vs. 9.3 ± 6.0] and percentage of MII oocytes [85.7% vs. 72.8%] available for cryopreservation). Multivariate linear regression demonstrated approximately three more MII oocytes and 2PN embryos available for cryopreservation in the GnRH-agonist trigger group, irrespective of cancer and COS protocol type. Utilization of a GnRH-agonist trigger increases the number of MII oocytes and 2PN embryos available for cryopreservation in cancer patients undergoing COS for fertility preservation. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2015-08-01

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

  15. Pregnancy outcomes decline in recipients over age 44: an analysis of 27,959 fresh donor oocyte in vitro fertilization cycles from the Society for Assisted Reproductive Technology.

    Science.gov (United States)

    Yeh, Jason S; Steward, Ryan G; Dude, Annie M; Shah, Anish A; Goldfarb, James M; Muasher, Suheil J

    2014-05-01

    To use a large and recent national registry to provide an updated report on the effect of recipient age on the outcome of donor oocyte in vitro fertilization (IVF) cycles. Retrospective cohort study. United States national registry for assisted reproductive technology. Recipients of donor oocyte treatment cycles between 2008 and 2010, with cycles segregated into five age cohorts: ≤34, 35 to 39, 40 to 44, 45 to 49, and ≥50 years. None. Implantation, clinical pregnancy, live-birth, and miscarriage rates. In donor oocyte IVF cycles, all age cohorts ≤39 years had similar rates of implantation, clinical pregnancy, and live birth when compared with the 40- to 44-year-old reference group. Patients in the two oldest age groups (45 to 49, ≥50 years) experienced statistically significantly lower rates of implantation, clinical pregnancy, and live birth compared with the reference group. Additionally, all outcomes in the ≥50-year-old group were statistically significantly worse than the 45- to 49-year-old group, demonstrating progressive decline with advancing age. Recent national registry data suggest that donor oocyte recipients have stable rates of pregnancy outcomes before age 45, after which there is a small but steady and significant decline. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro

    DEFF Research Database (Denmark)

    Færge, Inger; Strejcek, Frantisek; Laurincik, Jozef

    2006-01-01

    Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturaion is improved...

  17. The influence of in vitro fertilization and embryo culture on the embryo epigenetic constituents and the possible consequences in the bovine model.

    Science.gov (United States)

    Sirard, M-A

    2017-08-01

    Medically assisted reproductive technologies, such as in vitro embryo production, are increasingly being used to palliate infertility. Eggs are produced following a hormonal regimen that stimulates the ovaries to produce a large number of oocytes. Collected oocytes are then fertilized in vitro and allowed to develop in vitro until they are either frozen or transferred to mothers. There are controversial reports on the adverse impacts of these technologies on early embryos and their potential long-term effects. Using newly developed technological platforms that enable global gene expression and global DNA methylation profiling, we evaluated gene perturbations caused by such artificial procedures. We know that cells in the early embryo produce all cells in the body and are able to respond to their in vitro environment. However, it is not known whether gene perturbations are part of a normal response to the environment or are due to distress and will have long-term impacts. While the mouse is an established genetic model used for quality control of culture media in clinics, the bovine is a large mono-ovulating mammal with similar embryonic kinetics as humans during the studied developmental window. These model systems are critical to understand the effects of assisted reproduction without the confounding impact of infertility and without the limitations imposed by the scarcity of donated human samples and ethical issues. The data presented in this review come mostly from our own experimentation, publications, and collaborations. Together they demonstrate that the in vitro environment has a significant impact on embryos at the transcriptomic level and at the DNA methylation level.

  18. Protective effect of crocetin on bovine spermatozoa against oxidative stress during in vitro fertilization.

    Science.gov (United States)

    Sapanidou, V; Taitzoglou, I; Tsakmakidis, I; Kourtzelis, I; Fletouris, D; Theodoridis, A; Lavrentiadou, S; Tsantarliotou, M

    2016-11-01

    Oxidative stress is one of the major factors that contribute to poor semen quality and low rates of in vitro fertilization. Crocetin, a main constituent of saffron (Crocus sativus L.) possesses potent antioxidant activity, by scavenging reactive oxygen species (ROS) and/or enhancing the activity of intracellular antioxidant enzymes. The aim of this study was to investigate, for the first time, the effect of crocetin on the quality characteristics of bull spermatozoa and fertilization rate. For this reason, frozen/thawed bovine spermatozoa were incubated with crocetin (1, 2.5, and 5 μm), for 120 or 240 min, in the presence of a negative control, and evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. In order to evaluate the impact of crocetin on cleavage and blastocyst rate, the compound was added in the IVF medium at the previously identified optimal concentration (2.5 μm). The results indicate that incubation of spermatozoa with 2.5 μm of crocetin resulted in a statistically significant lower production of superoxide anion and hydrogen peroxide, lower lipid peroxidation, and in better maintenance of motility parameters, viability, and acrosomal integrity, with a very small number of cells with DNA fragmentation, compared to the other groups (p fertilization medium also resulted in a significant increase in acrosome-reacted spermatozoa and blastocyst production, compared to the control group (p fertilizing ability, directly and/or indirectly, by regulating ROS concentration and lipid peroxidation. © 2016 American Society of Andrology and European Academy of Andrology.

  19. Low doses of bovine somatotropin enhance conceptus development and fertility in lactating dairy cows.

    Science.gov (United States)

    Ribeiro, Eduardo S; Bruno, Ralph G S; Farias, Alexandre M; Hernández-Rivera, Juan A; Gomes, Gabriel C; Surjus, Ricardo; Becker, Luis F V; Birt, Alyssa; Ott, Troy L; Branen, Josh R; Sasser, R Garth; Keisler, Duane H; Thatcher, William W; Bilby, Todd R; Santos, José E P

    2014-01-01

    Objectives were to evaluate the effects of administering either one or two low doses of slow-release recombinant bovine somatotropin (bST) on hormone concentrations, conceptus development, and fertility in dairy cows. Cows from two farms were detected in estrus on or after 50 days postpartum (n = 1483), inseminated, and enrolled in the study (Day 0). Within farm, cows were blocked by parity and assigned randomly to receive a single placebo injection at insemination (control), a single injection with 325 mg of bST at insemination (S-bST), or two injections with 325 mg of bST administered on Days 0 and 14 (T-bST). From a subset of cows, blood was collected twice weekly from Day 0 to 42 for determination of hormone concentrations and on Day 19 for isolation of leucocytes and analysis of transcript abundance of selected interferon-stimulated genes. Pregnancy was diagnosed on Days 31 and 66, and ultrasonographic morphometry of the conceptus was performed on Days 34 and 48 in a subset of cows. Cows that received T-bST had increased plasma concentrations of GH and IGF1 for 4 wk, increased mRNA expression of ISG15 and RTP4 in leukocytes, earlier rise in the pregnancy-specific protein B in plasma of pregnant cows, increased conceptus size, and enhanced fertility. Cows that received S-bST had increased concentrations of GH and IGF1 for only 2 wk and it was insufficient to alter conceptus development and fertility. In conclusion, supplementation with low doses of bST during the pre- and peri-implantation periods enhanced conceptus development, reduced embryonic losses, and improved fertility in dairy cows.

  20. Vitrification of Germinal Vesicle Stage Oocytes

    OpenAIRE

    ABE, Yasuyuki; AONO, Nobuya; Hara, Kenshiro; Matsumoto, Hiromichi; BAKHTIYARI, Mehrdad; Sasada, Hiroshi; Sato, Eimei

    2004-01-01

    In order to cryopreserve germinal vesicle (GV) stage oocytes, we first need to develop a novel container for keeping large quantities of GV oocytes, because of collecting them as cumulus oocytes complexes (COCs) that have bigger size and larger volume than oocytes themselves, and second modify a protocol for optimizing vitrification of them. In this mini-review, we describe our recent progress for attaining these objectives. When 65 bovine COCs having GV oocytes could be placed on a sheet of ...

  1. Isolation of bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea virus (BVDV) in association with the in vitro production of bovine embryos.

    Science.gov (United States)

    Bielanski, A; Loewen, K S; Del Campo, M R; Sirard, M A; Willadsen, S

    1993-09-01

    The purpose of this study was to determine whether oocytes obtained from bovine ovaries collected at commercial abattoirs for use in in vitro fertilization programs would be contaminated with bovine herpesvirus-1 (BHV-1) and/or bovine viral diarrhea virus (BVDV). In total, of 85 samples tested containing 759 embryos produced by in vitro fertilization, 2 (2.4%) were positive for BHV-1 while none were positive for BVDV. The follicular fluid collected during oocyte aspiration tested positive in 11.8% for BVH-1 and in 4.7% for BVDV. Oviductal cells used to co-culture zygotes/embryos tested positive for BHV-1 and BVDV in 6.2% and 1.2% samples respectively.

  2. Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro.

    Science.gov (United States)

    Rizos, Dimitrios; Fair, Trudee; Papadopoulos, Serafeim; Boland, Maurice P; Lonergan, Patrick

    2002-07-01

    The objective of this study was to compare bovine and ovine oocytes in terms of (1) developmental rates following maturation, fertilization, and culture in vitro, (2) the quality of blastocysts produced in vitro, assessed in terms of their ability to undergo cryopreservation, and (3) the ultrastructural morphology of these blastocysts. In vitro blastocysts were produced following oocyte maturation/fertilization and culture of presumptive zygotes in synthetic oviduct fluid. In vivo blastocysts were used as a control from both species. In Experiment 1, the cleavage rate of bovine oocytes was significantly higher than that of ovine oocytes (78.3% vs. 58.0%, respectively, P bovine and ovine, respectively, P vitrification, there was no difference in survival between in vivo produced bovine and ovine blastocysts (72 hr: 85.7% vs. 75.0%). However, IVP ovine blastocysts survived at significantly higher rates than IVP bovine blastocysts at all time points (72 hr: 47.4% vs. 18.1%, P bovine IVP blastocysts, which also displayed electron-lucent mitochondria and large intercellular cavities. These observations may in part explain the species differences observed in terms of cryotolerance. In conclusion, the quality of ovine blastocysts was significantly higher than their bovine counterparts produced under identical in vitro conditions suggesting inherent species differences between these two groups affecting embryo quality. Copyright 2002 Wiley-Liss, Inc.

  3. Delaying the initiation of progesterone supplementation until the day of fertilization does not compromise cycle outcome in patients receiving donated oocytes: a randomized study.

    Science.gov (United States)

    Escribá, María-José; Bellver, José; Bosch, Ernesto; Sánchez, María; Pellicer, Antonio; Remohí, José

    2006-07-01

    To determine whether the initiation of P supplementation as artificial luteal phase support (day -1, day 0, or day +1 of egg donation) in extensive programs of ovum donation influences cycle cancellation, pregnancy outcome, and implantation rate in day 3 embryo transfers. Prospective randomized trial. Oocyte donation program at the Instituto Valenciano de Infertilidad, Valencia, Spain. Three hundred recipients with normal ovarian function, absence of uterine anomalies, and undergoing their first egg donation were recruited between September 2003 and September 2004. A computer-based randomization divided the recipients into three groups when hCG was administered to their matched donors. The first group (group A) started P supplementation the day before oocyte retrieval; the second group (group B) started P supplementation on the day of the oocyte retrieval; and the third group (group C) started P supplementation 1 day after the egg retrieval once fertilization was confirmed. Implantation, pregnancy, and ongoing pregnancy rates were the primary outcome measures considered. The secondary outcome measure was the cancellation rate, especially due to fertilization failure. Global cancellation rate and cancellation rate due to fertilization failure were significantly higher in group A (12.4% and 8.2%, respectively) than in group C (3.3% and 0%, respectively). Reproductive outcome was similar in all the groups except for a higher biochemical pregnancy rate in group A (12.9%) than in groups B (6.6%) and C (2.3%). Initiation of P on day +1 of embryo development decreases cancellation rates of day 3 embryo transfers in extensive programs of ovum donation without any deleterious effect on pregnancy outcome or implantation rate.

  4. Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen.

    Science.gov (United States)

    O'Meara, C M; Hanrahan, J P; Donovan, A; Fair, S; Rizos, D; Wade, M; Boland, M P; Evans, A C O; Lonergan, P

    2005-11-01

    No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 x 10(6) to 0.03125 x 10(6) spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 x 10(6) or 0.0625 x 10(6)/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6-8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 x 10(6) spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. (P>0.05). When the experiment was repeated using 0.0625 x 10(6) spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. (P>0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; Pfertility in vivo and cleavage rate in vitro was significant (P=0.013). Replicate of IVF was a source

  5. High FSH decreases the developmental potential of mouse oocytes and resulting fertilized embryos, but does not influence offspring physiology and behavior in vitro or in vivo.

    Science.gov (United States)

    Li, Min; Zhao, Yue; Zhao, Cui H; Yan, Jie; Yan, Ying L; Rong, Li; Liu, Ping; Feng, Huai-Liang; Yu, Yang; Qiao, Jie

    2013-05-01

    Do different concentrations of FSH in the assisted reproductive technology (ART) procedure in vitro or in vivo affect the developmental competence of oocytes, the embryos and the offspring conceived from these embryos? Improper FSH treatment (200 IU/l in vitro, 10 IU/ml in vivo and 200 IU/ml in vivo) impairs the development competence of oocyte and embryo, but does not influence offspring physiology and behavior. Exogenous FSH has been widely used in the field of ART. However, the effects of different concentrations of FSH on the developmental competence of oocytes, embryos and the offspring conceived from these embryos, are still unknown. In a prospective study, a total of 45 mice at 8-10 weeks of age were primed in vivo with different dosages of FSH (9 mice in the 10 IU/ml, 10 mice in the 50 IU/ml, 10 mice in the 100 IU/ml and 16 mice in the 200 IU/ml groups). Fresh MII oocytes were retrieved from ovaries: this was designated as in vivo group. Thirty six mice at 8-10 weeks of age were sacrificed by cervical dislocation to obtain ovaries without FSH treatment (9 mice in the 0 IU/l, 9 mice in the 50 IU/l, 8 mice in the 100 IU/l and 10 mice in the 200 IU/l groups), and then the immature oocytes were collected from these ovaries and cultured in vitro matured medium supplemented with 0, 50, 100 and 200 IU/l FSH: this was designated as in vitro group. Spindle assembly of matured MII oocytes was stained via an immunofluorescence method and the oocytes ratio of normal spindle was analyzed. The developmental competence of the resulting fertilized embryos in the pre- and post-implantation stages was examined in in vitro and in vivo groups. Furthermore, physiological index, including reproductive potential and body weight, of the offspring was investigated by mating experiments and behavior index, including learning, memory, probing and intelligence, was tested by Morris water maze in in vitro and in vivo groups. In the in vitro groups, the oocyte maturation competence

  6. Evaluation of fertilization-to-planting and fertilization-to-harvest intervals for safe use of noncomposted bovine manure in Wisconsin vegetable production.

    Science.gov (United States)

    Ingham, Steven C; Fanslau, Melody A; Engel, Rebecca A; Breuer, Jeffry R; Breuer, Jane E; Wright, Thomas H; Reith-Rozelle, Judith K; Zhu, Jun

    2005-06-01

    Fresh bovine manure was mechanically incorporated into loamy sand and silty clay loam Wisconsin soils in April 2004. At varying fertilization-to-planting intervals, radish, lettuce, and carrot seeds were planted; crops were harvested 90, 100, 110 or 111, and 120 days after manure application. As an indicator of potential contamination with fecal pathogens, levels of Escherichia coli in the manure-fertilized soil and presence of E. coli on harvested vegetables were monitored. From initial levels of 4.0 to 4.2 log CFU/g, E. coli levels in both manure-fertilized soils decreased by 2.4 to 2.5 log CFU/g during the first 7 weeks. However, E. coli was consistently detected from enriched soil samples through week 17, perhaps as a result of contamination by birds and other wildlife. In the higher clay silty clay loam soil, the fertilization-to-planting interval affected the prevalence of E. coli on lettuce but not on radishes and carrots. Root crop contamination was consistent across different fertilization-to-harvest intervals in silty clay loam, including the National Organic Program minimum fertilization-to-harvest interval of 120 days. However, lettuce contamination in silty clay loam was significantly (P fertilization-to-harvest interval. Increasing the fertilization-to-planting interval in the lower clay loamy sand soil decreased the prevalence of E. coli on root crops. The fertilization-to-harvest interval had no clear effect on vegetable contamination in loamy sand. Overall, these results do not provide grounds for reducing the National Organic Program minimum fertilization-to-harvest interval from the current 120-day standard.

  7. Three-step in vitro maturation culture of bovine oocytes imitating temporal changes of estradiol-17β and progesterone concentrations in preovulatory follicular fluid

    Directory of Open Access Journals (Sweden)

    M. Matsuo

    2017-10-01

    Full Text Available The objective of the article is to evaluate the effect of three-step in vitro maturation (IVM culture system imitating estradiol-17β (E2 and progesterone (P4 concentrations in preovulatory follicles on in vitro bovine embryo production. The cumulus–oocyte complexes (COCs were collected from follicles (2 to 8 mm in diameter of bovine ovaries obtained from a local slaughterhouse. For IVM, the COCs were cultured for 22 h in a three-step system: (1 culture in medium 199, containing 700 ng mL−1 E2 and 50 ng mL−1 P4, for 5 h, followed by the medium containing 150 ng mL−1 E2 and 150 ng mL−1 P4 for 11 h, and then the medium containing 20 ng mL−1 E2 and 300 ng mL−1 P4 for 6 h (EP group; (2 culture in the medium containing 700 ng mL−1 E2 for 5 h, followed by the medium containing 150 ng mL−1 E2 for 11 h, and then the medium containing 20 ng mL−1 E2 for 6 h (E group; or (3 culture in the medium containing 50 ng mL−1 P4 for 5 h, followed by the medium containing 150 ng mL−1 P4 for 11 h, and then the medium containing 300 ng mL−1 P4 for 6 h (P group. The COCs were cultured in the medium containing 1000 ng mL−1 E2 for 22 h (control group. After IVM, the COCs were co-incubated with sperm and further cultured. At 48 h after insemination, the cleavage rate of embryos was not different among the groups. At 192 h after insemination, the blastocyst formation rate of EP group was significantly higher than that of the other groups. The total cell number of blastocysts did not differ among the groups. In conclusion, these results demonstrate that the three-step IVM culture system of bovine oocytes imitating temporal changes of E2 and P4 concentrations in preovulatory follicular fluid improves the developmental potential of embryos in vitro.

  8. Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos.

    Science.gov (United States)

    Kropp, Jenna; Carrillo, José A; Namous, Hadjer; Daniels, Alyssa; Salih, Sana M; Song, Jiuzhou; Khatib, Hasan

    2017-04-05

    stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.

  9. Oocyte competence in in vitro fertilization and intracytoplasmic sperm injection patients suffering from endometriosis and its possible association with subsequent treatment outcome: a matched case-control study.

    Science.gov (United States)

    Shebl, Omar; Sifferlinger, Ida; Habelsberger, Alwin; Oppelt, Peter; Mayer, Richard B; Petek, Erwin; Ebner, Thomas

    2017-06-01

    Endometriosis affects up to 15% of women of reproductive age. There is an obvious lack of studies dealing with morphological parameters of oocyte morphology in endometriosis patients in assisted reproduction. One aim of the study is to describe oocyte morphology in patients undergoing intracytoplasmic sperm injection suffering from endometriosis. In addition, the impact of endometriosis on in vitro fertilization results is analyzed. Both in vitro fertilization and intracytoplasmic sperm injection patients are then matched with an endometriosis-free control group for highlighting the possible association of endometriosis with pregnancy outcome. Oocyte morphology of endometriosis patients was assessed in two groups. Both study group and control group consisted of 129 in vitro fertilization/intracytoplasmic sperm injection cycles each. Patients were matched according to anti-Müllerian hormone, female age, previous treatment cycles, and method of fertilization. Endometriosis was graded according to the revised American Society for Reproductive Medicine guidelines of 1997. Patients with endometriosis had a significantly lower rate of mature oocytes (p < 0.03) and morphologically normal oocytes (p < 0.001). In particular, brownish oocytes (p < 0.009; stage I-IV) and the presence of refractile bodies (p < 0.001; stage IV) were found to be increased. Endometriosis stage IV was associated with significantly worse-quality oocytes than stages I-III (p < 0.01). Fertilization was significantly reduced in conventional in vitro fertilization but not in intracytoplasmic sperm injection (p < 0.03). This was due to lower fertilization rates in stage III-IV endometriosis compared with stage I-II (p < 0.04). No difference was observed with respect to rates of implantation, clinical pregnancy, miscarriage, live birth, and malformation. Endometriosis patients, in particular those with severe endometriosis, present lower-quality oocytes. Once fertilized, no impairment

  10. Oviductal transcriptional profiling of a bovine fertility model by next-generation sequencing

    Directory of Open Access Journals (Sweden)

    Angela Maria Gonella-Diaza

    2017-09-01

    Full Text Available In cattle, the oviduct plays a fundamental role in the reproductive process. Oviductal functions are controlled by the ovarian sex steroids: estradiol and progesterone. Here, we tested the hypothesis that the exposure to contrasting sex steroid milieus differentially impacts the oviductal transcriptional profile. We manipulated growth of the pre-ovulatory follicle to obtain cows that ovulated a larger (LF group or a smaller (SF group follicle. The LF group presented greater proestrus/estrus concentrations of estradiol and metaestrus concentrations of progesterone (Gonella-Diaza et al. 2015 [1], Mesquita et al. 2014 [2]. Also, the LF group was associated with greater fertility in timed-artificial insemination programs (Pugliesi et al. 2016 [3]. Cows were slaughtered on day 4 of the estrous cycle and total RNA was extracted from ampulla and isthmus fragments and analyzed by RNAseq. The resulting reads were mapped to the bovine genome (Bos taurus UMD 3.1, NCBI. The differential expression analyses revealed that 325 and 367 genes in ampulla and 274 and 316 genes in the isthmus were up-regulated and down-regulated in LF samples, respectively. To validate the RNAseq results, transcript abundance of 23 genes was assessed by qPCR and expression patterns were consistent between the two techniques. A functional enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID software. Processes enriched in the LF group included tissue morphology changes (extracellular matrix remodeling, cellular changes (proliferation, and secretion changes (growth factors, ions and metal transporters. An overview of the gene expression data was deposited in the NCBI's Gene Expression Omnibus (GEO and is accessible through the accession number GSE65681. In conclusion, differences in the peri-ovulatory sex steroid milieu modify the oviductal gene expression profiles. Such differences may be associated with the greater fertility

  11. Conjugated linoleic acid improves oocyte cryosurvival through modulation of the cryoprotectants influx rate.

    Science.gov (United States)

    Matos, Joana E; Marques, Carla C; Moura, Teresa F; Baptista, Maria C; Horta, Antonio E M; Soveral, Graça; Pereira, Rosa M L N

    2015-06-12

    In cryopreservation, oocytes are subjected to extreme hyperosmotic conditions, inducing large volume changes that, along with an abrupt temperature drop, interfere with their developmental competence. Our objectives in this work were to find conditions enabling an increase in oocyte cryosurvival and subsequent development. Abattoir-derived bovine oocytes were cultured without (Control group) or with trans-10,cis-12 conjugated linoleic acid isomer (CLA group). Comparative observations were made for 1) the oocyte developmental competence after exposure to cryoprotectants followed or not by vitrification/warming, 2) the oocyte membrane permeability to water (using the non-permeant cryoprotectant sucrose) and 3) the oocyte membrane permeability to two cryoprotectants (ethylene glycol, EG, and dimethyl sulfoxide, DMSO). Mature oocytes cultured with or without CLA and vitrified/warmed or only exposed to cryoprotectants without vitrification were subjected to in vitro fertilization; embryo culture proceeded until the blastocyst stage. The oocyte membrane permeabilities to water and cryoprotectants were estimated using mature oocytes subjected to hyperosmotic challenges. For water permeability, 200 mM sucrose was used, whereas for the cryoprotectant permeability, a 10 % solution of both EG and DMSO was used. The data were analyzed using the MIXED procedure and Student's T-test. CLA supplementation improves the developmental competence of vitrified/warmed and cryoprotectants exposed oocytes (p < 0.01) and reduces their membrane permeability to water (37 %, p < 0.001) and to cryoprotectants (42 %, p < 0.001). By slowing the fluxes of water and of permeant cryoprotectants, CLA contributed to improved oocyte cryosurvival and post-thawed viability. This isomer supplementation to the maturation media should be considered when designing new protocols for oocyte cryopreservation.

  12. Factors affecting the outcome of in vitro bovine embryo production using ovum pick-up-derived cumulus oocyte complexes

    NARCIS (Netherlands)

    Merton, J.S.

    2013-01-01

    Optimization of bovine ovum pick up (OPU) followed by in vitro embryo production (IVP) has been driven by the desire of both beef and dairy cattle breeders to enhance genetic improvement. The work presented in this thesis focuses on optimizing the efficiency and efficacy of the OPU-IVP program.

  13. Partial characterization of the factor in theca-cell conditioned medium that inhibits the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to the membrana granulosa.

    Science.gov (United States)

    van Tol, H T; Bevers, M M

    2001-11-01

    A factor, secreted by theca cells, inhibits FSH induced resumption of meiosis in bovine oocytes that are surrounded by cumulus cells which are attached to a piece of the membrana granulosa (COCGs). In order to characterize this factor, theca cell conditioned medium (CMt) was heat-treated, filtered through a 5 kD spin off filter, charcoal treated, chloroform extracted and protease treated. To investigate whether the meiosis inhibiting factor produced by theca cells was also present in follicular fluid (FF), the same treatments were done with 50% bovine follicular fluid (bFF). COCGs, originating from 2 to 8 mm follicles of bovine ovaries collected at a slaughterhouse, were cultured in groups of 15 per 600 microl medium supplemented with 0.05 IU ml FSH for 22 hr at 39 degrees C in a humidified atmosphere of 5% CO(2). After culture the oocytes were denuded, stained with orcein, and the nuclear status assessed. Heat treatment did not affect the meiosis arresting capacity of CMt since a similar proportion of the oocytes remained at the GV stage after 22 hr of culture in heat treated CMt as compared to the proportion of oocytes in the GV stage after culture in untreated CMt. Filtering through a 5 kD spin-off filter revealed that the meiosis inhibiting action was maintained in the <5 kD fraction, although there was a significant (P < 0.05) loss of inhibiting activity compared to nonfiltered CMt. No significant decrease was observed in the meiosis arresting capacity of the <5 kD fraction after charcoal or protease treatment. Extraction of the <5 kD fraction with chloroform also did not affect the theca cell produced factor. The effect of the theca cell factor on the progression of meiosis of the oocytes that resumed meiosis, as demonstrated by a very low percentage of the oocytes that matured up to the M2 stage, was not affected following any of the treatments. With regard to bFF, the results show a lower percentage of the oocytes in the GV stage after culture in 50% bFF as

  14. Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos.

    Science.gov (United States)

    Pereira, A F; Melo, L M; Freitas, V J F; Salamone, D F

    2015-08-01

    In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.

  15. Effect of bovine sperm chromatin integrity evaluated using three different methods on in vitro fertility.

    Science.gov (United States)

    Castro, L S; Siqueira, A F P; Hamilton, T R S; Mendes, C M; Visintin, J A; Assumpção, M E O A

    2018-02-01

    In vitro fertility potential of individual bulls is still relatively uncharacterized. Classical sperm analysis does not include the evaluation of all sperm characteristics and thus, some cell compartments could be neglected. In humans, sperm DNA integrity has already proven to have major influence in embryo development and assisted reproduction techniques successfully. In bovine, some studies already correlated chromatin integrity with field fertility. However, none of those have attempted to relate DNA assessment approaches such as chromatin deficiency (CMA3), chromatin stability (SCSA; AO+) and DNA fragmentation (COMET assay) to predict in vitro bull fertility. To this purpose, we selected bulls with high and low in vitro fertility (n = 6/group), based on embryo development rate (blastocyst/cleavage rate). We then performed CMA3, SCSA test and COMET assay to verify if the difference of in vitro fertility may be related to DNA alterations evaluated by these assays. For the three tests performed, our results showed only differences in the percentage of cells with chromatin deficiency (CMA3+; high: 0.19 ± 0.03 vs low: 0.04 ± 0.04; p = 0.03). No difference for chromatin stability and any of COMET assay categories (grade I to grade IV) was observed between high and low in vitro fertility bulls. A positive correlation between AO + cells and grade IV cells was found. Despite the difference between groups in CMA3 analysis, our results suggest that protamine deficiency in bovine spermatozoa may not have a strong biological impact to explain the difference of in vitro fertility between the bulls used in this study. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Endometrial transcriptional profiling of a bovine fertility model by Next-Generation Sequencing

    Directory of Open Access Journals (Sweden)

    F.S. Mesquita

    2016-03-01

    Full Text Available Studying the multitude of molecular networks and pathways that are potentially involved in a complex trait such as fertility requires an equally complex and broad strategy. Here, we used Next-Generation Sequencing for the characterization of the transcriptional signature of the bovine endometrial tissue. Periovulatory endocrine environments were manipulated to generate two distinctly different fertility phenotypes. Cycling, non-lactating, multiparous Nelore cows were manipulated to ovulate larger (>13 mm; LF group; high fertility phenotype or smaller (<12 mm; SF group follicles. As a result, greater proestrus estrogen concentrations, corpora lutea and early diestrus progesterone concentrations were also observed in LF group in comparison to SF group. Endometrial cell proliferation was estimated by the protein marker MKI67 on tissues collected 4 (D4 and 7 (D7 days after induction of ovulation. Total RNA extracts from D7 were sequenced and compared according to the transcriptional profile of each experimental group (LF versus SF. Functional enrichment analysis revealed that LF and SF endometria were asynchronous in regards to their phenotype manifestation. Major findings indicated an LF endometrium that was switching phenotypes earlier than the SF one. More specifically, a proliferating SF endometrium was observed on D7, whereas the LF tissue, which expressed a proliferative phenotype earlier at D4, seemed to have already shifted towards a biosynthetically and metabolically active endometrium on D7. Data on MKI67 support the transcriptomic results. RNA-Seq-derived transcriptional profile of the endometrial tissue indicated a temporal effect of the periovulatory endocrine environment, suggesting that the moment of the endometrial exposure to the ovarian steroids, E2 and P4, regulates the timing of phenotype manifestation. Gene expression profiling revealed molecules that may be targeted to elucidate ovarian steroid-dependent mechanisms that

  17. 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

  18. 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. Copyright © 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  19. Diving into the oocyte pool

    DEFF Research Database (Denmark)

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

    2017-01-01

    PURPOSE OF REVIEW: The ovarian reserve comprises an enormous surplus of follicles. Despite this, some women produce insufficient numbers of oocytes by conventional fertility treatments. However, recent technical accomplishments may transform assisted reproductive technology (ART) in such a way...... of the signaling pathways activating dormant follicles and breakthroughs in techniques for autologous transfer of mitochondria have opened new doors to unexploited sources of oocytes and attractive ways of revitalizing oocytes. Extended numbers of mature oocytes may be obtained by in-vitro activation of dormant...... follicles in cortical biopsies or in-vitro maturation of immature oocytes during the natural or stimulated cycle, and used directly for fertility treatment or as a source of autologous mitochondria. SUMMARY: New approaches utilizing the abundant resources of immature oocytes combined with techniques...

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

    Science.gov (United States)

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

    2009-01-01

    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. METHODS AND RESULTS We examined follicles from human ovaries (n = 18) by electron microscopy and found that many follicles had additional layers of basal lamina. Oocytes (n = 222) from bovine follicles with normal or unusual basal laminas were isolated and their ability to undergo in vitro maturation, fertilization and culture to blastocyst was compared. Healthy bovine follicles with a single layer of basal lamina had oocytes with significantly (P < 0.01) greater developmental competence than healthy follicles with additional layers of follicular basal lamina (65% versus 28%). CONCLUSIONS These findings provide direct evidence that the phenotype of the follicular basal lamina is related to oocyte competence. PMID:19095662

  1. Endocrine profiles after triggering of final oocyte maturation with GnRH agonist after cotreatment with the GnRH antagonist ganirelix during ovarian hyperstimulation for in vitro fertilization

    NARCIS (Netherlands)

    B.C.J.M. Fauser (Bart); D. de Jong (Danielle); F. Olivennes; H. Wramsby; C. Tay; J. Itskovitz-Eldor; H.G. van Hooren

    2002-01-01

    textabstractIn a randomized multicenter study, the efficacies of two different GnRH agonists were compared with that of hCG for triggering final stages of oocyte maturation after ovarian hyperstimulation for in vitro fertilization. Ovarian stimulation was conducted by recombinant

  2. 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.

  3. Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors?

    Science.gov (United States)

    Gosálvez, Jaime; Caballero, Pedro; López-Fernández, Carmen; Ortega, Leonor; Guijarro, José Andrés; Fernández, José Luís; Johnston, Stephen D; Nuñez-Calonge, Rocío

    2013-11-01

    This study compared the potential of assessing sperm DNA fragmentation (SDF) from neat semen and the subsequent swim-up (SU) procedure to predict pregnancy when conducting ICSI of fertile donor oocytes. Infertile females (n=81) were transferred embryos resulting from intracytoplasmic sperm injection (ICSI) of their partner's spermatozoa and proven donor oocytes. This model normalized the impact of female factor in putative sperm DNA repair. Semen was blindly assessed for SDF using Halosperm immediately following ejaculation (NS) and after swim-up at the time of ICSI fertilisation. There was a decrease in SDF values of the ejaculated semen sample following the swim-up protocol (P=0.000). Interestingly, pregnancy could be equally predicted from SDF values derived from either neat or swim-up semen samples. Receiver operator curves and the derived Youden's indices determined SDF cutoff values for NS and SU of 24.8% and 17.5%, respectively. Prediction of pregnancy from NS SDF had a sensitivity of 75% and a specificity of 69%, whereas for SU SDF was 78% and 73%, respectively. While increased levels of SDF negatively impact reproductive outcome, we have shown that a reduction in SDF following sperm selection using ICSI with proven donor oocytes is not mandatory for achieving pregnancy. This suggests that a certain level of DNA damage that is not detectable using current technologies could be impacting on the relative success of assisted reproductive technology (ART) procedures. Consequently, we propose a modification of the so called 'iceberg model' as a possible rationale for understanding the role of SDF in reproductive outcome.

  4. Toxicity evaluation of ethanol treatment during in vitro maturation of porcine oocytes and subsequent embryonic development following parthenogenetic activation and in vitro fertilization.

    Science.gov (United States)

    Lee, Sanghoon; Kim, Eunhye; Hyun, Sang-Hwan

    2014-11-01

    Ethanol is frequently used as a solvent in several techniques for in vitro production (IVP). It is also used for the parthenogenetic activation (PA) of oocytes. Although a number of studies have suggested that ethanol has detrimental effects on fibroblasts and neuronal cells, little attention has been paid to the effects of ethanol on porcine oocytes. Thus, the aim of this study was to evaluate the effects of the addition of ethanol to in vitro maturation (IVM) medium. We investigated the effects of ethanol (0, 1 and 3%) on the following parameters: nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic development following PA and in vitro fertilization (IVF). After 44 h of IVM, the 3% group showed a significant (Pethanol group showed significantly (Pethanol group had significantly (P1% ethanol during IVM exerts a toxic effect on the developmental potential of PA and IVF porcine embryos by decreasing the intracellular GSH level, thereby increasing the intracellular ROS level and upregulating the expression of apoptosis‑related genes.

  5. What is the quality of information on social oocyte cryopreservation provided by websites of Society for Assisted Reproductive Technology member fertility clinics?

    Science.gov (United States)

    Avraham, Sarit; Machtinger, Ronit; Cahan, Tal; Sokolov, Amit; Racowsky, Catherine; Seidman, Daniel S

    2014-01-01

    To evaluate adequacy and adherence to American Society for Reproductive Medicine (ASRM) guidelines of internet information provided by Society for Assisted Reproductive Technology (SART)-affiliated clinics regarding social oocyte cryopreservation (SOC). Systematic evaluation of websites of all SART member fertility clinics. The internet. None. All websites offering SOC services were scored using a 0-13 scale, based on 10 questions designed to assess website quality and adherence to the ASRM/SART guidelines. The websites were analyzed independently by two authors. Whenever disagreement occurred, a third investigator determined the score. Scores defined website quality as excellent, ≥9; moderate, 5-8; or poor, ≤4 points. Of the 387 clinics registered as SART members, 200 offered oocyte cryopreservation services for either medical or social reasons; 147 of these advertised SOC. The average website scores of those clinics offering SOC was 3.4 ± 2.1 (range, 2-11) points. There was no significant difference in scores between private versus academic clinics or clinics performing more or less than 500 cycles per year. The majority of the websites do not follow the SART/ASRM guidelines for SOC, indicating that there is a need to improve the type and quality of information provided on SOC by SART member websites. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Developmental competence of ovine oocyte following vitrification: effect of oocyte developmental stage, cumulus cells, cytoskeleton stabiliser, FBS concentration, and equilibration time.

    Science.gov (United States)

    Shirazi, Abolfazl; Taheri, Fatemeh; Nazari, Hassan; Norbakhsh-Nia, Maryam; Ahmadi, Ebrahim; Heidari, Banafsheh

    2014-05-01

    The aim of the present study was to examine the effects of fetal bovine serum (FBS) concentration, equilibration time, and oocyte pre-treatment with cytochalasin B (CCB) on subsequent development of vitrified-warmed ovine immature (GVCOCs) and matured (MII) oocytes with (MIICOCs) or without cumulus cells (MIIDOs). In Experiment 1, the effects of FBS concentrations (10 and 20%) during the vitrification-warming procedure were examined. Survival rates after warming were not different between GVCOCs, MIICOCs and MIIDOs oocytes. After in vitro fertilization, rate of cleaved embryos in MIICOCs group at the presence of 20%FBS was higher than MIIDOs and GVCOCs groups. In Experiment 2, the effects of equilibration times (5, 7, and 10 min) were examined. There was no difference in survival rate of vitrified-warmed oocytes equilibrated at different times. Although, the rate of cleavage in MIICOCs and MIIDOs oocytes equilibrated for 10 and 7 min, respectively, was higher than 5 min equilibrated MIIDOs and 7 and 10 min equilibrated GVCOCs oocytes. In Experiment 3, the effects of oocyte pre-treatment with CCB were examined. Despite the insignificant difference in survival rate of vitrified-warmed ovine immature and matured oocytes, the rates of cleavage in CCB pretreated groups were significantly lower than untreated groups. Moreover, the blastocysts were only derived from those cumulus enclosed vitrified-warmed germinal vesicle (GV) and MII oocytes that had been exposed to 10% FBS in the absence of CCB. In conclusion, the presence of cumulus cells, 10% FBS, and the omission of CCB were beneficial for post-warming development of vitrified ovine oocytes.

  7. Expression profile of PLCζ, PAWP, and TR-KIT in association with fertilization potential, embryo development, and pregnancy outcomes in globozoospermic candidates for intra-cytoplasmic sperm injection and artificial oocyte activation.

    Science.gov (United States)

    Tavalaee, M; Nasr-Esfahani, M H

    2016-09-01

    Sperm-mediated oocyte activation critically depends upon appropriate expression and assembly of sperm-borne oocyte-activating factors (SOAFs) during spermiogenesis. Several factors have been considered as candidate for SOAFs over the recent years. However, little is known about the expression profile of these candidates and their potential contribution to the clinical outcomes of intra-cytoplasmic sperm injection (ICSI), particularly in globozoospermia. This study investigated the expression profile of PLCζ, PAWP, and TR-KIT and clinical outcomes of ICSI in 12 men with total globozoospermia and compared with 12 fertile individuals. Levels of PLCζ, PAWP, and TR-KIT mRNA in the spermatozoa of fertile men were significantly higher than the corresponding values of the globozoospermic subjects. Interestingly, at protein level, expressions of these factors in the cases assessed were low in globozoospermic individuals. Fertilization rates following artificial oocyte activation (AOA) in the majority of globozoospermic couples were higher than the expected 30% cut-off value reported for individuals with failed or low fertilization rate. Clinical outcomes of ICSI-AOA were dependent on inter-individual variation in globozoospermic couples. None of the SOAFs assessed could provide a greater prediction value with respect to fertilization rate in globozoospermic couples which underwent ICSI-AOA. High fertilization (56.06%) and pregnancy (41.7%) rates accomplished in this study following ICSI-AOA indicated that expression profiles of PLCζ, PAWP, and TR-KIT were low in globozoospermic individuals, and ICSI combined with artificial oocyte activation could mimic physiological calcium changes which occur during fertilization. © 2016 American Society of Andrology and European Academy of Andrology.

  8. [Recent knowledge on follicle and oocyte maturation. 2. Oocyte development and maturation].

    Science.gov (United States)

    Sudik, R; Fliess, F R

    1984-01-01

    A review is given about the present knowledge in oocyte development and oocyte maturation. The four parts of the review contain: development of the oocyte in the fetal ovary, morphology and metabolism during meiotic arrest, oocyte maturation, and the relations between oocyte maturation and in vitro-fertilization in the human. The morphological and biochemical changes in the maturation process and present hypotheses about maturation regulation are described especially. The increasing knowledge in this field supports the progress of in vitro-fertilization in the human. On the other hand this technique contributes importantly to new directions in oocyte research.

  9. Highly efficient vitrification method for cryopreservation of human oocytes.

    Science.gov (United States)

    Kuwayama, Masashige; Vajta, Gábor; Kato, Osamu; Leibo, Stanley P

    2005-09-01

    Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.

  10. bovine

    African Journals Online (AJOL)

    of various breeds under local conditions of management. (Hale, 1974b). AdditionaIly, this procedure has been used to assess the production of LH by the bovine anterior pituitary in vitro and to study the relationships between this production and the activity of the pineal- hypothalamic axis (Hayes, Knight & Symington, 1974;.

  11. Human oocyte cryopreservation.

    Science.gov (United States)

    Tao, Tao; Zhang, Wenling; Del Valle, Alfonso

    2009-06-01

    This review summarized the clinical breakthroughs in the human oocyte cryopreservation field in the past 2 years and gave special emphasis on the role of vitrification method. Human oocyte cryopreservation is an attractive strategy to preserve female fertility, as it offers more opportunities to the future destination of the female gametes and also raises fewer legal and ethical questions compared with embryo cryopreservation. It became promising in recent years because of dramatic improvement in cryopreservation technologies. Human oocyte cryopreservation would not become a clinical routine until the availability of reliable cryopreservation methods and long-term follow-up results of the babies born by this technique. Oocyte cryopreservation produced very exciting results with pregnancy and implantation rates comparable to embryo cryopreservation and in some cases comparable to fresh in-vitro fertilization cycles with both modified slow-freezing and vitrification methods. A cancer patient conceived and delivered her own babies by this technology after recovery from the disease. Oocyte cryopreservation became a new focus in assisted reproductive technology. We witnessed the advanced development of human oocyte cryopreservation in the past years because of increasing demand, medically, legally and ethically, and also because of the dramatic improvement of the freezing technique. There is still a long way to go to integrate it into a routine clinical procedure to benefit more patients and encourage clinicians to follow the standard protocols.

  12. Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm.

    Science.gov (United States)

    Rodríguez-Villamil, P; Hoyos-Marulanda, V; Martins, J A M; Oliveira, A N; Aguiar, L H; Moreno, F B; Velho, A L M C S; Monteiro-Moreira, A C; Moreira, R A; Vasconcelos, I M; Bertolini, M; Moura, A A

    2016-02-01

    The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 μg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 μg/mL BSP1 (77.8 ± 3.1%), or 20 μg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 μg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 μg/mL BSP1 (22.4 ± 2.9%) and 40 μg/mL BSP1 (19.3 ± 4.1%; P epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 μg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma. Copyright

  13. The equine oocyte: factors affecting meiotic and developmental competence.

    Science.gov (United States)

    Hinrichs, Katrin

    2010-08-01

    There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli (Cp oocytes). Cp oocytes originate in viable follicles but are largely juvenile. Recovery and culture of equine oocytes immediately after slaughter yields a higher maturation rate than that obtained from oocytes after ovary storage; this is related to damage to chromatin in Cp oocytes during storage. In contrast, developmental competence (rate of blastocyst development in vitro) is higher in oocytes recovered from the ovary after a delay. The optimum duration of maturation varies based on cumulus morphology and time of recovery from the ovary, but there is no difference in developmental competence between Ex and Cp oocytes. Because standard in vitro fertilization is not repeatable in the horse, oocyte transfer (surgical transfer of oocytes to the oviducts of inseminated mares) has been developed to allow fertilization of isolated oocytes. Fertilization in vitro may be achieved using intracytoplasmic sperm injection; culture of injected oocytes in a medium with high glucose can yield over 30% blastocyst development. (c) 2010 Wiley-Liss, Inc.

  14. 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

  15. Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes.

    Science.gov (United States)

    Wiesak, Teresa; Wasielak, Marta; Złotkowska, Aleksandra; Milewski, Robert

    2017-06-01

    The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes. The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these oocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Forced collapse of the blastocoel enhances survival of cryotop vitrified bovine hatching/hatched blastocysts derived from in vitro fertilization and somatic cell nuclear transfer.

    Science.gov (United States)

    Min, Sung-Hun; Lee, Enok; Son, Hyeong-Hoon; Yeon, Ji-Yeong; Koo, Deog-Bon

    2013-04-01

    Freezing of bovine blastocysts has been widely used to improve the feasibility of cattle production by the embryo transfer technique. However, the low survival of vitrified-warmed embryos and their further development are crucial problems. Particularly, the production of offspring in vitrified-warmed bovine hatching/hatched blastocysts derived from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) is very low. Thus, we examined the effects of forced blastocoel collapse (FBC) before vitrification of bovine IVF- and SCNT-derived hatching/hatched embryos on the survival rate and apoptosis index after warming. Under optimal conditions, the overall survival rates in vitrified-warmed bovine IVF- and SCNT-derived hatching/hatched blastocysts were higher in FBC groups than in non-FBC groups (pvitrification of bovine IVF- and SCNT-derived hatching/hatched blastocysts. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. The effectiveness of earlier oocyte retrieval in the case of a premature luteinizing hormone surge on hCG day in in vitro fertilization-embryo transfer cycles.

    Science.gov (United States)

    Choi, Min Hye; Cha, Sun Hwa; Park, Chan Woo; Kim, Jin Young; Yang, Kwang Moon; Song, In Ok; Koong, Mi Kyoung; Kang, Inn Soo; Kim, Hye Ok

    2013-06-01

    To evaluate the efficacy of earlier oocyte retrieval in IVF patients with a premature LH surge on hCG day. One hundred forty IVF patients (164 cycles) with premature LH surge on hCG day were included, retrospectively. We divided them into 2 study groups: LH surge with timed ovum pick-up (OPU) 36 hours after hCG injection (group B, 129 premature cycles), and LH surge with earlier OPU within 36 hours after hCG injection (group C, 35 cycles). Control groups were tubal factor infertility without premature LH surge (group A, 143 cycles). The mean age (year) was statistically higher in group C than in groups A or B (38.2±5.4 vs. 36.2±4.2 vs. 36.8±4.9, respectively; p=0.012). The serum LH levels (mIU/mL) on hCG day were significantly higher in group B and C than in group A (22.7±14.9 vs. 30.3±15.9 vs. 3.2±2.9, respectively; p>0.001). Among groups A, B, and C, 4.9%, 31.7%, and 51.4% of the cycles, respectively, had no oocytes, and the overall rates of cycle cancellation (OPU cancellation, no oocyte, or no embryos transferrable) were 15.4%, 65.9%, and 74.3%, respectively. The fertilization rate (%) was significantly higher in group B than in group C (73.2±38.9 vs. 47.8±42.9, p=0.024). The clinical pregnancy rate was significantly higher in group C than in groups A and B (44.4% vs. 27.3% vs. 9.1%, respectively, p=0.021). However, the miscarriage rate was also higher in group C than in group B (22% vs. 0%, respectively, p=0.026). Earlier OPU may not be effective in reducing the risk of cycle cancellation in patients with premature LH surge on hCG day. A larger scale study will be required to reveal the effectiveness of earlier ovum retrieval with premature LH surge.

  18. 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

  19. Cysteamine supplementation during in vitro maturation of slaughterhouse- and opu-derived bovine oocytes improves embryonic development without affecting cryotolerance, pregnancy rate, and calf characteristics.

    Science.gov (United States)

    Merton, J S; Knijn, H M; Flapper, H; Dotinga, F; Roelen, B A J; Vos, P L A M; Mullaart, E

    2013-09-01

    Optimization of ovum pick up (OPU) followed by in vitro embryo production (IVP) is strongly driven by the needs of both beef and dairy cattle breeders to enhance genetic improvement. The rapidly growing use of genomic selection in cattle has increased the interest in using OPU-IVP technology to increase the number of embryos and offspring per donor, thus allowing enhanced selection intensity for the next generation. The aim of this study was to optimize embryo production through supplementation of cysteamine during in vitro maturation (IVM) and in vitro culture (IVC) of both slaughterhouse- and OPU-derived oocytes. The effects on embryo production and on embryo cryotolerance, post-transfer embryo survival, and calf characteristics, including gestation length, birth weight, perinatal mortality, and sex ratio were studied. In study 1, immature slaughterhouse-derived cumulus-oocyte complexes (COCs) were matured in IVM medium supplemented with or without 0.1 mM cysteamine, fertilized and cultured for 7 days in 0.5 ml SOFaaBSA. In study 2, cysteamine was present during both IVM (0.1 mM) and IVC (0.01, 0.05, 0.1 mM) from Days 1 to 4. In study 3, OPU-derived COCs were matured in medium supplemented with or without 0.1 mM cysteamine in a 2 × 2 factorial design (OPU week and cysteamine treatment). Embryos were evaluated for stage and grade on Day 7 and, depending on the number of transferable embryos and recipients available, the embryos were transferred either fresh or frozen-thawed at a later date. The presence of cysteamine during IVM significantly increased the embryo production rate with slaughterhouse-derived COCs (24.0% vs. 19.4%). The higher number of embryos at Day 7 was due to an increased number of blastocysts, whereas the distribution of embryos among different quality grades and cryotolerance was not affected. Embryo production rate was negatively affected when cysteamine was present during both the processes of IVM and IVC during Days 1 to 4 of culture (13

  20. Enhancement of Bovine oocyte maturation by leptin is accompanied by an upregulation in mRNA expression of leptin receptor isoforms in cumulus cells

    NARCIS (Netherlands)

    van Tol, Helena T A|info:eu-repo/dai/nl/313871817; van Eerdenburg, Frank J C M|info:eu-repo/dai/nl/072455993; Colenbrander, Ben; Roelen, Bernard A J|info:eu-repo/dai/nl/109291859

    In this study, the mechanisms of supposed leptin action on oocyte maturation were examined. Expression of leptin mRNA, as determined with RT-PCR, was present in oocytes but not in cumulus cells. The long isoform of the leptin receptor (ObR-L) was expressed exclusively in cumulus cells after 7 and 23

  1. Oocyte cryopreservation: where are we now?

    Science.gov (United States)

    Argyle, Catrin E; Harper, Joyce C; Davies, Melanie C

    2016-06-01

    Since the first live birth from oocyte cryopreservation three decades ago, oocyte cryopreservation has become an important component of ART. Cryopreservation techniques have evolved, leading to higher success rates and the introduction of oocyte cryopreservation into IVF clinics worldwide. Concurrently, there has been an increase in patient demand, especially for so-called 'social egg freezing' that allows women to preserve their fertility in anticipation of age-related fertility decline. This review addresses a need to evaluate the current status of oocyte cryopreservation. It explores current techniques and success rates, clinical applications, the rise of elective oocyte cryopreservation, and future implications. A search was performed using Web of Science and PubMed databases for publications between January 1980 and December 2015. Keywords used included 'egg freezing', 'oocyte freezing', 'oocyte cryopreservation', 'oocyte vitrification', and 'fertility preservation'. The success rate of oocyte cryopreservation has risen, and the increasing use of vitrification offers has improved outcomes, with IVF pregnancy rates now similar to those achieved with fresh oocytes. There are conflicting opinions about the comparative success rates of open and closed vitrification. Patients are accessing and receiving oocyte cryopreservation for a wide range of indications, and there has been a marked increase in patient numbers and oocyte cryopreservation cycles. Oocyte cryopreservation for circumventing age-related infertility is becoming more widely accepted. Oocyte cryopreservation is an established component of ART, with vitrification now being the cryopreservation technique of choice. Increasing numbers of women undergo oocyte cryopreservation for both medical and social reasons. It is important to continue auditing outcomes and reporting long-term follow-up of children born from frozen-thawed oocytes. © The Author 2016. Published by Oxford University Press on behalf of

  2. Comet assay of cumulus cells and spermatozoa DNA status, and the relationship to oocyte fertilization and embryo quality following ICSI.

    Science.gov (United States)

    Abu-Hassan, D; Koester, F; Shoepper, B; Schultze-Mosgau, A; Asimakopoulos, B; Diedrich, K; Al-Hasani, S

    2006-04-01

    It has been postulated that apoptosis may affect cumulus cell and sperm DNA integrity, and therefore influence the outcome of assisted reproductive techniques. This study investigates apoptotic levels in both cumulus cells and spermatozoa, and their relationship with fertilization and embryo quality after intracytoplasmic sperm injection (ICSI). The neutral comet assay was performed on cumulus cells and semen samples from 55 couples with male factor infertility undergoing ICSI treatment. Cells were fixed in agarose on comet assay slides, lysed in a neutral buffer and submitted to electrophoresis. The cells were stained with SYBR green fluorescent dye, which binds to double-stranded DNA and upon excitation emits light. Analysis showed that there was no correlation between apoptosis levels and the outcome of ICSI (fertilization and embryo quality).

  3. Cytogenetic Analysis of Sperm Nucleous Components of Iranian Normal and Sub-Fertile Individuals Using Zona Free Hamster Oocytes

    OpenAIRE

    Mozdarani, H.; Mohseni.Meybodi, A.

    2004-01-01

    Purpose: The purpose of this study was to investigate whether infertility is affected by sperm chromatin and cytogenetic abnormalities. To this purpose, the frequency of sperm premature chromosome condensation (PCC) induction and numerical chromosome abnormalities in the sperm of normal and sub-fertile men were analyzed. PCC rate was studied for evaluating the role of sperm chromatin abnormalities in the process of nuclear decondensation.

  4. Associação entre morfologia do ovócito e taxa de fertilização após ICSI Relationship between oocyte morphology and fertilization rate after ICSI

    Directory of Open Access Journals (Sweden)

    Alexandros Aggelis

    2006-04-01

    Full Text Available OBJETIVO: verificar a possibilidade de selecionar ovócitos que resultariam em maior taxa de fertilização. MÉTODOS: estudo retrospectivo que analisou a taxa de fertilização após ICSI de 957 ovócitos em metáfase II segundo três parâmetros da morfologia ovocitária: granulações citoplasmáticas, espaço perivitelino e fragmentação do primeiro corpúsculo polar. Os ovócitos foram obtidos de 115 ciclos realizados em 107 mulheres atendidas no CRHC, entre abril e dezembro de 2004. Para a análise estatística das diferenças na taxa de fertilização entre ovócitos "normais" e os que apresentavam cada alteração, utilizou-se o teste de chi2, com nível de confiança de 5 e 10%. RESULTADOS: não se observou diferença significativa na taxa de fertilização segundo as características do corpúsculo polar ou espessura do espaço perivitelino. A taxa de fertilização dos ovócitos com espaço perivitelino apresentando debris foi quase 14 pontos percentuais inferior ao dos ovócitos com espaço "ausente" (p=0,055 e a dos ovócitos com citoplasma granular foi sete pontos percentuais inferior à obtida pelos ovócitos com citoplasma de aspecto normal (p0,05. CONCLUSÕES: os parâmetros da morfologia do ovócito atualmente utilizados não permitem distinguir claramente aqueles que serão fertilizados dos que não serão.PURPOSE: to verify the possibility of identifying oocytes that would result in a higher fertilization rate. METHODS: retrospective analysis of the fertilization rate after ICSI of 957 oocytes in metaphase II according to three morphology parameters: cytoplasm inclusions, thickness of the perivitelline space, and fragmentation of the first polar body. Oocytes were obtained from 115 cycles performed among 107 women attended at the "Centro de Reprodução Humana de Campinas", from April to December of 2004. For the statistical analysis of differences in the fertilization rate between 'normal' oocytes and those presenting each

  5. Improved bovine embryo production in an oviduct-on-a-chip system : prevention of poly-spermic fertilization and parthenogenic activation

    NARCIS (Netherlands)

    Ferraz, Marcia A.M.M.; Henning, Heiko H.W.; Costa, Pedro F.; Malda, Jos; Melchels, Ferry P.W.; Wubbolts, Richard; Stout, Tom A.E.; Vos, Peter L.A.M.; Gadella, Bart M.

    2017-01-01

    The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to

  6. Effect of the exposure to methyl-β-cyclodextrin prior to chilling or vitrification on the viability of bovine immature oocytes.

    Science.gov (United States)

    Sprícigo, J F W; Morais, K S; Yang, B S; Dode, M A N

    2012-12-01

    The present study aimed to evaluate the effect of methyl-β-cyclodextrin (MβCD) as a cholesterol loader to change oocyte plasma membrane and increase its tolerance toward cryopreservation. The first and second experiments were conducted to investigate if MβCD could improve nuclear and cytoplasmic maturation after oocyte exposure to cold stress for 10 or 30 min, respectively. No differences (P>0.05) in either experiment in the metaphase II (MII) rate of oocytes exposed to MβCD and cold stress; but these oocytes presented lower maturation rates than control groups. In the second experiment, a lower percentage of oocytes showed degenerated chromatin (P0.05). Chromatin degeneration was higher in the vitrification groups. We conclude that MβCD improved nuclear maturation by reducing oocyte degeneration after cold stress or vitrification; however, more studies are required to clarify the usefulness of MβCD use in oocyte cryopreservation. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Heterologous murine and bovine IVF using bottlenose dolphin (Tursiops truncatus) spermatozoa.

    Science.gov (United States)

    Sánchez-Calabuig, M J; de la Fuente, J; Laguna-Barraza, R; Beltrán-Breña, P; Martínez-Nevado, E; Johnston, S D; Rizos, D; Gutiérrez-Adán, A; Pérez-Gutiérrez, J F

    2015-10-01

    Assisted reproductive technologies are of great importance for increasing the genetic diversity in captive animals. The use of bovine or murine oocytes in heterologous IVF provides advantages compared to homologous IVF in nondomestic animals, such as the accessibility to oocytes and the availability of well-developed in vitro maturation systems. The aim of this study was to determine the heterologous IVF parameters using cryopreserved dolphin spermatozoa and zona-intact bovine or murine oocytes and to examine the nuclear chromatin status of the dolphin spermatozoa. All the processes involved in the fertilization including embryo cleavage were observed by confocal microscopy and hybrid embryo formation was confirmed by polymerase chain reaction. Heterologous bovine IVF showed no polyspermy, lower percentages of pronuclear formation, and a lower cleavage rate compared to homologous IVF group (34.8% vs. 89.3%). Heterologous murine IVF showed a lower cleavage rate than homologous IVF (9.6% vs. 77.1%). With respect to dolphin sperm chromatin, it was more stable, i.e. more resistant to EDTA-SDS decondensation than the bovine sperm chromatin. This study revealed the stability of the dolphin sperm chromatin and the ability of the dolphin spermatozoa to penetrate zona-intact bovine and murine oocytes, leading to hybrid embryo formation. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Theca cells and theca-cell conditioned medium inhibit the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

    Science.gov (United States)

    van Tol, H T; Bevers, M M

    1998-11-01

    The effect of follicular cells and their conditioned media on the FSH-induced oocyte maturation of oocytes surrounded by cumulus cells connected to the membrana granulosa (COCGs) was investigated. COCGs and cumulus oocyte complexes (COCs) were cultured for 22 hr in M199 supplemented with 0.05 IU FSH/ml in either the presence of pieces of theca cell layer or in the presence of pieces of membrana granulosa. COCGs and COCs were also cultured for 22 hr in either theca-cell conditioned medium (CMt) or in granulosa cell conditioned medium (CMg), both supplemented with 0.05 IU FSH/ml. To investigate the importance of cell-cell contacts between granulosa cells and cumulus cells, oocytes were cultured as COCs in CMt, as COCs in CMt supplemented with pieces of membrana granulosa, or as COCGs in CMt. In all groups the medium was supplemented with 0.05 IU FSH/ml. After culture the nuclear status of the oocytes was assessed using orcein staining. Culture of COCGs in the presence of theca cells as well as in CMt resulted in a significantly decreased proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) at the end of the culture period as compared to the control. Of the oocytes that resumed meiosis in the presence of theca cells or in CMt, the proportion of oocytes that progressed up to the MII stage was significantly reduced. This indicates the production of a meiosis-inhibiting factor by theca cells. Culture with COCs instead of COCGs resulted in comparable results although the effect was less pronounced. The significant effect on the progression of meiosis of oocytes cultured as COCGs or as COCs, obtained in the presence of granulosa cells or in CMg, was much weaker than the effect of theca cells or culture in CMt. Culture of COCs in CMt supplemented with layers of membrana granulosa and 0.05 IU FSH/ml, resulted in significantly less oocytes that resumed meiosis as compared to culture of COCs in CMt. Of the oocytes that showed GVBD, the proportion that

  10. A comparative study of saffron aqueous extract and its active ingredient, crocin on the in vitro maturation, in vitro fertilization, and in vitro culture of mouse oocytes.

    Science.gov (United States)

    Mokhber Maleki, Elham; Eimani, Hussein; Bigdeli, Mohammad Reza; Ebrahimi, Bita; Shahverdi, Abdol Hossein; Golkar Narenji, Afsane; Abedi, Reyhane

    2014-03-01

    Reactive oxygen species have effects on gamete quality and gamete interaction; they influence spermatozoa, oocytes, embryos, and their environment. In this study, we evaluated the antioxidant effect of different concentrations of saffron (Crocus sativus L.) aqueous extract (SAE) and its ingredient, crocin, on the improvement of in vitro maturation (IVM) and subsequent in vitro fertilization (IVF) and embryo development of mouse oocytes. Cumulus oocyte complexes were collected from ovaries, and germinal vesicle oocytes were cultured in the presence of SAE and crocin. SAE was added at dosages of 5 μg/mL, 10 μg/mL, and 40 μg/mL; dosages of crocin were 50 μg/mL, 100 μg/mL, and 400 μg/mL. All dosages were added to maturation medium and a group without SAE or crocin was considered as the control group. Following IVM, metaphase II oocytes were fertilized and cultured in vitro in order to observe embryo development. Both SAE and crocin improved the rate of IVM, IVF, and in vitro culture. Addition of 40 μg/mL SAE to maturation medium significantly increased the rate of IVM, IVF, and in vitro culture (p < 0.05). Furthermore 100 μg/mL crocin significantly increased the IVM rate compared to the control group (p < 0.05). Use of SAE during IVM can affect on IVM, IVF, and early embryo development in a dose-dependent manner. SAE appears to have a stronger effect than pure crocin. Copyright © 2014. Published by Elsevier B.V.

  11. 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.

    Science.gov (United States)

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

    2014-08-01

    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 days. Activation rate, pronucleus formation, cleavage rate, and developmental potential of parthenotes during culture were evaluated. A total of 170 unfertilized oocytes were activated; 65 developed to cleavage stage, 19 developed to greater than the eight-cell stage, and five blastocysts were obtained. The activation rate of the calcium ionophore A23187 group was higher than that of the strontium chloride group (75.4% and 46.8%, respectively; pstrontium chloride group, six developed to the two- to four- cell stage, 10 developed to the five- to eight-cell stage, four developed to greater than the eight-cell stage, and one blastocyst was obtained. Three-day-old unfertilized human oocytes after IVF or ICSI could be activated by the calcium ionophore A23187 or strontium chloride, and a small part of parthenogenetic embryos developed into blastocysts. The treatment with the calcium ionophore A23187 was better than that of strontium chloride in respect to the activation rate of 3-day-old unfertilized human oocytes after IVF or ICSI.

  12. Vacuum-cooled liquid nitrogen increases the developmental ability of vitrified-warmed bovine oocytes Nitrogênio super resfriado por vácuo melhora a capacidade de desenvolvimento de oócitos bovinos após vitrificação

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    Rodrigo Marques dos Santos

    2006-10-01

    Full Text Available The objective of this study was to determine the effects of vacuum-cooled liquid nitrogen on the development of vitrified immature (germinal vesicle stage; GV and mature (metaphase II; MII bovine oocytes after re-warming. Liquid nitrogen was exposed to either atmospheric pressure or to a vacuum (300mm Hg for 45sec; the latter decreased the temperature of the liquid nitrogen to -200°C. Partially denuded oocytes were vitrified either just after selection (GV or after 22 hours of in vitro maturation (MII in TCM 199 medium + 10% of estrous mare serum. For vitrification, oocytes were firstly exposed to an intermediate solution (10% EG + 10% DMSO for 30sec, followed by the vitrification solution (20% EG + 20% DMSO + 0.5M sucrose for 20sec. Groups of three or four oocytes were loaded into an open-pulled-straw and directly plunged into liquid nitrogen. Oocytes were subsequently re-warmed by exposure to air (25°C for 4sec, followed by 5 min exposure to decreasing concentrations (0.3 and 0.15M of sucrose. Fertilization (Day 0 was done with 2 x 106 spermatozoa mL-1 (selected by a swim-up procedure and incubated for 18 to 22 hours. Presumptive zygotes were cultured at 39°C in four-well dishes with SOFaaci medium, under 5% CO2 and saturated humidity. Cleavage (Day 2 and blastocyst rates (Day 8 were 33.9 and 4.2%, respectively, for GV stage oocytes at atmospheric pressure, 41.2 and 8.8% for GV oocytes under vacuum, 43.5 and 6.7% for MII oocytes at atmospheric pressure, and 53.6 and 10.6% for MII oocytes under vacuum. In conclusion, vacuum-cooled liquid nitrogen improved developmental rates of vitrified-thawed bovine oocytes.O objetivo deste estudo foi determinar o efeito do nitrogênio liquido super resfriado por vácuo no desenvolvimento, após reaquecimento, de oócitos bovinos vitrificados imaturos ou maturados. O nitrogênio líquido foi mantido em atmosfera normal ou submetido ao vácuo (300mm Hg por 45s este último reduzindo a temperatura do nitrog

  13. 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

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    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.

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

    Science.gov (United States)

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

    2017-03-28

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

  15. Antioxidant effect of crocin on bovine sperm quality and in vitro fertilization.

    Science.gov (United States)

    Sapanidou, V; Taitzoglou, I; Tsakmakidis, Ι; Kourtzelis, I; Fletouris, D; Theodoridis, A; Zervos, I; Tsantarliotou, M

    2015-11-01

    Reactive oxygen species (ROS) production above critical levels affects the genetic and functional integrity of spermatozoa by causing oxidative stress. Spermatozoa are susceptible to oxidative stress in terms of motility and fertilization capacity. Crocin (crocetin di-gentiobiose ester), a main constituent of Crocus Sativus L. (saffron), is known for its antioxidant activity by scavenging ROS, especially superoxide anion. The aim of the present study is to evaluate the effect of crocin on the quality characteristics of spermatozoa and fertilization rate. Frozen-thawed and washed spermatozoa from four different bulls were incubated with three different concentrations of crocin (0.5, 1, and 2 mM), for 120 and 240 minutes, in the presence of a negative control, and were evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. The most potent concentration of crocin (1 mM) was also added in the fertilization medium to test its impact on fertilization outcome. The results indicate that the incubation of spermatozoa with 1 mM of crocin resulted in a statistically significant lower production of ROS, lower lipid peroxidation and in better maintenance of motility, viability, and acrosomal integrity, with a very small number of fragmented cells, compared to the control and the other treated groups (P quality and its fertilization capability, directly and/or indirectly, by modulating ROS concentration. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. The Role of Oocyte Organelles in Determining Developmental Competence.

    Science.gov (United States)

    Reader, Karen L; Stanton, Jo-Ann L; Juengel, Jennifer L

    2017-09-18

    The ability of an oocyte to undergo successful cytoplasmic and nuclear maturation, fertilization and embryo development is referred to as the oocyte's quality or developmental competence. Quality is dependent on the accumulation of organelles, metabolites and maternal RNAs during the growth and maturation of the oocyte. Various models of good and poor oocyte quality have been used to understand the essential contributors to developmental success. This review covers the current knowledge of how oocyte organelle quantity, distribution and morphology differ between good and poor quality oocytes. The models of oocyte quality are also described and their usefulness for studying the intrinsic quality of an oocyte discussed. Understanding the key critical features of cytoplasmic organelles and metabolites driving oocyte quality will lead to methods for identifying high quality oocytes and improving oocyte competence, both in vitro and in vivo.

  17. 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.

  18. The quality after culture in vitro or in vivo of porcine oocytes matured and fertilized in vitro and their ability to develop to term.

    Science.gov (United States)

    Nakamura, Yoshiyuki; Tajima, Sigeyuki; Kikuchi, Kazuhiro

    2017-12-01

    The quality of porcine blastocysts produced in vitro is poor in comparison with those that develop in vivo. We examined the quality of in vitro-matured and fertilized (IVM/IVF) oocytes, their abilities to develop to blastocysts under in vivo and in vitro conditions, and the potential of the embryos to develop to term after transfer. IVM/IVF oocytes were either transferred and the embryos recovered on Days 5 and 6 (100% and 87.5%, respectively) ('ET-vivo' embryos), or cultured in vitro for 5 or 6 days ('IVC' embryos). The proportion of blastocysts differed significantly between the two groups on Day 5 (20.6% and 8.0%, respectively), but not on Day 6 (23.8% and 21.2%, respectively). The mean number of cells in ET-vivo blastocysts on Days 5 or 6 was significantly higher (72.8 and 78.7, respectively) than that in IVC blastocysts (22.1 and 39.7, respectively). When IVM/IVF oocytes and IVC blastocysts on Day 6 were transferred, all (three and three, respectively) developed to piglets (16 and 16, respectively), without any difference in the rates of development to term (2.1% and 2.6%, respectively). These data suggest that, although blastocyst production differs between the two culture conditions, IVM/IVF oocytes possess the same ability to develop to term. © 2017 Japanese Society of Animal Science.

  19. 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

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    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.

  20. Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes.

    Science.gov (United States)

    Tatone, Carla; Carbone, Maria Cristina

    2006-09-25

    Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes. An In-Vitro-Fertilization assay (IVF) was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val), was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val), were evaluated. The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P exocytosis of cortical granules. These data support the view that RGD-binding receptors may function as signalling receptors giving rise integrated signalling not sufficient for a full oocyte activation response. This study may contribute to the understanding of possible negative effects of skipping gamete interaction in IVF techniques.

  1. Aminopeptidase-like protease released from oocytes affects oocyte surfaces and suppresses the acrosome reaction in establishment of polyspermy block in oocytes of the mussel Mytilus edulis.

    Science.gov (United States)

    Togo, T; Morisawa, M

    1997-02-15

    Suppression of the acrosome reaction of sperm on fertilized oocytes inhibits sperm-oocyte binding and is considered one of the three mechanisms responsible for polyspermy block in oocytes of the mussel Mytilus edulis (Togo et al., 1995). When unfertilized oocytes were inseminated in the presence of aminopeptidase inhibitors and the fertilized oocytes were inseminated again, neither the acrosome reaction nor sperm binding to fertilized oocytes were suppressed, suggesting that aminopeptidase-like protease participates in suppression of the acrosome reaction. The supernatant solution obtained after centrifuging a suspension of fertilized oocytes hydrolyzed aminopeptidase substrates, and these activities were inhibited strongly or effectively by aminopeptidase inhibitors. When unfertilized oocytes were incubated with this supernatant solution and inseminated, both the number of sperm bound and the acrosome reaction rate decreased, and these effects were reversed by conducting this treatment in the presence of aminopeptidase inhibitors. These results suggest that aminopeptidase-like protease released from the oocyte at fertilization affects the oocyte surface to suppress the acrosome reaction and consequently inhibits sperm-oocyte binding.

  2. Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry.

    Science.gov (United States)

    Abeydeera, L R; Johnson, L A; Welch, G R; Wang, W H; Boquest, A C; Cantley, T C; Rieke, A; Day, B N

    1998-11-01

    The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.

  3. Successful ongoing pregnancies after vitrification of oocytes.

    Science.gov (United States)

    Lucena, Elkin; Bernal, Diana Patricia; Lucena, Carolina; Rojas, Alejandro; Moran, Abby; Lucena, Andrés

    2006-01-01

    To demonstrate the efficiency of vitrifying mature human oocytes for different clinical indications. Descriptive case series. Cryobiology laboratory, Centro Colombiano de Fertilidad y Esterilidad-CECOLFES LTDA. (Bogotá, Colombia). Oocyte vitrification was offered as an alternative management for patients undergoing infertility treatment because of ovarian hyperstimulation syndrome, premature ovarian failure, natural ovarian failure, male factor, poor response, or oocyte donation. Mature oocytes were obtained from 33 donor women and 40 patients undergoing infertility treatment. Oocytes were retrieved by ultrasound-guided transvaginal aspiration and vitrified with the Cryotops method, with 30% ethylene glycol, 30% dimethyl sulfoxide, and 0.5 mol/L sucrose. Viability was assessed 3 hours after thawing. The surviving oocytes were inseminated by intracytoplasmic sperm injection. Fertilization was evaluated after 24 hours. The zygotes were further cultured in vitro for up to 72 hours until time of embryo transfer. Recovery, viability, fertilization, and pregnancy rates. Oocyte vitrification with the Cryotop method resulted in high rates of recovery, viability, fertilization, cleavage, and ongoing pregnancy. Vitrification with the Cryotop method is an efficient, fast, and economical method for oocyte cryopreservation that offers high rates of survival, fertilization, embryo development, and ongoing normal pregnancies, providing a new alternative for the management of female infertility.

  4. 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.

  5. Fbos, a novel oocyte-specific protein, interacts with proteins important for oocyte development in rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    Oogenesis is characterized by a series of developmentally regulated events, which result in the matured oocyte that can give rise to a new organism after fertilization. Oocyte-specific genes play critical roles in oogenesis; however, the molecular details of oocyte-specific genes are poorly defined....

  6. High survival and hatching rates following vitrification of embryos at blastocyst stage: a bovine model study.

    Science.gov (United States)

    Huang, Jack Y J; Chung, Jin-Tae; Tan, Seang Lin; Chian, Ri-Cheng

    2007-04-01

    Cryopreservation of embryos at the blastocyst stage may provide an effective method to increase the cumulative pregnancy rate for each treatment cycle of ovarian-stimulated IVF. The objective of this study was to evaluate the survival rate and hatching rate of bovine blastocysts following vitrification using a method designed for oocytes, with a view to introducing this methodology into human assisted reproduction technology and reproductive medicine. Bovine blastocysts were produced from abattoir materials subjected to in-vitro maturation and in-vitro fertilization. Survival rate of the bovine blastocysts was 100% (94/94) following vitrification using a method designed for oocyte cryopreservation. There was no difference in the hatching rate of the bovine blastocysts between control (62.5%: 60/96) and vitrified (61.7%: 58/94) groups. The number of dead cells in the blastocysts was not significantly different between control (5.0 +/- 2.9) and vitrified (9.5 +/- 4.0) groups. In conclusion, the results of this study indicate that bovine blastocysts can be vitrified successfully using a procedure designed for oocyte cryopreservation. It is possible that this method may also be successful for the cryopreservation of human embryos. A further study into this is currently being organized.

  7. Comparison of in-vitro outcomes from cryopreserved oocytes and sibling fresh oocytes.

    Science.gov (United States)

    Chamayou, S; Alecci, C; Ragolia, C; Storaci, G; Maglia, E; Russo, E; Guglielmino, A

    2006-06-01

    In Italy, the restrictive IVF law generalizes the indication for oocyte freezing for surplus oocytes in 78.5% of in-vitro assisted reproductive cycles. With a view to understanding better what the prospects for intracytoplasmic sperm injection (ICSI) on frozen-thawed oocytes might be, the consequences of freeze-thaw procedures on fertilization, cleavage rates and embryo quality obtained from frozen-thawed oocytes were studied and compared with the results obtained from sibling fresh oocytes. Eleven IVF and 29 ICSI on 76 and 169 fresh oocytes were performed and the corresponding 40 ICSI on 221 sibling frozen-thawed oocytes. There was no difference in terms of fertilization rate between fresh and sibling frozen-thawed oocytes. The cleavage rate (98.0 and 94.4% with fresh oocytes in IVF and ICSI; 77.3% with frozen-thawed oocytes in ICSI; P cryoconservation induces irreversible damage to microtubule repolymerization. The consequences of oocyte cryopreservation procedures on embryo development are reviewed.

  8. Current trends and progress in clinical applications of oocyte cryopreservation

    Science.gov (United States)

    Cil, Aylin P.; Seli, Emre

    2013-01-01

    Purpose of review To delineate the current trends in the clinical application of oocyte cryopreservation. Recent findings Although the first live birth from oocyte cryopreservation was reported approximately three decades ago, significant improvement in the clinical application of oocyte cryopreservation took place only over the past decade. On the basis of the available evidence suggesting that success rates with donor oocyte vitrification are similar to that of IVF with fresh donor oocytes, the American Society of Reproductive Medicine has recently stated that oocyte cryopreservation should no longer be considered experimental for medical indications, outlying elective oocyte cryopreservation. Meanwhile, a few surveys on the attitudes toward oocyte cryopreservation revealed that elective use for the postponement of fertility is currently the most common indication for oocyte cryopreservation. Most recently, a randomized controlled trial revealed important evidence on the safety of nondonor oocyte cryopreservation, and confirmed that the clinical success of vitrification is comparable to that of IVF with fresh oocytes. Summary The evidence suggesting similar IVF success rates with both donor and nondonor cryopreserved oocytes compared with fresh oocytes will increase the utilization of elective oocyte cryopreservation. Appropriate counseling of women for oocyte cryopreservation requires the establishment of age-based clinical success rates with cryopreserved oocytes for various indications. PMID:23562954

  9. How does polyspermy happen in mammalian oocytes?

    Science.gov (United States)

    Wang, Wei-Hua; Day, Billy N; Wu, Guang-Ming

    2003-07-01

    Polyspermy is one of the most commonly observed abnormal types of fertilization in mammalian oocytes. In vitro fertilization (IVF) provides approaches to study the mechanisms by which oocytes block polyspermic fertilization. Accumulated data indicate that oocyte, sperm and insemination conditions are all related to the occurrence of polyspermic fertilization. A high proportion of immature and aged oocytes showed polyspermy as compared with mature oocytes. Preincubation of oocytes and/or sperm with oviductal epithelial cells or collected oviductal fluid before IVF reduces polyspermic penetration. Recently, it was found that an abnormal zona pellucida is one of main causes of polyspermy in human eggs. A high proportion of polyspermy has resulted from the use of a high concentration of capacitated spermatozoa at the site of fertilization, irrespective of in the in vivo or in vitro environment. Oviductal secretions or oviductal epithelial cells themselves can regulate the number of spermatozoa reaching or binding to the zona pellucida thus reducing multiple sperm penetration. Suboptimal in vitro conditions, such as supplementations in IVF media, pH, and temperature during IVF, also induce polyspermic fertilization in some mammals. Species-specific differences are present regarding the relationship between insemination conditions and polyspermy. Copyright 2003 Wiley-Liss, Inc.

  10. [Oocyte vitrification in an ART laboratory].

    Science.gov (United States)

    Boyer, P; Montjean, D; Tourame, P; Gervoise-Boyer, M

    2013-09-01

    Oocyte vitrification has been authorized in France after the modification of French bioethics law in July 2011. This evolution will bring a dramatic change in patients' management since, from 2011, infertile couples, oocyte donation and fertility preservation programs will benefit this technique in France. We have introduced oocyte vitrification in our ART laboratory through a validation of the method using Evidence-Based Medicine model: open system Cryotop, Ethylène-glycol 15% and DMSO 15%. Based on our 1-year experience, oocyte vitrification upgrades our daily practice while also minimizing embryo cryoconservation as recommended by the law. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  11. A novel oocyte maturation trigger using 1500 IU of human chorionic gonadotropin plus 450 IU of follicle-stimulating hormone may decrease ovarian hyperstimulation syndrome across all in vitro fertilization stimulation protocols.

    Science.gov (United States)

    Anaya, Yanett; Mata, Douglas; Letourneau, Joseph; Cakmak, Hakan; Cedars, Marcelle I; Rosen, Mitchell P

    2017-10-30

    Modification of the trigger used to induce final oocyte maturation in in vitro fertilization (IVF) is a major strategy used to reduce the risk of ovarian hyperstimulation syndrome (OHSS). A novel trigger composed of 1500 IU of human chorionic gonadotropin (hCG) plus 450 IU of follicle-stimulating hormone (FSH) has been developed to reduce OHSS risk. This study compares outcomes of the novel trigger to conventional triggers used in high-risk OHSS patients undergoing IVF. In this retrospective cohort study, IVF cycles at high risk for OHSS based on a serum estradiol > 5000 pg/ml on trigger day conducted between January 2008 and February 2016 were evaluated. Oocyte maturation was induced with the novel trigger (1500 IU hCG plus 450 IU FSH) or a conventional trigger [3300 IU hCG, gonadotropin-releasing hormone agonist (GnRHa) alone, or GnRHa plus 1500 IU hCG]. IVF cycle outcomes were compared. Trigger strategies were examined for associations with OHSS development using logistic regression. Among 298 eligible IVF cycles identified, there were no differences in oocyte maturation, fertilization, embryo quality, or pregnancy outcomes among all triggers. After adjusting for serum estradiol level and number of follicles, the novel trigger was associated with lower odds of OHSS symptom development compared to the 3300 IU hCG and GnRHa plus hCG 1500 IU triggers (p = 0.007 and 0.04, respectively). This study suggests that 1500 IU hCG plus 450 IU FSH may be associated with decreased OHSS symptoms compared to conventional triggers, while producing similar IVF and pregnancy outcomes. More important, this novel trigger may provide a superior alternative in down-regulated cycles and in patients with hypothalamic dysfunction where GnRHa triggers cannot be utilized.

  12. Physiology and Endocrinology Symposium: influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature.

    Science.gov (United States)

    Paula-Lopes, F F; Lima, R S; Satrapa, R A; Barros, C M

    2013-03-01

    High environmental temperatures during the hot months of the year reduce reproductive performance in cattle. Summer heat stress depression in fertility is a multifactorial problem; however, there is evidence that the bovine germinal vesicle and maturing oocyte, as well as the early embryo, are major targets of the deleterious effects of heat stress. Such adverse effects are less pronounced in heat-tolerant breeds (Bos indicus) than heat-sensitive breeds (Bos taurus). This genetic variation results from the greater thermoregulatory ability and cellular thermoresistance of heat-tolerant breeds. Heat-induced oocyte cellular damage occurs in both cytoplasmic and nuclear compartments. Heat shock has been shown to reduce oocyte nuclear maturation, induce apoptosis, compromise oocyte cytoskeleton, and impair oocyte mitochondrial function and developmental competence. However, the oocyte cytoplasm is more susceptible to heat shock than the nucleus. This effect is greater for Bos taurus than Bos indicus oocytes. The detrimental effects of heat shock are also critical during the first cleavage divisions when most of the embryonic genome is inactive; however, the bovine embryo becomes more resistant to increased temperature as it proceeds through development. Several studies demonstrated that Bos indicus embryos are more thermotolerant than Bos taurus embryos. Adaptive changes involved in acquisition of thermotolerance are likely derived from changes in gene expression and (or) activity of biochemical molecules that control cellular functions against stress. Recently, molecules such as IGF-I and caspase inhibitor z-DEVD-fmk have been shown to exert a thermoprotective role, rescuing heat-induced oocyte and embryo cellular damage and developmental competence. Therefore, cattle genotype and thermoprotective molecules can be considered as an alternative to modulate the effects of increased temperature in reproductive function.

  13. 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)....

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

    Science.gov (United States)

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

    2017-04-01

    Older women are often the most challenging group of patients in fertility clinics due to a decline in both number and overall quality of oocytes. The quality of oocytes has been linked to mitochondrial dysfunction. In this mini-review, we discuss this hypothesis and suggest alternative treatment options using autologous mitochondria to potentially augment pregnancy potential in ART. Autologous transfer of mitochondria from the patient's own germline cells has attracted much attention as a possible new treatment to revitalize deficient oocytes. IVF births have been reported after transfer of oogonial precursor cell-derived mitochondria; however, the source and quality of the mitochondria are still unclear. In contrast, fully grown oocytes are loaded with mitochondria which have passed the genetic bottleneck and are likely to be of high quality. An increased supply of such oocytes could potentially be obtained by in vitro follicle activation of ovarian cortical biopsies or from surplus immature oocytes collected from women undergoing ART or fertility preservation of ovarian tissue. Taken together, autologous oocytes are not necessarily a limiting resource in ART as fully grown oocytes with high quality mitochondria can be obtained from natural or stimulated ovaries and potentially be used to improve both quality and quantity of oocytes available for fertility treatment. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Successful pregnancy and delivery after ICSI with artificial oocyte activation by calcium ionophore in in-vitro matured oocytes: a case report.

    Science.gov (United States)

    Kim, Jun-Woo; Yang, Seong-Ho; Yoon, San-Hyun; Kim, Sang-Don; Jung, Jae-Hoon; Lim, Jin-Ho

    2015-04-01

    The achievement of a successful pregnancy and delivery after oocyte activation with calcium ionophore is reported in a couple having low fertilization rates after intracytoplasmic sperm injection (ICSI) of in-vitro matured oocytes. A couple, in which the wife had polycystic ovary syndrome and the husband had moderate oligoteratozoospermia, showed a low fertilization rate in a previous in-vitro maturation cycle (2/11 [18.2%]). The most likely cause of complete fertilization failure or low fertilization rates is failure of oocyte activation. Therefore, artificial oocyte activation by calcium ionophore was combined with ICSI to achieve viable fertilized oocytes. Oocytes were stimulated with calcium ionophore for 30 min after ICSI. The fertilization rate of oocytes activated with calcium ionophore (13/15 [86.7%] and 7/9 [77.8%]) was higher than that of the non-activated oocytes. In the latest cycle, three embryos derived from the activated oocytes were transferred into the uterus on day 3. Subsequently, two gestational sacs were identified on ultrasound. The patient delivered dizygotic twins (girl 2260 g and boy 2760 g) at 35 weeks and 6 days gestation by caesarean section. This result suggests that calcium ionophore could be useful for oocyte fertilization in couples with low fertilization rates after ICSI of in-vitro matured oocytes. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    Older women are often the most challenging group of patients in fertility clinics due to a decline in both number and overall quality of oocytes. The quality of oocytes has been linked to mitochondrial dysfunction. In this mini-review, we discuss this hypothesis and suggest alternative treatment ...

  17. Effects of melatonin during IVM in defined medium on oocyte meiosis, oxidative stress, and subsequent embryo development.

    Science.gov (United States)

    Rodrigues-Cunha, Maria Carolina; Mesquita, Lígia G; Bressan, Fabiana; Collado, Maite Del; Balieiro, Júlio C C; Schwarz, Kátia R L; de Castro, Fernanda C; Watanabe, Osnir Y; Watanabe, Yeda F; de Alencar Coelho, Lia; Leal, Cláudia L V

    2016-10-15

    Melatonin may have beneficial effects when used in oocyte maturation and embryo development culture. The effect of melatonin during IVM on meiosis resumption and progression in bovine oocytes and on expression of antioxidant enzymes, nuclear fragmentation and free radicals, as well as on embryo development were assessed. Cumulus-oocyte complexes were matured in vitro with melatonin (10(-9) and 10(-6) M), FSH (positive control), or without hormones (negative control) in defined medium. Maturation rates were evaluated at 6, 12, 18, and 24 hours. Transcripts for antioxidant enzymes (CuZnSOD, MnSOD, and glutathione peroxidase 4 (GPX4)) in oocytes and cumulus cells, nuclear fragmentation in cumulus cells (TUNEL) and reactive oxygen species levels in oocytes (carboxy-H2 difluorofluorescein diacetate) were determined at 24 hours IVM. Effect of treatments on embryo development was determined after in vitro fertilization and culture. At 12 hours, meiosis resumption rates in FSH and melatonin-treated groups were similar (69.6%-81.8%, P > 0.05). At 24 hours, most oocytes were in metaphase II, with FSH showing highest rates (90.0%, P  0.05). In cumulus cells, MnSOD expression was higher in FSH group (P  0.05). In conclusion, although melatonin during IVM in a defined medium does not stimulate nuclear maturation progression it does stimulate meiosis resumption and such treated oocytes support subsequent embryo development. Melatonin also shows cytoprotective effects on cumulus-oocyte complexes. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. In vitro production of bovine embryos: cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence

    DEFF Research Database (Denmark)

    Mazzoni, Gianluca; Razza, Eduardo; Pedersen, Hanne S.

    2017-01-01

    In vitro production (IW) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level, a...

  19. The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development.

    Science.gov (United States)

    Mingoti, Gisele Zoccal; Castro, Viviane Sggobi Dias Caiado; Méo, Simone Cristina; Sá Barretto, Letícia Siqueira; Garcia, Joaquim Mansano

    2011-06-01

    Aiming to standardize in vitro production of bovine embryos and to obtain supplements to replace serum in culture media, this study evaluated the nuclear maturation kinetics and embryonic development in bovine after in vitro maturation (IVM) and culture (IVC) with several macromolecules (animal origin: bovine serum albumin (BSA), fetal calf serum (FCS); synthetic: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), Ficoll, and Knockout) at two oxygen tensions (20% and 5% O(2)). Regarding nuclear kinetics, neither the presence of the expected stage (metaphase I, transition anaphase to telophase, and metaphase II) at each evaluation moment (6, 18, and 24 h after IVM, respectively) nor the accelerated polar body emission (at 18 h after IVM) related developmental competence to blastocyst stage when different supplements were compared. Independently of supplement, cleavage rates at 20% O(2) (61.6-79.2%) were higher than at 5% O(2) (38.9-58.7%). At 20% O(2), higher blastocyst and hatching rates, respectively, were obtained in treatments BSA, FCS, Knockout, and control group (IVM with FCS and IVC with BSA + FCS, 14.0-23.5% and 6.8-15.4%) in comparison to PVA, PVP, and Ficoll (0%). The same was observed at 5% O(2) for blastocyst rates with BSA, FCS, Knockout, and control (5.4-16.8%) and for hatching rates with BSA, FCS, and control (2.0-11.1%). We can conclude that producing bovine embryos at 20% O(2) during the entire IVP process resulted in higher developmental rates than at 5% O(2). In addition, while defined macromolecules PVA, PVP, and Ficoll were not suitable for embryonic development, the synthetic serum Knockout was able to replace serum and albumin for IVP in bovine at 20% O(2).

  20. Rendimento de feijão-caupi cultivado com esterco bovino e adubo mineral Yield of cowpea-beans cultivated with bovine manure and mineral fertilization

    Directory of Open Access Journals (Sweden)

    Ademar P. Oliveira

    2001-03-01

    Northeast as 'macassar-bean' or rope bean is one of the main crops of this region being consumed as dry beans or green beans (pods and/or immature grain. In Paraíba, it is cultivated in almost all regions, representing 75% of the area cultivated with common dry beans. The low yield is due to the lack of a program of mineral nutrition. This experiment was carried out to evaluate the effect of different levels of bovine manure in the presence or absence of mineral fertilizer on pods and green and dry grains yield of the cowpea-bean, cv. IPA 206. The experiment was performed in the Federal University of Paraíba, in Brazil, from September/1998 to January/1999, in a randomized blocks design, where the treatments were distributed in a factorial scheme 5 x 2, with the first factor corresponding to levels of bovine manure (0, 10, 20, 30 and 40 t/ha and, the second factor, the presence or absence of mineral fertilizer, in four replications. Each plot consisted of 20 plants, spaced 0.80 x 0.40 m apart. The estimated maximum yield of pods (9.64 t/ha was obtained with 25 t/ha of bovine manure in the presence of mineral fertilizer, while in the absence of mineral fertilizer, the yield of pods, increased with the increasing levels of bovine manure, in the order of 49.3 kg/ha to each ton of bovine manure added to the soil. The yield of green grains in the presence of mineral fertilizer reached estimated maximum value (6.8 t/ha in the estimated optimum level of 17 t/ha of bovine manure. In the absence of mineral fertilizer, the yield of green grains, increased with the increasing of the levels of bovine manure in the order of 47.9 kg/ha to each ton of bovine manure added to soil. The yield of dry grains in the presence of mineral fertilizer reached estimated maximum value (3.03 t/ha in the level of 21 t/ha of bovine manure. In the absence of mineral fertilizer, the level of 25 t/ha of bovine manure was responsible for the maximum yield of dry grains (2.00 t/ha.

  1. Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes

    Directory of Open Access Journals (Sweden)

    Carbone Maria

    2006-09-01

    Full Text Available Abstract Background Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC and the exocytosis of cortical granules in mouse oocytes. Methods An In-Vitro-Fertilization assay (IVF was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val, was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val, were evaluated. Results The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P Conclusion The presents results provide evidence that a cyclic RGD peptide highly effective in inhibiting sperm-oocyte interaction stimulates in mouse oocytes the activation of PKC and the exocytosis of cortical granules. These data support the view that RGD-binding receptors may function as signalling receptors giving rise integrated signalling not sufficient for

  2. (LH) on in vitro maturation of Egyptian buffalo oocytes

    African Journals Online (AJOL)

    Microsoft Corporation

    2012-03-08

    Mar 8, 2012 ... immature bovine oocytes. Gamete Res. 24:197-204. Deshmukh SP, Pawshe CH, Ingawale MV, Deshmukh SG (2010). In vitro maturation of buffalo immature oocyte after Vitrification with combination of Ethylene Glycol and Dimethyl Sulfoxide. Proceedings. 9th World Buffalo Congress, pp. 911-921. Duncan ...

  3. Japanese Society for Animal Reproduction: award for outstanding research 2002. Cryopreservation of follicular oocytes and preimplantation embryos in cattle and horses.

    Science.gov (United States)

    Hochi, Shinichi

    2003-02-01

    Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both in vitro and in vivo when the embryos were exposed to vitrification solution in a stepwise manner. The vitrification procedure was also applied to the relatively large expanded blastocysts. (2) Bovine embryos produced in vitro have been considered to be highly sensitive to the process of cryopreservation. To solve this problem, Day-7 blastocysts produced in a serum-free system were cooled at 0.3 C/min rather than 0.6 C/min before being plunged into liquid nitrogen, resulting in no loss of the post-thaw viability. The supplementation of LAA in IVM/IVF media or IVC medium was effective in producing pronuclear-stage zygotes or morula-stage embryos relatively tolerable to freezing, respectively. (3) Transmission electron microscopic observation of immature equine oocytes showed that cellular injury occurred near the sites of gap-junctions between cumulus cells and the oocyte. In cattle, higher fertilization rates of oocytes were obtained when the oocytes were subjected to cryopreservation at an intermediate stage during IVM (GVBD for freezing, Met-I for vitrification). Vitrification of bovine Met-II oocytes in open-pulled glass capillaries, characterized by an ultra-rapid cooling rate (3,000-5,000 C/min), was found to avoid any harmful influence of vitrification and warming.

  4. Buffalo embryos produced by handmade cloning from oocytes selected using brilliant cresyl blue staining have better developmental competence and quality and are closer to embryos produced by in vitro fertilization in terms of their epigenetic status and gene expression pattern.

    Science.gov (United States)

    Mohapatra, Sushil K; Sandhu, Anjit; Neerukattu, Venkata S; Singh, Karn P; Selokar, Naresh L; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat

    2015-04-01

    We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB+) or colorless cytoplasm (BCB-). The blastocyst rate was higher (p<0.001) for BCB+ than for BCB- oocytes (43.41 ± 2.54 vs. 22.74 ± 1.76%). BCB+ blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher (p<0.05) than that of BCB- blastocysts. The global level of H3K9me2, which was similar in BCB+ and BCB- blastocysts, was higher (p<0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher (p<0.05) and that of GATA2 was lower (p<0.05) in BCB+ than that in BCB- blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower (p<0.05) and that of CDX2 was higher (p<0.05) in BCB+ than in IVF blastocysts. In conclusion, because BCB+ blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB- blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.

  5. Effect of Female Body Mass Index on Oocyte Quantity in Fertility Treatments (IVF): Treatment Cycle Number Is a Possible Effect Modifier. A Register-Based Cohort Study

    DEFF Research Database (Denmark)

    Christensen, Mette Wulf; Ingerslev, Hans Jakob; Degn, Birte

    2016-01-01

    treatment-cycles and a more favorable outcome. The main objective was to explore if treatment cycle number modifies the outcome when investigating the effect of female Body Mass Index (BMI) on oocyte quantity in IVF. MATERIAL AND METHODS: A historical cohort study was conducted on 5,342 treatment......-cycles during the period 1999-2009. Exclusion criteria were missing information on BMI or treatment type. Further, women were excluded if they had ovulated before oocyte retrieval. According to baseline BMI, women were divided into four categories following the World Health Organization standards. Multiple...... linear regressions analyses were performed accounting for the non-independence of ≥2 cycles in a woman. RESULTS: Stratification according to cycle number revealed a more suboptimal outcome in the first treatment- cycles than in the following cycles, suggesting a possible interaction or effect...

  6. Equine zona protein synthesis and ZP structure during folliculogenesis, oocyte maturation, and embryogenesis.

    Science.gov (United States)

    Kölle, Sabine; Dubois, Clotilde S; Caillaud, Maud; Lahuec, Cécile; Sinowatz, Fred; Goudet, Ghylène

    2007-07-01

    In the equine, the zona pellucida (ZP) is the major barrier to successful in vitro fertilization. Therefore the aim of our studies was to analyze species-specific features of the equine ZP in regard to structure and glycoprotein ZPB and ZPC expression sites during oocyte development and embryogenesis. The equine ZP revealed high immunological cross-reactivity to porcine ZPB and ZPC. In the ovary, the distribution of ZPB and ZPC was co-localized and correlated with the developmental stage of the follicle. ZPB and ZPC expression started in the oocyte of the late primordial and primary follicle. In the secondary follicle, both the oocyte and the cumulus cells contributed to ZPB and ZPC synthesis. After in vivo maturation the oocyte stopped ZPB and ZPC production whereas the cumulus cells continued synthesis. Contrary, in vitro matured (IVM) cumulus-oocyte-complexes (COCs) revealed a reverse expression pattern. This was correlated to alterations in the distribution, number, and size of pores in the ZP. In the zona, N-acetylglucosamine residues were co-localized with ZPC. The acellular glycoprotein capsule surrounding early equine embryos was negative for ZPB and ZPC. Our results imply that in the horse ZPB and ZPC glycoprotein expression is differentially regulated during folliculogenesis, oocyte maturation, and embryogenesis. Contrary to the bovine and porcine, zona protein synthesis during in vivo maturation is completely overtaken by the cumulus cells implying that in the horse these cells are crucial for zona integrity. During IVM, the cumulus cells lose their ability to synthesize glycoproteins leading to alterations in the zona structure.

  7. Antihyaluronidase action of ellagic acid effectively prevents polyspermy as a result of suppression of the acrosome reaction induced by sperm-zona interaction during in vitro fertilization of porcine oocytes.

    Science.gov (United States)

    Tokeshi, Isao; Yoshimoto, Teppei; Muto, Norio; Nakamura, Satoshi; Ashizawa, Koji; Nakada, Tadashi; Tatemoto, Hideki

    2007-08-01

    The present study was conducted to examine the effects of three tannin relatives (tannic acid, TA; gallic acid, GA; and ellagic acid, EA) on antihyaluronidase and reactive oxygen species (ROS) scavenging activity, in vitro fertilization (IVF) parameters, and the acrosome reaction (AR) induced by sperm-zona interaction. Among the three tannin relatives, TA and EA showed the strongest potency for blocking the hyaluronidase activity of boar sperm, with concentration-dependent inhibition over the range of 2-10 microg/ml. In contrast, ROSs were effectively scavenged by TA and GA, but not EA. When cumulus-free oocytes were inseminated in IVF medium containing 5 microg/ml of the tannin relatives, polyspermy was significantly reduced by TA and EA (32 and 29%, respectively) compared with oocytes treated with or without GA (51 and 69%, respectively) under conditions that maintained a high sperm penetration rate (Ppolyspermy by suppression of AR functionality induced by sperm-zona interaction and that hyaluronidase intervention is therefore required during porcine IVF.

  8. Morphological markers to select populations of oocytes with different cultural needs for dedicated pre-maturation protocols

    Directory of Open Access Journals (Sweden)

    Cecilia Dieci

    2017-05-01

    Full Text Available Oocyte’s chromatin gradually becomes more compacted during the final stage of oocyte development and the level of chromatin compaction is considered a marker of oocyte differentiation [Luciano et al, 2014]. Moreover, several studies demonstrate that in vitro pre-maturation treatments (Pre-IVM, aimed to improve the developmental capability of immature oocytes, might behave differently depending on the oocyte metabolic status, when it is isolated from follicle [Luciano et al., 2011]. This study aims at identifying correlations between cumulus-oocyte complex (COC morphology and oocyte chromatin configuration and secondly at testing the hypothesis that only fully grown oocytes at earlier stages of differentiation with loosely compacted chromatin  (GV1 can benefit from Pre-IVM treatment.   COCs were collected from bovine 2-6mm ovarian follicles, and further divided in three groups according to their morphology (Class-1, 2 and 3 as previously described [Blondin & Sirard, 1995]. Analysis of chromatin configuration revealed that only Class-1 COC was enriched in GV1 oocyte, while Class-2 and 3 presented a similar distribution of GV1, GV2 and GV3 oocytes, where GV2 and 3 oocytes are characterized by increased chromatin compaction [Lodde et al., 2007]. Then COCs were divided into two groups, one containing Class-1 COCs and the other containing Class-2 and 3 COCs and subjected to pre-IVM for 6 hours in presence of cilostamide and 10-4 UI/ml rhFSH. Finally, COCs underwent standard in vitro maturation (IVM for 22 hours, in vitro fertilization and embryo culture. Blastocyst rate and embryos cell number were assessed at day 7. Pre-IVM positively affected developmental competences of Class-1, while in Classes 2 and 3 Pre-IVM had detrimental effects.In conclusion COCs morphology could be used as a non-invasive approach to select population of oocyte with different cultural needs. These data could be useful in setting-up dedicated IVM protocols considering

  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. 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.

  11. Comparison Of The Effect Of Cytochalasin B And Paclitaxel (Taxol Tm On Cryopreservation Of Icr Mouse Oocytes

    Directory of Open Access Journals (Sweden)

    Po-Ning Lee

    2005-03-01

    Conclusion: Cumulus-free oocytes have greater developmental competence than cumulus-enclosed ones. Adding paclitaxel significantly improves the survival rate and fertilization rates of post-thaw cumulus-free ICR mouse oocytes. The addition of CCB also improves the fertilization rate of post-thaw oocytes.

  12. A quantitative assessment of follicle size on oocyte developmental competence

    Science.gov (United States)

    Rosen, Mitchell P.; Shen, Shehua; Dobson, Anthony T.; Rinaudo, Paolo F.; McCulloch, Charles E.; Cedars, Marcelle I.

    2015-01-01

    Objective To quantitatively assess the impact of follicle size on oocyte maturation, fertilization, and embryo quality. Design Prospective study. Setting Academic medical center. Patient(s) Couples undergoing ovarian stimulation and in vitro fertilization (IVF). Intervention(s) A total of 235 cycles were monitored prospectively, and 2934 oocytes were collected from five groups of follicle size. Repeated measures multivariate analyses were used to compare the smaller follicle sizes with the lead follicle. Main Outcome Measure(s) Oocyte maturation, fertilization, and embryo quality. Result(s) Compared with the lead follicular group (>18 mm), the odds of a mature oocyte from a 16 to 18 mm size follicle were 37% and declined progressively with each size. The odds of fertilization of oocytes from follicles 16 to 18 mm in size was 28% less than the lead group and decreased with each size. The rate of polyspermy with conventional insemination was increased for the smaller follicular groups (adjusted odds ratio =2.37). Follicle size did not predict embryo cell number, but embryos from smaller follicles had a statistically significantly higher fragmentation compared with the lead group. Conclusion(s) The lead follicular group was most likely to have a mature oocyte that was capable of fertilization and best suited for development into a high-quality embryo. The smaller follicles were capable of producing metaphase II oocytes that could fertilize, but at rates approaching only 60% that of the lead follicular group. PMID:18249377

  13. Calcium signals and oocyte maturation in marine invertebrates.

    Science.gov (United States)

    Deguchi, Ryusaku; Takeda, Noriyo; Stricker, Stephen A

    2015-01-01

    In various oocytes and eggs of animals, transient elevations in cytoplasmic calcium ion concentrations are known to regulate key processes during fertilization and the completion of meiosis. However, whether or not calcium transients also help to reinitiate meiotic progression at the onset of oocyte maturation remains controversial. This article summarizes reports of calcium signals playing essential roles during maturation onset (=germinal vesicle breakdown, GVBD) in several kinds of marine invertebrate oocytes. Conversely, other data from the literature, as well as previously unpublished findings for jellyfish oocytes, fail to support the view that calcium signals are required for GVBD. In addition to assessing the effects of calcium transients on GVBD in marine invertebrate oocytes, the ability of maturing oocytes to enhance their calcium-releasing capabilities after GVBD is also reviewed. Furthermore, possible explanations are proposed for the contradictory results that have been obtained regarding calcium signals during oocyte maturation in marine invertebrates.

  14. Collection of human oocytes at laparoscopy and laparotomy.

    Science.gov (United States)

    Lopata, A; Johnston, I W; Leeton, J F; Muchnicki, D; Talbot, J M; Wood, C

    1974-12-01

    In order to determine whether ovarian follicular aspiration at laparoscopy would provide enough oocytes for adequate in vitro studies, oocytes were collected during the periovulatory stage of the normal menstrual cycle from 45 women undergoing laparoscopy and from 25 women undergoing laparotomy. A larger average number of oocytes was recovered per patient at laparotomy due to better oocyte recoveries from ovarian wedges. However, the average number of oocytes rocovered per patient was the same at both procedures, providing that a suction vacuum of 200 mm Hg was used at laparoscopy, and under these conditions a greater percentage of follicles yielded oocytes at laparoscopy. Overall, 498 follicles were aspirated and 217 oocytes collected; the average recovery was 3 per patient. Moreover, the mean follicular diameter was 9.1 mm in infertile and 8.0 mm in fertile patients (p less than .05).

  15. 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.

  16. High exposure to progesterone between the end of menstruation and the day of triggering final oocyte maturation is associated with a decreased probability of pregnancy in patients treated by in vitro fertilization and intracytoplasmic sperm injection.

    Science.gov (United States)

    Kyrou, Dimitra; Kolibianakis, Efstratios M; Fatemi, Human M; Camus, Michel; Tournaye, Herman; Tarlatzis, Basil C; Devroey, Paul

    2011-10-01

    To investigate the association between the probability of pregnancy and hormone exposure between the end of menstruation and the day of triggering final oocyte maturation (menstruation-free interval). Prospective study. University. One hundred women (aged ≤ 39 years) stimulated with a fixed dose of recombinant follicle-stimulating hormone (200 IU). Daily gonadotropin-releasing hormone antagonist (GnRH, 0.25 mg) used from day 6 of stimulation onward, final oocyte maturation triggered by administration of 10,000 IU of human chorionic gonadotropin (hCG) as soon as ≥ 3 follicles ≥ 17 mm were present, and hormone assessment performed at initiation of stimulation, on the first day after menstruation had stopped, on the day of antagonist initiation, and on the day of hCG administration. The association between hormone exposure during the menstruation-free interval and the probability of ongoing pregnancy. The exposure to progesterone during the menstruation-free interval was statistically significantly higher in patients who did not become pregnant compared with those who did (4.20 ± 2.54 vs. 3.13 ± 1.14, respectively). Binary logistic regression confirmed the adverse effect of the increased exposure to progesterone for the achievement of pregnancy. In recombinant follicle-stimulating hormone/gonadotropin-releasing hormone antagonist in vitro fertilization/intracytoplasmic sperm injection cycles, a lower probability of pregnancy is associated with a higher exposure to progesterone during the menstruation-free interval. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  17. Effect of collection techniques on cumulus oocyte complexes (COCs ...

    African Journals Online (AJOL)

    The experiment was undertaken to study the effect of collection techniques on cumulus oocyte complexes (COCs) recovery, in vitro maturation (IVM) and in vitro fertilization (IVF) of goat oocytes. COCs were collected by three techniques viz. puncture, slicing and aspiration of goat ovaries obtained at slaughterhouse.

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

    Directory of Open Access Journals (Sweden)

    Nayara López Carpintero

    2014-01-01

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

  19. 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.

  20. 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

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

    Directory of Open Access Journals (Sweden)

    Jung Kyu Choi

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

  2. The potential for improving the growth and development of cultured farm animal oocytes.

    Science.gov (United States)

    Hansel, W

    2003-12-15

    Previous predictions that the technologies for producing genetically engineered large animal embryos containing genes for faster growth rates, leaner carcasses, greater disease resistance and improved lactational performance would be available early in the twenty-first century have been, for the most part, realized. The animal industries have been slow to adopt these technological advances and it cannot be said that any of them are currently having great impact on animal agriculture worldwide. A major reason for this is the inefficiencies of the techniques for superovulation, ovum recovery, in vitro fertilization, nuclear transfer, cloning and embryo transfer. Although improvements in these techniques can be expected, the best hope for increasing the impact of embryo transfer technologies on the animal industries lies in developing ways to mature, harvest, store and fertilize in vitro the large numbers of primordial oocytes present in the ovaries of all farm animals. Although limited progress has been made in the culture of bovine primordial oocytes, it is clear that much more research is needed to achieve success in this important area.

  3. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan.

    Science.gov (United States)

    Lane, Michelle; Maybach, Jeffrey M; Hooper, Kathy; Hasler, John F; Gardner, David K

    2003-01-01

    Recombinant albumin can be used to supplement culture medium for the maturation and fertilization of bovine oocytes and subsequent embryo development to the blastocyst stage. Recombinant albumin was able to support blastocyst development at rates equivalent to that of bovine serum albumin (BSA) supplemented media. Supplementation of media containing recombinant albumin and citrate stimulated blastocyst expansion. Culture with recombinant albumin and citrate significantly increased the ability of the resultant blastocysts to re-expand and hatch following cryopreservation. The further addition of the glycosaminoglycan hyaluronan to the culture medium containing either BSA or recombinant albumin also increased the ability of blastocysts to survive cryopreservation. Inclusion of recombinant albumin and hyaluronan in culture media facilitates the development of physiological defined culture conditions. For bovine embryos this has implications for both research and commercial applications where defined reproducible conditions are desirable. Copyright 2003 Wiley-Liss, Inc.

  5. A matched case-control study comparing udder health, production and fertility parameters in dairy farms before and after the eradication of Bovine Virus Diarrhoea in Switzerland.

    Science.gov (United States)

    Tschopp, A; Deiss, R; Rotzer, M; Wanda, S; Thomann, B; Schüpbach-Regula, G; Meylan, M

    2017-09-01

    An obligatory eradication programme for Bovine Virus Diarrhoea (BVD) was implemented in Switzerland in 2008. Between 2008 and 2012, all bovines were tested for antigen or antibodies against BVDV. By the year 2012, eradication was completed in the majority of farms. A decrease of the prevalence of persistently infected (PI) newborn calves was observed from 1.4% in 2008 to study was to assess the effects of BVD eradication on different parameters of animal health, production and fertility in Swiss dairy herds which had completed the eradication programme. A matched case-control study was carried out using data from two periods, before (Period 1) and after (Period 2) the active phase of eradication. Case farms had at least two PI animals detected before or during the eradication; controls were BVD-free and matched for region, herd size and use of alpine pasture. A total of 110 farmers (55 pairs) were recruited. During a phone interview, a questionnaire about farm characteristics, animal health and appreciation of the BVD eradication programme was filled in. Breeding data and milk test day records were also analyzed. Parameters were first compared between (i) case and control herds before eradication, and (ii) Period 1 and Period 2 for case herds only. Milk yield (MY), bulk milk somatic cell count (BMSCC), prevalence of subclinical mastitis (SCM), and non-return rate (NRR) showed a p-valuecase-control) was created (IA). Except for MY, the IA was significant for all parameters modelled. Despite an overall p-value of 0.27, case herds tended to have a higher MY after eradication (β=0.53, p=0.050). For BMSCC and SCM, case herds had higher values than controls in both periods; udder health was significantly improved in control herds and it remained stable in case herds, with a slight decrease of BMSCC (β=-0.19, p=0.010). Finally, among fertility parameters, NRR showed a general improvement but it was significant only in control herds (β=0.29, p=0.019). Even though the

  6. Effect of B-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws.

    Science.gov (United States)

    Takahashi, H; Kuwayama, M; Hamano, S; Takahashi, M; Okano, A; Kadokawa, H; Kariya, T; Nagai, T

    1996-10-15

    Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.

  7. Effects of trehalose vitrification and artificial oocyte activation on the development competence of human immature oocytes.

    Science.gov (United States)

    Zhang, Zhiguo; Wang, Tianjuan; Hao, Yan; Panhwar, Fazil; Chen, Zhongrong; Zou, Weiwei; Ji, Dongmei; Chen, Beili; Zhou, Ping; Zhao, Gang; Cao, Yunxia

    2017-02-01

    Sucrose and trehalose are conventional cryoprotectant additives for oocytes and embryos. Ethanol can artificially enhance activation of inseminated mature oocytes. This study aims to investigate whether artificial oocyte activation (AOA) with ethanol can promote the development competence of in vitro matured oocytes. A total of 810 human immature oocytes, obtained from 325 patients undergoing normal stimulated oocyte retrieval cycles, were in vitro maturated (IVM) either immediately after collection (Fresh group n = 291)) or after being vitrified as immature oocytes (Vitrified group n = 519). These groups were arbitrarily assigned. All fresh and vitrified oocytes which matured after a period of IVM then underwent intra-cytoplasmic sperm injection (ICSI). Half an hour following ICSI, they were either activated by 7% ethanol (AOA group) or left untreated (Non-AOA group). Fertilization, cleavage rate, blastocyst quality and aneuploidy rate were then evaluated. High-quality blastocysts were only obtained in both the fresh and vitrified groups which had undergone AOA after ICSI. Trehalose vitrification slightly, but not significantly, increased the formation rates of high-quality embryos (21.7% VS 15.4%, P > 0.05) and blastocysts (15.7% VS 7.69%, P > 0.05)) when compared with sucrose vitrification. Aneuploidy was observed in 12 of 24 (50%) of the AOA derived high quality blastocysts. High-quality blastocysts only developed from fresh or vitrified immature oocytes if the ICSI was followed by AOA. This information may be important for human immature oocytes commonly retrieved in normal stimulation cycles and may be particularly important for certain patient groups, such as cancer patients. AOA with an appropriate concentration of ethanol can enhance the developmental competence of embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Ultrastructure of human mature oocytes after vitrification

    Directory of Open Access Journals (Sweden)

    M.A. Khalili

    2012-08-01

    Full Text Available Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morphofunctional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microscopy has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.

  9. Female age, serum antimüllerian hormone level, and number of oocytes affect the rate and number of euploid blastocysts in in vitro fertilization/intracytoplasmic sperm injection cycles.

    Science.gov (United States)

    La Marca, Antonio; Minasi, Maria Giulia; Sighinolfi, Giovanna; Greco, Pierfrancesco; Argento, Cindy; Grisendi, Valentina; Fiorentino, Francesco; Greco, Ermanno

    2017-11-01

    To study the relative role of female age and ovarian reserve, measured through serum antimüllerian hormone (AMH) in determining the rate and number of euploid blastocysts in in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Retrospective analysis of cycles performed in 2014-2015. Tertiary referral IVF center. A total of 578 infertile couples undergoing IVF/ICSI and preimplantation genetic screening (PGS) analysis. All embryos were cultured and biopsied at the blastocyst stage. The method involved whole-genome amplification followed by array comparative genome hybridization. Serum AMH was measured by means of the modified Beckman Coulter AMH Gen II assay. The rate and number of euploid blastocysts and their correlation with ovarian reserve and response to stimulation. The mean (±SD) age of patients was 37.6 ± 4.1 years, and the mean number of blastocysts per patient was 3.1 ± 2. The total number of blastocysts available to the analysis was 1,814, and 36% of them were euploid after PGS. Age and serum AMH were significantly and independently related to the rate of euploid blastocysts available for patients. As an effect of the cohort size, the number of mature oocytes positively affected the total number of euploid blastocysts per patient. A strong positive age-independent relationship between AMH level and the rate of euploid blastocysts was found. This confirms that the measurement of ovarian reserve by means of AMH has high relevance when counseling infertile patients. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Calcium transients during early development in single starfish (Asterias forbesi) oocytes

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    Eisen, A.; Reynolds, G.T.

    1984-11-01

    Maturation and fertilization of the starfish oocyte are putative calcium-dependent events. The authors have investigated the spatial distribution and temporal dynamics of this calcium dependence in single oocytes of Asterias forbesi. They used the calcium photoprotein, aequorin, in conjunction with a microscope-photomultiplier and microscope-image intensifier. Surprisingly, in contrast to earlier work with Marasthenias glacialis, there is no detectable increase in intracellular-free calcium in the oocyte of A. forbesi in response to the maturation hormone 1-methyl adenine. During fertilization of the same, matured, A. forbesi oocyte there is a large increase in intracellular-free calcium. The calcium concentration increases to approx.1 ..mu..M at the point of insemination and the region of elevated free calcium expands across the oocyte in approx.20 s (17-19/sup 0/C). After the entire oocyte reaches an elevated concentration of free calcium, the concentration decreases uniformly throughout the oocyte over the next several minutes.

  11. Requirement of sperm-oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes.

    Science.gov (United States)

    Sengoku, K; Tamate, K; Takaoka, Y; Horikawa, M; Goishi, K; Okada, R; Tsuchiya, K; Ishikawa, M

    1999-02-01

    We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida-free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5+/-0.6 and 4.3+/-0.6 for ICSI; 3.0+/-0.3 and 3.8+/-0.4 for activated and 0.2+/-0.1 and 0.6+/-0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore-puromycin-activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes.

  12. Vitrification of human immature oocytes before and after in vitro maturation: a review.

    Science.gov (United States)

    Khalili, Mohammad Ali; Shahedi, Abbas; Ashourzadeh, Sareh; Nottola, Stefania Annarita; Macchiarelli, Guido; Palmerini, Maria Grazia

    2017-08-18

    The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some 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. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.

  13. 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 < 0.05) stimulatory effects. Ovine GDF9 or oBMP15 alone inhibited progesterone production by bovine granulosa cells, whereas in ovine cells only oGDF9 was inhibitory. The effects of oGDF9 and oBMP15 together were often cooperative and not always the same as those observed for each factor alone. Active immunisation of ewes with BMP15 and/or GDF9 peptides affected ovarian follicular development and ovulation rate. Depending on the GDF9 and/or BMP15 vaccine formulation, ovulation rate was either increased or suppressed. A primary and single booster immunisation of ewes with a BMP15 peptide in a water-based adjuvant has led to 19-40% increases in lambs born per ewe lambing. Collectively, the evidence suggests that oocyte

  14. Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification.

    Science.gov (United States)

    Nedambale, T L; Dinnyés, A; Groen, W; Dobrinsky, J R; Tian, X C; Yang, X

    2004-08-01

    The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.

  15. 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

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    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

  16. Dual trigger of final oocyte maturation with a combination of GnRH agonist and hCG versus a hCG alone trigger in GnRH antagonist cycle for in vitro fertilization: A Systematic Review and Meta-analysis.

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    Ding, Nan; Liu, Xingchen; Jian, Qiliang; Liang, Zhongzhen; Wang, Fang

    2017-11-01

    Increasing evidence indicates that a dual trigger (a gonadotrophin-releasing hormone agonist [GnRH-a] with a human chorionic gonadotrophin [hCG] trigger) is the best choice for final oocyte maturation in the GnRH antagonist (GnRH-ant) cycle. However, this conclusion remains controversial. Therefore, we performed this meta-analysis to systematically evaluate the efficacy of a GnRH-a combined with a standard hCG trigger in comparison with hCG alone for final oocyte maturation in the GnRH-ant cycle for in vitro fertilization. Complete electronic databases, including PubMed, Embase, The Cochrane Library, and Web of Science, were searched for relevant randomized controlled trials (RCT). The search was not restricted by language or publication time. Two reviewers selected trials and assessed trial quality independently by using the Cochrane Handbook 5.1.0. Four eligible RCT studies involving 527 women were included. The results of this meta-analysis indicated that the dual trigger group had a significantly higher pregnancy rate (relative risk [RR], 1.55; 95% confidence interval [CI], 1.17-2.06) than the hCG-only trigger group. No significant differences were found in the number of oocytes retrieved (weighted mean difference [WMD], 0.47; 95% CI, -0.42 to 1.37), number of mature oocytes retrieved (WMD, 0.41; 95% CI, -0.48 to 1.30), number of fertilized oocytes (WMD, 0.47; 95% CI, -0.32 to 1.26), number of good-quality embryos (WMD, 0.17; 95% CI, -0.29 to 0.64), or implantation rate (RR, 1.17; 95% CI, 0.69-2.00) between the two groups. GnRH-a and hCG as dual trigger was equivalent to hCG in triggering oocyte maturation and may be beneficial in improving reproductive outcomes. Further intensive randomized-controlled studies should be conducted to investigate the efficacy of the dual trigger. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2013-08-01

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

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

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

    1990-12-05

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

  19. Porcine oocyte maturation in vitro: role of cAMP and oocyte-secreted factors – A practical approach

    Science.gov (United States)

    APPELTANT, Ruth; SOMFAI, Tamás; MAES, Dominiek; VAN SOOM, Ann; KIKUCHI, Kazuhiro

    2016-01-01

    Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling. PMID:27349308

  20. Porcine oocyte maturation in vitro: role of cAMP and oocyte-secreted factors - A practical approach.

    Science.gov (United States)

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

    2016-10-18

    Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling.

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

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    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

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

    Science.gov (United States)

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

    2015-01-01

    The golden hamster is an excellent animal experimental model for oocyte research. The hamster oocytes are very useful in clinical examination of human spermatozoan activity. Non-fertile oocytes can lead to time-dependent processes of aging, which will affect the results of human spermatozoa examination. As a consequence there is a need to investigate the aging and anti-aging processes of golden hamster oocytes. In order to study the aging processes and parthenogenetic activation of golden hamster oocytes, in vivo oocytes, oocytes cultured with or without cumulus cells, and oocytes treated with Trichostatin A (TSA) or caffeine were collected and investigated. We found that: (1) spontaneous parthenogenetic activation, developmental potential (cleavage rate), and zona pellucida (ZP) hardening undergo age-dependent changes in in vivo, in vitro, and after TSA or caffeine treatment; (2) in vivo, oocytes became spontaneously parthenogenetic 25 h post-hCG treatment; (3) in vitro, cumulus cells did not significantly increase the parthenogenetic activation rate of cultured hamster oocytes; and (4) TSA or caffeine could delay spontaneous oocyte parthenogenetic activation and the aging processes by at least 5h, but also accelerated the hardening of the ZP. These results define the conditions for the aging and anti-aging processes in golden hamster oocytes. TSA and caffeine play roles in controlling spontaneous activation, which could facilitate the storage and use of golden hamster oocytes for studying processes relevant to human reproduction. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

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    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.

  4. Antral follicle count (AFC) can be used in the prediction of ovarian response but cannot predict the oocyte/embryo quality or the in vitro fertilization outcome in an egg donation program.

    Science.gov (United States)

    Melo, Marco Antonio Barreto; Garrido, Nicolás; Alvarez, Claudio; Bellver, José; Meseguer, Marcos; Pellicer, Antonio; Remohí, José

    2009-01-01

    To verify whether the antral follicle count (AFC) could predict ovarian response, oocyte/embryo quality, and IVF outcome. Prospective study. Instituto Universitario-Instituto Valenciano de Infertilidad, Valencia, Spain. One thousand seventy-four donors and 975 oocyte recipient cycles. Controlled ovarian hyperstimulation (COH), endometrial preparation, IVF/intracytoplasmic sperm injection, ET. COH and oocyte/embryo quality parameters and IVF outcome. We observed lower E(2) levels and fewer mature retrieved oocyte numbers among donors who showed an AFC that was <10. These donors also showed significantly higher cancellation and no-donation rates; poor and/or insufficient response was the principal cause (82%). However, there were no differences among the groups regarding embryo development parameters and IVF outcome. AFC is a noninvasive and simple tool that can improve the oocyte donor's selection of an egg donation program. This study suggests that AFC is a good predictor of ovarian response but cannot be used to predict oocyte/embryo quality or IVF outcome.

  5. In Vitro Maturation and Embryo Development to blastocyst Mouse Germinal Vesicle Oocytes after Vitrification

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    M Nikseresht

    2013-05-01

    Full Text Available Abstract Background & aim: Vitrification is a simple and ultra rapid technique for the conservation of fertility. Improving pregnancy rate associate with the use of cryopreserved oocytes would be an important advanced in human assisted reproductive technology (ART. The purpose of this study was to evaluate survival, oocytes maturation and embryo development to the blastocyst stage after vitrification of oocytes germinal vesicle-stage and multi stage Methods: In the present experimental study, germinal vesicle oocytes with or without cumulus cells were transferred to vitrification solution containing 30% (v/v ethylene glycol, 18% (w/v Ficoll-70, and 0.3 M sucrose, either by single step or in a step-wise way. After vitrification and storage in liquid nitrogen, the oocytes were thawed and washed twice in culture medium TCM119, and then subjected to in vitro maturation, fertilization, and culture. Data analysis was performed by using One-way variance and Tukey tests. Results: Oocytes survival, metaphase 2 stage oocyte maturation, fertilization and embryo formed blastocyst in vitrification methods multistage were significantly higher than the single step procedure (P<0/05 Conclusion: The Germinal vesicle stage oocytes vitrified with cumulus cells and stepwise procedure had positive effect on the survival, maturation and developmental rate on blastocyst compared to oocytes without cumulus cell and single step procedure. Key words: Germinal Vesicle Oocyte, Blastocyst, Vitrification, Ethylene glycol

  6. SPECIFICITY OF ANTIBODY BOVINE ZONNA PELLUCIDAE 3 (ANTI-bZP3 TO RABBIT ZP3 BASED ON bZP3 AS CONTRACEPTIVE ANTIGENS

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    Edwin Widodo

    2010-06-01

    Full Text Available Zonna pellucidae can be develop as antigen potential candidates based on reversible immunocontraceptive vaccines. Immunogenic sites of bovine zonna pellucidae 3 (bZP3 could stimulated the presence of anti-bZP3 which be located on rabbit ZP and inhibit sperm-egg interaction on fertilization process. Purpose of this research is to detect spesific binding anti-bZP3 to rabbit oocytes using dot blotting and ELISA method. Sub cutan induction of bZP3 with Freund's adjuvant, CFA (Complete Freund's Adjuvant for initial immunization and following by IFA (Incomplete Freund's Adjuvant at the 14th day and 39th day. Control female rabbit injected by Tris-Cl buffer diluted in Freund's adjuvant without bZP3 antigen. Rabbit serum injected to rat for producing Rat Anti Rabbit Anti-bZP3. This research concludes spesific binding of anti-bZP3 with increasing purple colour on dot blotting methods. Anti-bZP3 increasing on 24th day and 31th day and still until 48th day. Measurement with ELISA methods showed increased titer on OD405. Highest titer showed on 31th day post immunization. Anti-bZP3 synthetized by bZP3 induced on rabbit detectable by immunohistochemistry methods on late primary oocytes, early secondary oocytes, growing secondary oocytes, and oocytes on de Graaf folicular phase. Keywords: Dot blotting, ELISA, bZP3, anti-bZP3

  7. Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization.

    Science.gov (United States)

    Sánchez-Calabuig, María Jesús; García-Vázquez, Francisco Alberto; Laguna-Barraza, Ricardo; Barros-García, Carlos; García-Parraga, Daniel; Rizos, Dimitrios; Gutiérrez Adan, Alfonso; Pérez-Gutíerrez, José Félix

    2017-08-21

    The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

  8. Repeated collection of conjoined oocytes from a patient with polycystic ovary syndrome, resulting in one successful live birth from frozen thawed blastocyst transfer: a case report.

    Science.gov (United States)

    Yano, Kohji; Hashida, Naoko; Kubo, Toshiko; Ohashi, Ikuko; Koizumi, Azusa; Kageura, Rumi; Furutani, Kouichi; Yano, Chieko

    2017-08-05

    Few cases have been reported in which the aspiration of a single follicle led to the recovery of two conjoined oocytes surrounded by a single zona pellucida. This report describes a successful embryo transfer with subsequent live birth derived from conjoined oocytes, and a later pair of conjoined oocytes in the same patient. After oocyte retrieval from a patient with polycystic ovary syndrome, two pairs of conjoined oocytes were collected. One oocyte was fertilized using in vitro fertilization (IVF) and developed to the blastocyst stage. This blastocyst was cryopreserved and later transferred to the uterus after separating the unfertilized conjoined oocyte. A successful pregnancy and healthy live birth was achieved. Two years later, the patient returned for a second IVF; one pair of conjoined oocytes was detected. One of the pair was fertilized and developed to a blastocyst, but was not transferred. We demonstrate that selective fertilization of a mature oocyte from conjoined oocytes by IVF can lead to the development of a blastocyst and subsequent pregnancy and live birth. To our knowledge, this is the second case report of successful live birth from conjoined oocytes. It may be the first case of repeated fertile conjoined oocytes from the same patient.

  9. [Fertility preservation in oncology].

    Science.gov (United States)

    Chaput, Laure; Grémeau, Anne-Sophie; Vorilhon, Solène; Pons, Hanae; Chabrot, Cécile; Grèze, Victoria; Pouly, Jean-Luc; Brugnon, Florence

    2018-01-01

    Since the improvement of cancer diagnosis and treatment, survival rates of these patients increase. Gonadal damages are frequent consequences of cancer treatments with different evidence of impaired fertility. In this context, fertility preservation should be proposed to patients exposed to potentially gonadotoxic treatments. Different preservation approaches may be proposed depending on patient age, sex, cancer type and type of treatment. The indications of fertility preservation depend on sexual maturity. In young girls, ovarian cortex cryopreservation is the only technique feasible in order to preserve their reproductive potential. Vitrification of oocytes which needs ovarian stimulation or oocytes in vitro maturation is becoming more commonly performed for pubertal women to preserve their fertility. Ovarian cortex freezing could be offered to emergency fertility preservation of adult female cancer patients. In prepubertal boys, testicular tissue cryopreservation is the only line treatment for fertility preservation. For future use, various approaches are being evaluated such as spermatogonial stem cell injection or in vitro maturation. Cryopreservation of spermatozoa is, today, an established and successful technique for male adults. When there are no spermatozoa in ejaculate, sperm can be retrieved after treatment of testicular biopsy. The French bioethics law clearly indicates that fertility preservation should be proposed to patients exposed to potentially gonadotoxic treatment. Today, many approaches are possible. Fertility preservation indications are based on multidisciplinary consultations within platforms for the fertility preservation in order to optimize the patient care. Copyright © 2017 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

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

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    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.

  11. 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

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    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

  12. Structural Changes in Cattle Immature Oocytes Subjected to Slow Freezing and Vitrification

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    H. Wahid*, M. Thein1, E.A. El-Hafez2, M.O. Abas3, K. Mohd Azam4, O. Fauziah5, Y. Rosnina and H. Hajarian

    2012-05-01

    Full Text Available This study was conducted to evaluate the effect of different cryopreservation methods (slow-freezing and vitrification on structural changes of bovine immature oocytes. Bovine ovaries were collected from local abattoirs. Cumulus-oocyte-complexes (COCs were retrieved using aspiration method from 2-6 mm follicles. In Experiment 1, selected oocytes were randomly divided into 4 treatment groups namely freezing solution-exposed, frozen-thawed, vitrification solution-exposed and vitrified-thawed and then oocytes abnormalities were examined under a stereomicroscope. In Experiment 2, oocytes were randomly allocated to the same grouping as experiment 1 plus control group. Following freezing or vitrification, all oocytes were fixed in glutaraldehyde and processed for transmission electron microscopy. In experiment 1, there was a higher incidence of abnormalities in the frozen-thawed and vitrified-warmed oocytes compared to those in freezing solution and vitrification solution-exposed groups (P<0.05. In experiment 2, there were marked alterations in the perivitelline space, microvilli and vesicles of frozen-thawed and vitrified-warmed oocytes characterized by loss of elasticity and integrity of cytoplasmic processes and microvilli following cooling and warming. In conclusion, ethylene glycol-based freezing and vitrification solutions are suitable choices for cryopreservation of immature oocytes and most organelles are able to retain their normal morphology following cryopreservation and thawing processes.

  13. A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep.

    Science.gov (United States)

    Mullen, S F; Fahy, G M

    2012-11-01

    Vitrification as a means of cryopreservation has become a standard approach for oocytes from livestock. This paradigm shift occurred primarily as a result of the demonstration in 1996 that bovine oocytes are extremely susceptible to chilling injury. Since that early work, numerous devices have been used as supports for oocytes during so-called "ultra-rapid cooling", and occasionally, trials involving the deposition of small volumes of media containing oocytes directly into liquid nitrogen to facilitate cooling have been reported. Results reporting blastocyst development exceeding 10% are common, but variability remains high, and a standard method for bovine oocytes remains to be established. Oocytes from pigs are particularly difficult to cryopreserve, even with the use of ultrarapid cooling approaches. Few reports have demonstrated blastocyst development exceeding 5%. The application of hydrostatic pressure before vitrification appears to impart stress tolerance to porcine oocytes, as the results of some treatments have shown development to blastocysts at proportions >10%. Work on sheep oocyte vitrification is relatively new, and a few articles have reported blastocyst development at 10% or more. Messenger RNA levels are reportedly altered in sheep oocytes as a result of vitrification, and damage to the cytoskeleton is common across species. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa.

    Science.gov (United States)

    Bedford, S J; Kurokawa, M; Hinrichs, K; Fissore, R A

    2003-10-01

    In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.

  15. Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring.

    Science.gov (United States)

    Schurmann, Anita; Wells, David N; Oback, Björn

    2006-12-01

    Cloning by somatic cell nuclear transfer (SCNT) subverts sperm-mediated fertilization that normally leads to physiological activation of the oocyte. Therefore, artificial activation is required and it is presently unclear what developmental consequences this has. In this study, we aimed to improve cattle cloning efficiency by utilizing a more physiological method of activating SCNT reconstructs. We carried out in vitro fertilization (IVF) of zona-intact bovine oocytes before SCNT. We removed the zona pellucida 4 h after insemination, stained the fertilized eggs with Hoechst 33342 and mechanically removed both male and female chromatin. The enucleated pre-activated cytoplasts were fused with male adult ear skin fibroblasts ("IVF-NT" group). Chemically activated SCNT embryos, produced according to our standard operating procedure for zona-free SCNT, served as controls. After 7 days, in vitro development to blastocysts of morphological grade 1-3 or grade 1-2 was very similar in both groups (39 vs 40% and 20 vs 21% respectively). However, post-implantation development was improved after sperm-mediated activation. Across four replicate runs, pregnancy establishment at day 35 was significantly higher for IVF-NT than for control SCNT embryos (30/49 = 61 vs 17/41 = 42% respectively; P calves at term or weaning was also higher in the IVF-NT group compared with control SCNT (9/49 = 18 vs 3/41 = 7% and 6/49 = 12 vs 3/41 = 7%; P = 0.11 and 0.34 respectively).

  16. Effect of seminal plasma removal before cryopreservation of bovine semen obtained by electroejaculation on semen quality and in vitro fertility.

    Science.gov (United States)

    Campanholi, Suzane Peres; Monteiro, Fabio Morato; Ribeiro Dias, Erika Aline; Mercadante, Maria Eugênia Zerlotti; de Paz, Claudia Cristina Paro; Dell'Aqua Junior, José Antonio; Papa, Frederico Ozanam; Dell'Aqua, Camila de Paula Freitas; Vantini, Roberta; Garcia, Joaquim Mansano

    2017-02-01

    Cryopreservation of bull semen is a common biotechnology procedure in cattle breeding. However, when the ejaculate is obtained by electroejaculation, wide variation is observed in the sperm/seminal plasma (SP) ratio that can affect the freezability of semen in this species. The removal of SP may improve the quality of frozen bull semen. The objective of this study was to evaluate the effect of SP removal from the ejaculate on the cryopreservation of semen from 38 Nellore bulls collected by electroejaculation. After collection, the ejaculate was divided into three aliquots: (1) control (N) diluted to a concentration of 60 × 10(6) spermatozoa/mL and frozen with SP; (2) centrifugation (C) at ×600g for 10 minutes and the pellet resuspended and frozen at the same concentration as N; and (3) filtration (F) through SpermFilter and sperm recovered and frozen at the same concentration as N. After thawing, sperm kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, oxidative stress, and in vitro fertility were evaluated. Statistical analysis was performed using the SAS 9.2 package, and differences were considered significant when P < 0.05. Higher average path velocity and straight-line velocity were observed in the groups submitted to SP removal compared to the control group (P < 0.01). In contrast, filtered samples exhibited higher beat cross frequency, straightness, and linearity compared to the other groups. Plasma membrane integrity was reduced when SP was removed, but lower oxidative stress was observed in groups C and F (34.91 ± 2.95% and 31.63 ± 2.95%, respectively) compared to group N (57.39 ± 2.95%). However, the percentage of hatched blastocysts was similar in the N and F groups (21.22 ± 1.05% and 24.00 ± 1.05%, respectively) and higher compared to group C (18.83 ± 1.05%). In conclusion, removal of SP by centrifugation for bull semen freezing reduced the rate of in vitro-produced embryos, whereas filtration of

  17. 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.

  18. Comparing the effects of different in vitro maturation media on IVM outcomes of MI oocytes

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    Farzaneh Fesahat

    2017-09-01

    Conclusion: While the immature oocytes rescued from stimulated cycles based on specific conditions of patients can be useful for an alternative IVM intervention, it seems that different commercial culture media and longer incubation time has no beneficial effects on maturation, fertilization and embryo development on oocytes at MI stage.

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

    Science.gov (United States)

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Guang-Zhong Jiao

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

  2. Calcium and actin in the saga of awakening oocytes

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-04-24

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

  3. The Role of Mitochondria from Mature Oocyte to Viable Blastocyst

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    Scott Chappel

    2013-01-01

    Full Text Available The oocyte requires a vast supply of energy after fertilization to support critical events such as spindle formation, chromatid separation, and cell division. Until blastocyst implantation, the developing zygote is dependent on the existing pool of mitochondria. That pool size within each cell decreases with each cell division. Mitochondria obtained from oocytes of women of advanced reproductive age harbor DNA deletions and nucleotide variations that impair function. The combination of lower number and increased frequency of mutations and deletions may result in inadequate mitochondrial activity necessary for continued embryo development and cause pregnancy failure. Previous reports suggested that mitochondrial activity within oocytes may be supplemented by donor cytoplasmic transfer at the time of intracytoplasmic sperm injection (ICSI. Those reports showed success; however, safety concerns arose due to the potential of two distinct populations of mitochondrial genomes in the offspring. Mitochondrial augmentation of oocytes is now reconsidered in light of our current understanding of mitochondrial function and the publication of a number of animal studies. With a better understanding of the role of this organelle in oocytes immediately after fertilization, blastocyst and offspring, mitochondrial augmentation may be reconsidered as a method to improve oocyte quality.

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

    Science.gov (United States)

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

    2012-09-01

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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    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 impro...

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

    Directory of Open Access Journals (Sweden)

    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

  7. Ca2+ homeostasis regulates Xenopus oocyte maturation.

    Science.gov (United States)

    Sun, Lu; Hodeify, Rawad; Haun, Shirley; Charlesworth, Amanda; MacNicol, Angus M; Ponnappan, Subramaniam; Ponnappan, Usha; Prigent, Claude; Machaca, Khaled

    2008-04-01

    In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte's competence to undergo maturation in preparation for fertilization and embryonic development.

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

    Directory of Open Access Journals (Sweden)

    Evangelia Kasapi

    2017-03-01

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

  9. Social oocyte cryopreservation: a portrayal of Brazilian women.

    Science.gov (United States)

    Santo, Elisangela V Espirito; Dieamant, Felipe; Petersen, Claudia G; Mauri, Ana L; Vagnini, Laura D; Renzi, Adriana; Zamara, Camila; Oliveira, João Batista A; Baruffi, Ricardo L R; Franco, José G

    2017-06-01

    This study aimed to determine what Brazilian childless women of reproductive age think about oocyte cryopreservation to postpone pregnancy and their reasons for performing or not performing this procedure. Women of reproductive age were randomly selected from the general population using different e-mail lists and were invited to participate in the study by completing an online web survey regarding social oocyte cryopreservation. The survey was also distributed through social media to women of reproductive age. Although most of the responders had a partner (86.9%) and had already planned the pregnancy of their first child (69.6%), 85.4% (379) considered the potential of social oocyte freezing to improve their chances of giving birth later in life. Those that had already planned pregnancy were two times more likely to intend to freeze their oocytes (p=0.03). The most important barrier for not undergoing oocyte cryopreservation was cost. The women who indicated that they could not currently undergo the procedure now because of cost were two times (p=0.03) more likely to intend to cryopreserve their oocytes than women who thought that they would not need to delay pregnancy. Brazilian women who think that they are not ready to have a family are discovering the option of oocyte cryopreservation. Most participants considered safeguarding their reproductive potential. Making the procedure more accessible could give women the opportunity to make proactive decisions about the future of their fertility.

  10. Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti-polyspermy defense, and assisted reproduction.

    Science.gov (United States)

    Sutovsky, Peter; Manandhar, Gaurishankar; Wu, Alex; Oko, Richard

    2003-07-01

    Perinuclear theca (PT) is the cytoskeletal coat of mammalian sperm nucleus that is removed from the sperm head at fertilization. PT harbors the sperm borne, oocyte-activating factor (SOAF), a yet-to-be-characterized substance responsible for triggering the signaling cascade of oocyte activation, thought to be dependent on intra-oocyte calcium release. The present article reviews the current knowledge on the biogenesis and molecular composition of sperm PT. Possible functions of sperm PT during natural and assisted fertilization, and in the initiation of embryonic development are discussed. Furthermore, evidence is provided that SOAF is transferred from the sperm PT to oocyte cytoplasm through the internalization and rapid solubilization of the post-acrosomal PT. It is shown that during natural fertilization the sperm PT dissolves in the oocyte cytoplasm concomitantly with sperm nuclear decondensation and the initiation of pronuclear development. SOAF activity is preserved in the differentially extracted sperm heads only if the integrity of PT is maintained. After intracytoplasmic sperm injection (ICSI), activation occurs only in those oocytes in which the injected spermatozoon displays complete or partial dissolution of PT. In the latter case, the residual PT of the sub-acrosomal and/or post-acrosomal sperm region may persist on the apical surface of the sperm nucleus/male pronucleus and may cause a delay or arrest of zygotic development. We propose that the sperm PT harbors SOAF in the post-acrosomal sheath, as this is the first part of the sperm cytosol to enter the oocyte cytoplasm and its disassembly appears sufficient to initiate the early events of oocyte activation. Dissolution of the sub-acrosomal part of the PT, on the other hand, appears necessary to insure complete DNA decondensation in the internalized sperm nucleus and initiate DNA synthesis of both pronuclei. The release of the SOAF from the sperm head into oocyte cytoplasm at fertilization ultimately

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

    Science.gov (United States)

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

    2014-01-01

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

  12. Pelvic abscess complicating transvaginal oocyte retrieval: A case ...

    African Journals Online (AJOL)

    Pelvic abscess complicating transvaginal oocyte retrieval for in vitro fertilization is uncommon. Difficulties and/or delays in diagnosis, attributable to the rarity of the pathology, are associated with complications that lead to severe maternal and perinatality morbidity and mortality. In this report, we present a 37 year old ...

  13. Understanding fertilization through intracytoplasmic sperm injection (ICSI)

    Science.gov (United States)

    Neri, Queenie V.; Lee, Bora; Rosenwaks, Zev; Machaca, Khaled; Palermo, Gianpiero D.

    2014-01-01

    Summary Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described. PMID:24290744

  14. Chemical composition of lipids present in cat and dog oocyte by matrix-assisted desorption ionization mass spectrometry (MALDI- MS).

    Science.gov (United States)

    Apparicio, M; Ferreira, C R; Tata, A; Santos, V G; Alves, A E; Mostachio, G Q; Pires-Butler, E A; Motheo, T F; Padilha, L C; Pilau, E J; Gozzo, F C; Eberlin, M N; Lo Turco, E G; Luvoni, G C; Vicente, W R R

    2012-12-01

    The aim of the present study was to investigate the level of information on the chemical structures and relative abundances of lipids present in cat and dog oocytes by matrix-assisted laser desorption mass spectrometry (MALDI-MS). The MALDI-MS approach requires a simple analysis workflow (no lipid extraction) and few samples (two or three oocytes per analysis in this work) providing concomitant profiles of both intact phospholipids such as sphingomyelins (SM) and phosphatidylcholines (PC) as well as triacylglycerols (TAG). The lipids were detected in oocytes by MALDI using dihydroxybenzoic acid (DHB) as the matrix. The most abundant lipid present in the MS profiles of bitch and queen oocytes was a PC containing 34 carbons and one unsaturation [PC (34:1)]. Oocytes of these two species are characterized by differences in PC and TAG profiles detected qualitatively as well as by means of principal component analysis (PCA). Cat oocytes were mainly discriminated by more intense C52 and C54 TAG species and a higher number of unsaturations, indicating predominantly linoleic and oleic fatty acyl residues. Comparison of the lipid profile of bitch and queen oocytes with that of bovine oocytes revealed some similarities and also some species specificity: TAG species present in bovine oocytes were also present in bitches and queens; however, a more pronounced contribution of palmitic, stearic and oleic fatty acid residues was noticed in the lipid profile of bovine oocytes. MALDI-MS provides novel information on chemical lipid composition in canine and feline oocytes, offering a suitable tool to concomitantly monitor, in a nearly direct and simple fashion the composition of phospholipids and TAG. This detailed information is highly needed to the development of improved protocols for in vitro culture and cryopreservation of cat and dog oocytes. © 2012 Blackwell Verlag GmbH.

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

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

    2000-05-01

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

  16. Significant Down-Regulation of “Biological Adhesion” Genes in Porcine Oocytes after IVM

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    Joanna Budna

    2017-12-01

    Full Text Available Proper maturation of the mammalian oocyte is a compound processes determining successful monospermic fertilization, however the number of fully mature porcine oocytes is still unsatisfactory. Since oocytes’ maturation and fertilization involve cellular adhesion and membranous contact, the aim was to investigate cell adhesion ontology group in porcine oocytes. The oocytes were collected from ovaries of 45 pubertal crossbred Landrace gilts and subjected to two BCB tests. After the first test, only granulosa cell-free BCB+ oocytes were directly exposed to microarray assays and RT-qPCR (“before IVM” group, or first in vitro matured and then if classified as BCB+ passed to molecular analyses (“after IVM” group. As a result, we have discovered substantial down-regulation of genes involved in adhesion processes, such as: organization of actin cytoskeleton, migration, proliferation, differentiation, apoptosis, survival or angiogenesis in porcine oocytes after IVM, compared to oocytes analyzed before IVM. In conclusion, we found that biological adhesion may be recognized as the process involved in porcine oocytes’ successful IVM. Down-regulation of genes included in this ontology group in immature oocytes after IVM points to their unique function in oocyte’s achievement of fully mature stages. Thus, results indicated new molecular markers involved in porcine oocyte IVM, displaying essential roles in biological adhesion processes.

  17. Vitrification of mouse MII oocytes: Developmental competency using paclitaxel.

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    Fesahat, Farzaneh; Faramarzi, Azita; Khoradmehr, Arezoo; Omidi, Marjan; Anbari, Fatemeh; Khalili, Mohammad Ali

    2016-12-01

    Oocyte cryopreservation provides an important alternative for fertility preservation for women who will be treated with cytotoxic drugs. However, it can cause spindle disorganization of microtubules, putting the zygote at risk for aneuploidy. Paclitaxel is known to stabilize the microtubules that constitute the spindle. The aim of this study was to investigate the suitable concentration of paclitaxel for adding to the vitrification media to improve the developmental potential of post-thawed mature oocytes to blastocyst formation in mice. A total of 300 MII oocytes were retrieved from superovulated mice, and were divided into three groups of control, Experimental I, and Experimental II. Oocytes in Experimental I and Experimental II were cryopreserved in the presence of 0.5μM or 1μM of paclitaxel in vitrification media, respectively. After thawing, all oocytes were incubated in G-IVF medium for 1 hour. From each group,12 oocytes were selected for viability evaluation by Hoechst/propidium iodide nuclear staining. Standard in vitro fertilization was performed on the rest of the oocytes and embryo development was followed to the blastocyst stage. Fertilization rate was not significantly different between the three groups. However, the cleavage rate (55%) in Experimental II group was significantly lower compared to Experimental I (88%) and control groups (83%). There was a detectable difference between the three groups at the blastocyst rate (Experimental I and control groups, p = 0.004; Experimental II vs. control and Experimental I, p < 0.001). The highest rates of parthenogenesis and arrest were in Experimental II (16% and 21%, respectively) compared with control (6% and 5%, respectively) and Experimental I (5% and 3%, respectively). There was also a significant decrease in viability rate of oocytes in Experimental II compared to the other groups. A high concentration of paclitaxel, an anticancer drug, interrupted the mouse oocyte competency when supplemented to

  18. Meiotic maturation and developmental capability of ovine oocytes at germinal vesicle stage following vitrification using different cryodevices.

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    Quan, Guo Bo; Wu, Guo Quan; Wang, Ya Jing; Ma, Yuan; Lv, Chun Rong; Hong, Qiong Hua

    2016-02-01

    In order to assess effects of vitrification on ovine oocytes at the germinal vesicle (GV) stage, the conventional plastic straw (CS), the open-pulled straw (OPS), and Cryoloop were used to vitrify ovine oocytes. Oocytes were randomly divided into five groups: (1) Control; (2) Oocytes exposed to vitrification and dilution solutions without any cryopreservation (toxicity); (3) Oocytes vitrified using CS (CS); (4) Oocytes vitrified using OPS (OPS), and (5) Oocytes vitrified using Cryoloop (Cryoloop). The viability, cumulus cell expansion, nuclear maturation after in vitro maturation (IVM), and developmental capability of vitrified oocytes following parthenogenetic activation (PA) or in vitro fertilization (IVF) were assessed. The pretreatment in the vitrification and dilution solutions without any freezing or thawing did not adversely influence oocytes. The viability of vitrified oocytes were significantly declined compared to unfrozen oocytes (P straws or Cryoloop was significantly higher than that in the CS group (P plastic straws was significantly less than those of the other freezing groups (P straws. However, the cleavage rate of vitrified oocytes in the CS group was significantly less than that in the OPS or Cryoloop group (P plastic straw developed to the blastocyst stage following IVF. There was no significant difference existing between OPS and Cryoloop with respect to the blastocyst rate. After staining with cFDA and PI, cumulus cells surrounding oocytes were partly damaged by vitrification and thawing while the membrane of vitrified oocyte still remained intact. In conclusion, vitrification can seriously damage ovine immature oocytes and cumulus cells surrounding oocytes, which may subsequently affect their developmental capability. Finally, this study further proves that increasing the freezing and thawing velocity benefits survival of vitrified immature oocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5

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    Brenner Mariana PC

    2012-07-01

    Full Text Available Abstract Background Bovine Herpesvirus type-5 (BoHV-5 is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. Methods For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions, in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1, anti-oxidant like protein 1 (AOP-1, heat shock protein 70.1 (Hsp 70.1 and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1, antioxidant protection (AOP-1 and stress response (Hsp70.1 were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR. MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. Results The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357

  20. Evaluation of the Cryotech Vitrification Kit for bovine embryos.

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    Gutnisky, C; Alvarez, G M; Cetica, P D; Dalvit, G C

    2013-12-01

    The purpose of this work was to assess commercially available Cryotech Vitrification Kit, in terms of survival, in vitro development and pregnancy rate for bovine embryos. Cumulus-oocyte complexes (COCs) were recovered from ovaries obtained from slaughtered cows and then matured in vitro for 22 h. COCs were fertilized by sex-sorted sperm in IVF-mSOF and cultured in IVC-mSOF for 7 days to the blastocyst stage. Blastocysts were vitrified with the Cryotech Vitrification Kit(®) and then either warmed to check viability or transferred to synchronized heifers. We observed 100% survival of the in vitro produced blastocysts and obtained the same pregnancy rate (46.8%) as that obtained using fresh in vitro produced blastocysts. We thus conclude that the Cryotech vitrification method is a valid alternative to other vitrification or slow-cooling methods in the bovine species and that it is ready for livestock production. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Oocyte cryopreservation for donor egg banking.

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    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

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

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    Trounson Alan

    2008-12-01

    Full Text Available Abstract Background The present study highlights basic physiological differences associated with oocyte maturation and ageing. The study explores the fertilizing capacity and resistance to injury of mouse oocytes at different stages of maturation and ageing following IVF and ICSI. Also, the study examines the developmental competence of embryos obtained from these oocytes. The outcome of the study supports views that the mouse can be a model for human IVF suggesting that utilizing in-vitro matured and failed fertilized oocytes to produce embryos mainly when limited number of oocytes is retrieved in a specific cycle, should be carefully considered. Methods Hybrid strain mouse oocytes were inseminated by in-vitro fertilization (IVF or intracytoplasmic sperm injection (ICSI. Oocytes groups that were used were germinal vesicle (GV in-vitro matured metaphase II (IVM-MII, freshly ovulated MII (OV-MII, 13 hrs in-vitro aged MII (13 hrs-MII and 24 hrs in-vitro aged MII (24 hrs-MII.