Histone deacetylase inhibitor trichostatin A affects porcine oocyte maturation in vitro

Previous studies show that porcine oocyte aging resulting from asynchronised IVM impairs embryo developmental competence. In the present study we investigated whether trichostatin A (TSA; an inhibitor of histone deacetylation) prolongs the maturation time and prevents the aging of oocytes. Porcine oocytes were cultured in medium containing increasing concentrations of TSA (300 nM) for 24, 44 or 64 h. The percentage of oocytes that underwent germinal vesicle breakdown was significantly lower in the TSA-treated group (300 nM) than in the control group. TSA did not affect oocyte quality at MII based on levels of maturation-promoting factor, the phosphorylation status of mitogen-activated protein kinase or histone H3K9 acetylation analysis. We also compared the preimplantation developmental competence and the viability of pathenogenetic embryos treated with 100 nM TSA for 24 h and then continuously cultured for another 24 h in TSA free condition. No significant differences were observed for either parameter between the TSA-treated and control groups. These results indicate that TSA prolongs the IVM of porcine oocytes but that oocyte quality and aging are not affected. These findings provide a feasible option by which to adjust the initiation time of downstream experiments based on porcine matured oocytes.

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Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes

Animals ◽

10.3390/ani9040163 ◽

2019 ◽

Vol 9 (4) ◽

pp. 163

Author(s):

Payungsuk Intawicha ◽

Li-Kuang Tsai ◽

Shih-Ying Yen ◽

Neng-Wen Lo ◽

Jyh-Cherng Ju

Keyword(s):

Oocyte Maturation ◽

Mammalian Cells ◽

In Vitro Maturation ◽

Germinal Vesicle ◽

Mitogen Activated Protein Kinase ◽

Meiotic Maturation ◽

Germinal Vesicle Breakdown ◽

Mitogen Activated Protein ◽

Porcine Oocyte

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

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Early germinal vesicle breakdown is a predictor of high preimplantation developmental competent oocytes in mice

Zygote ◽

10.1017/s0967199416000290 ◽

2016 ◽

Vol 25 (1) ◽

pp. 41-48 ◽

Cited By ~ 2

Author(s):

Shogo Higaki ◽

Masao Kishi ◽

Keisuke Koyama ◽

Masashi Nagano ◽

Seiji Katagiri ◽

...

Keyword(s):

Time Course ◽

Evaluation Method ◽

Germinal Vesicle ◽

Oocyte Quality ◽

Developmental Competence ◽

Germinal Vesicle Breakdown ◽

Nuclear Maturation ◽

Non Invasive ◽

Germinal Vesicle Break Down

SummaryThe preselection of highly developmentally competent oocytes for in vitro maturation (IVM) is crucial for improving assisted reproductive technology. Although several intrinsic markers of oocyte quality are known to be closely related to the onset of nuclear maturation (germinal vesicle break down, GVBD), a direct comparison between GVBD timing and oocyte quality has never been reported. In this study, we established a non-invasive oocyte evaluation method based on GVBD timing for preselecting more developmental competent oocytes in mice. Because the O2 concentration during IVM may affect the nuclear kinetics, all experiments were performed under two distinct O2 concentrations: 20% and 5% O2. First, we determined the time course of changes in nuclear maturation and preimplantation developmental competence of in vitro-matured oocytes to estimate GVBD timing in high developmental competent oocytes. Two-thirds of oocytes that underwent GVBD in early IVM seemed to mainly contribute to the blastocyst yield. To confirm this result, we compared the preimplantation developmental competence of the early and late GVBD oocytes. Cleavage and blastocyst formation rates of early GVBD oocytes (80.2% and 52.7% under 20% O2, respectively, and 67.6% and 47.3% under 5% O2, respectively) were almost double those of late GVBD oocytes (44.8% and 26.0% under 20% O2, respectively, and 40.4% and 17.9% under 5% O2, respectively). With no observable alterations by checking the timing of GVBD in preimplantation developmental competence, oocyte evaluation based on GVBD timing can be used as an efficient and non-invasive preselection method for high developmental competent oocytes.

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Cycloheximide speeds the porcine oocyte nuclear kinetics and retains embryonic developmental potential in the presence of gonadotrophins

Canadian Journal of Animal Science ◽

10.4141/cjas09066 ◽

2010 ◽

Vol 90 (2) ◽

pp. 189-196

Author(s):

X -L. Sun ◽

W -Z. Ma ◽

Y -B. Zhu ◽

Z -H. Wu ◽

L. An ◽

...

Keyword(s):

Embryo Development ◽

Germinal Vesicle ◽

Chorionic Gonadotrophin ◽

Developmental Competence ◽

Electrical Activation ◽

Control Group ◽

Germinal Vesicle Breakdown ◽

Developmental Potential ◽

Oocyte Meiosis

Animal embryo engineering requires large amounts of synchronized mature oocytes in vitro. However, porcine cumulus-oocyte complexes aspirated from 3-8 mm follicles are at different germinal vesicle stages. They reach metaphase II stages asynchronously when cultured in vitro. In this study, we examined the effects of pretreatment with or without cycloheximide (CHX), equine chorionic gonadotrophin (eCG), human chorionic gonadotrophin (hCG), and their combinations on meiotic synchronization and the developmental competence of porcine oocytes in vitro following electrical activation. The COCs were pretreated for 12 h with either control medium (TCM 199), CHX (TCM 199 + CHX), eCG/hCG (TCM 199 + eCG/hCG) or eCG/hCG + CHX (TCM 199 + CHX + eCG/hCG), and then cultured for up to 32 h with TCM199 + eCG/hCG. After 12 h pretreatment, the rates of germinal vesicle breakdown (GVBD) were lower (P < 0.05) in the CHX (8.4%) and eCG/hCG + CHX (1.5%) groups compared with control (55.4%) and eCG/hCG (27.2%) groups. After removal of CHX and culture for an additional 12 h in vitro, the majority of the oocytes were synchronized at the GVBD stage in CHX (75.6%) and eCG/hCG + CHX (65.0%) groups. At additional 32 h of culture, the rate of oocytes in metaphase II in eCG/hCG + CHX group (68.3%) was significantly (P < 0.05) higher than the eCG/hCG group (54.8%), but did not differ from other groups (control: 61.3%, CHX: 58.8%). After electrical activation, the cleavage and blastocyst formation rates in the CHX group (80.3%; 19.5%) were significantly (P < 0.05) lower than those in the control group (95.5%; 45.3%), while no difference was found between eCG/hCG + CHX (82.2%; 34.4%) and control groups. Our data, hence, demonstrate pretreatment with CHX hastened nuclear kinetics of porcine oocytes cultured in vitro; however, embryo development potential was retained only when gonadotrophins is present in the in vitro maturation (IVM) medium. Thus, CHX should be used in the two-step culture systems in combination with gonadotrophins. Key words: Oocyte meiosis, synchronization, cycloheximide, embryo development, pig

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Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

Zygote ◽

10.1017/s0967199405003412 ◽

2005 ◽

Vol 13 (4) ◽

pp. 303-308 ◽

Cited By ~ 13

Author(s):

H. Iwata ◽

T. Hayashi ◽

H. Sato ◽

K. Kimura ◽

T. Kuwayama ◽

...

Keyword(s):

Granulosa Cells ◽

Germinal Vesicle ◽

Storage Period ◽

Cell Number ◽

Developmental Competence ◽

Germinal Vesicle Breakdown ◽

Storage Solutions ◽

Stock Solutions ◽

Phosphate Buffered Saline

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 °C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 °C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.

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Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media

Reproduction ◽

10.1530/rep.0.1240557 ◽

2002 ◽

pp. 557-564 ◽

Cited By ~ 47

Author(s):

M Shimada ◽

N Kawano ◽

T Terada

Keyword(s):

Steroid Hormones ◽

In Vitro Maturation ◽

Germinal Vesicle ◽

Mineral Oil ◽

Developmental Competence ◽

Cdc2 Kinase ◽

Germinal Vesicle Breakdown ◽

High Concentrations ◽

P34 Cdc2

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.

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Supplementation of c-type natriuretic peptide during the whole in vitro growth period benefits the development of murine secondary follicles

10.21203/rs.3.rs-16148/v1 ◽

2020 ◽

Author(s):

Ang Li ◽

Haixia Cao ◽

Hongxia Li ◽

Ruijiao Li ◽

Huaixiu Wang ◽

...

Keyword(s):

Natriuretic Peptide ◽

Ovulation Induction ◽

Germinal Vesicle ◽

Antral Follicle ◽

Control Group ◽

Follicle Development ◽

In Vitro Growth ◽

Germinal Vesicle Breakdown ◽

Preovulatory Follicles

Abstract Background Supplementation of c-type natriuretic peptide (CNP) in the culture medium shortly before in vitro maturation (IVM) has been reported to be effective in delaying meiotic resumption of murine oocyte. The present study investigated the effect of CNP supplementation during the whole period of in vitro growth (IVG) on the development of murine secondary ovarian follicles.Methods Late secondary ovarian follicles isolated from ovaries of Kunming mice were cultured in vitro with and without supplementation of CNP. In experiment 1, CNP was supplemented at the early stage and the follicle development was evaluated. In experiment 2 and 3, CNP was supplemented during the whole period of IVG. In experiment 2, follicle development and oocyte maturity were evaluated. In group 3, follicle development and rate of cleaved embryos after in vitro fertilization (IVF) was assessed.Results In control group in all 3 experiments, granulosa cells migrated from within follicle and adhered to the plate at different degrees. The follicles flattened and could not reach antral stage. About 39.8% (39/98) of the oocytes ovulated nakedly. As no antral follicle was obtained, IVF was not performed in control group in experiment 3. In experiment group in all 3 experiments, no migration of guanulosa cells was observed and the follicles grew three-dimensionally. Ovulation of naked oocyte decreased substantially. The rate of antral stage follicle were 45% (18/40) in experiment 1. This parameter was 75.9% (44/58) in experiment 2 and 3 combined. In experiment 2, in preovulatory follicles without ovulation induction, oocytes at germinal vesicle (GV) stage and germinal vesicle breakdown (GVBD) stage were 87.5% (14/16) and 12.5% (2/16), respectively. In preovulatory follicles with ovulation induction, no GV stage oocyte was retrieved, oocytes at GVBD and metaphase II (MII) stage were 50% (8/16), respectively. In experiment 3, among 18 follicles cultured, 12 cumulus-oocyte complexes (COC) ovulated automatically after ovulation induction. Eleven oocytes were fertilized and cleaved. Compared with control groups, the follicle development assessed by naked oocyte ovulation and follicle stage (preantral follicle and antral follicle) in experiment groups were significantly superior (p<0.0001). CNP effectively maintained oocytes’ meiotic arrest and enhanced fertilization competency.Conclusions The supplementation of CNP in culture system of murine late secondary follicle during the whole period of IVG could sustain the 3-dimensional structure of follicle, increase the antral formation rate. As a result, the oocyte’s competency to be fertilized was greatly improved.

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Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are not required for the global histone deacetylation observed after germinal vesicle breakdown (GVBD) in porcine oocytes

Reproduction ◽

10.1530/rep.1.00924 ◽

2006 ◽

Vol 131 (3) ◽

pp. 439-447 ◽

Cited By ~ 12

Author(s):

Tsutomu Endo ◽

Kunihiko Naito ◽

Sachi Kume ◽

Yukio Nishimura ◽

Koji Kashima ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Protein Kinase ◽

Germinal Vesicle ◽

Histone Deacetylation ◽

Mitogen Activated Protein Kinase ◽

Germinal Vesicle Breakdown ◽

Immature Oocytes ◽

Mitogen Activated Protein ◽

Maturation Promoting Factor

The acetylation of nuclear core histone has been suggested to work as an epigenetic mark for transmitting gene expression patterns to daughter cells. Global histone deacetylations, presumably involved in the reprogramming of the gene expression, have been observed after germinal vesicle breakdown (GVBD) in a cell cycle-dependent manner during meiotic maturation of mouse and porcine oocytes, although the regulation mechanism of histone deacetylation has not been studied well. In the present study, we examined the involvement of a crucial cell-cycle-regulator, maturation-promoting factor (MPF), and a meiosis-related kinase, mitogen-activated protein kinase (MAPK), in the global histone deacetylation during porcine oocyte maturation. In order to know whether the activities of MPF and MAPK were required, or the breakdown of GV membrane was sufficient, for the global histone deacetylation observed after GVBD, we artificially destroyed the GV membrane of the porcine immature oocytes. The artificial GV destruction (AGVD) induced histone deacetylation without the activation of MPF and MAPK. This deacetylation after AGVD was not affected by an MPF inhibitor, roscovitine, or an inhibitor of protein synthesis, cycloheximide, but was completely prevented by an inhibitor of histone deactylases (HDACs), trichostatine A. HDAC1 was present in the GV of the immature oocytes and localized on chromosomes after GVBD and AGVD. These results suggest that the MPF and MAPK activities were dispensable and the breakdown of the GV membrane was sufficient for the global histone deacetylation, which was catalyzed by HDAC activity

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Treatment of allicin improves maturation of immature oocytes and subsequent developmental ability of preimplantation embryos

Zygote ◽

10.1017/s0967199417000302 ◽

2017 ◽

Vol 25 (4) ◽

pp. 480-488 ◽

Cited By ~ 1

Author(s):

Sang-Gi Jeong ◽

Seung-Eun Lee ◽

Yun-Gwi Park ◽

Yeo-Jin Son ◽

Min-Young Shin ◽

...

Keyword(s):

Oocyte Maturation ◽

Polar Body ◽

Treated Group ◽

Developmental Competence ◽

Mitogen Activated Protein Kinase ◽

Preimplantation Embryos ◽

Maturation Medium ◽

Mitogen Activated Protein ◽

Porcine Oocyte

SummaryAllicin (AL) regulates the cellular redox, proliferation, viability, and cell cycle of different cells against extracellular-derived stress. This study investigated the effects of allicin treatment on porcine oocyte maturation and developmental competence. Porcine oocytes were cultured in medium supplemented with 0 (control), 0.01, 0.1, 1, 10 or 100 μM AL, respectively, during in vitro maturation (IVM). The rate of polar body emission was higher in the 0.1 AL-treated group (74.5% ± 2.3%) than in the control (68.0% ± 2.6%) (P < 0.1). After parthenogenetic activation, the rates of cleavage and blastocyst formation were significantly higher in the 0.1 AL-treated group than in the control (P < 0.05). The reactive oxygen species level at metaphase II did not significantly differ among all groups. In matured oocytes, the expression of both BAK and CASP3, and BIRC5 was significantly lower and higher, respectively, in the 0.1 AL-treated group than in the control. Similarly, the expression of BMP15 and CCNB1, and the activity of phospho-p44/42 mitogen-activated protein kinase (MAPK), significantly increased. These results indicate that supplementation of oocyte maturation medium with allicin during IVM improves the maturation of oocytes and the subsequent developmental competence of porcine oocytes.

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Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

Zygote ◽

10.1017/s0967199403002247 ◽

2003 ◽

Vol 11 (3) ◽

pp. 199-206 ◽

Cited By ~ 40

Author(s):

Tamás Somfai ◽

Kazuhiro Kikuchi ◽

Akira Onishi ◽

Masaki Iwamoto ◽

Dai-ichiro Fuchimoto ◽

...

Keyword(s):

Cyclic Amp ◽

Germinal Vesicle ◽

Developmental Competence ◽

Meiotic Maturation ◽

Intracellular Level ◽

Dibutyryl Cyclic Amp ◽

Oocyte Collection ◽

Porcine Oocyte ◽

Blastocyst Rate

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 μg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.

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Meiosis and embryo technology: renaissance of the nucleolus

Reproduction Fertility and Development ◽

10.1071/rd04108 ◽

2005 ◽

Vol 17 (2) ◽

pp. 3 ◽

Cited By ~ 24

Author(s):

Poul Maddox-Hyttel ◽

Bolette Bjerregaard ◽

Jozef Laurincik

Keyword(s):

Topoisomerase I ◽

De Novo ◽

Germinal Vesicle ◽

Developmental Competence ◽

Rdna Transcription ◽

Morphological Marker ◽

Germinal Vesicle Breakdown ◽

Oocyte Growth ◽

Embryonic Genome

The nucleolus is the site of rRNA and ribosome production. This organelle presents an active fibrillogranular ultrastructure in the oocyte during the growth of the gamete but, at the end of the growth phase, the nucleolus is transformed into an inactive remnant that is dissolved when meiosis is resumed at germinal vesicle breakdown. Upon meiosis, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities harbour the development of fibrillogranular nucleoli and re-establishment of nucleolar function in conjunction with the major activation of the embryonic genome. This so-called nucleologenesis occurs at a species-specific time of development and can be classified into two different models: one where nucleolus development occurs inside the NPBs (e.g. cattle) and one where the nucleolus is formed on the surface of the NPBs (e.g. pigs). A panel of nucleolar proteins with functions during rDNA transcription (topoisomerase I, RNA polymerase I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) are localised to specific compartments of the oocyte nucleolus and those engaged in late processing are, to some degree, re-used for nucleologenesis in the embryo, whereas the others require de novo embryonic transcription in order to be allocated to the developing nucleolus. In the oocyte, inactivation of the nucleolus coincides with the acquisition of full meiotic competence, a parameter that may be of importance in relation to in vitro oocyte maturation. In embryo, nucleologenesis may be affected by technological manipulations: in vitro embryo production apparently has no impact on this process in cattle, whereas in the pig this technology results in impaired nucleologenesis. In cattle, reconstruction of embryos by nuclear transfer results in profound disturbances in nucleologenesis. In conclusion, the nucleolus is an organelle of great importance for the developmental competence of oocytes and embryos and may serve as a morphological marker for the completion of oocyte growth and normality of activation of the embryonic genome.

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