In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.     

Evaluation of fluids from cystic follicles for in vitro maturation and fertilization of bovine oocytes

Follicular cysts are defined as cystic structures derived from unovulated follicles. The formation of the cysts appears to be related to failure of the oocyte to resume meiosis. The aim of this study was to evaluate in the bovine: 1) the ability of the fluid from cystic follicles to promote in vitro oocyte maturation and fertilization, 2) the predictive value of the morphology of oocytes derived from cystic follicles on the ability of the follicular fluid to promote in vitro maturation/fertilization as well as the oocytes to undergo maturation and fertilization. In Experiment 1, the ability of fluid from cystic (and normal) follicles from live and slaughtered cows (to promote) in vitro maturation and fertilization of bovine cumulus-oocyte-complexes (COC's) was assessed by cumulus expansion, sperm penetration, male pronucleus formation and polyspermy rates. Concentrations of progesterone (P4) and estradiol-17 beta (E2) were measured in the fluid from cystic follicles collected from live and slaughtered cows. In Experiment 2, we investigated the relationship of the morphology of COC's from cystic follicles, and the effect of the follicular fluids on oocyte maturation as well as P4 and E2 concentrations. In Experiment 1, although sperm penetration and male pronucleus formation were inhibited significantly by fluid from some cystic follicles collected from live and slaughtered cows, there were no significant differences in sperm penetration, male pronucleus formation and polyspermy rates between fluid from cystic follicles collected from live cows, from slaughtered cows and from control groups, regardless of the P4/E2 ratio. In Experiment 2, the morphology of cumulus-oocyte complexes from cystic follicles varied and the pronucleus formation of oocytes after in vitro fertilization was abnormal. On the other hand, the male pronucleus formation rates were not significantly different between the cystic follicular fluids and control, regardless, of the P4/E2 ratio. The results of this study suggest that many of the bovine follicular fluids from cystic follicles possess the ability to induce cumulus expansion, nuclear maturation and male pronucleus formation following in vitro maturation and fertilization of bovine oocytes. The morphology of the cumulus-oocytes complexes from cystic follicles seems not to relate to the ability of the cystic follicular fluids to induce oocyte maturation, and oocytes from cystic follicles possess the ability to form male pronucleus even though most were abnormal after in vitro fertilization.     

Effect of duration of in vitro maturation on nuclear maturation and fertilizability of feline oocytes

This study was conducted to improve in vitro production of embryos from domestic cats using TCM-199 as an IVM medium. The time sequence of nuclear maturation and the optimal timing of in vitro insemination were examined. Most oocytes were at the germinal vesicle stage immediately after collection; however, 8.3% had already resumed meiosis before IVM culture. After 30 h of IVM culture, the percentage of oocytes at metaphase II (MII) reached a peak (75.5%) and did not change (P>0.05) from 30 to 48 h after IVM culture. The percentage of oocytes with two pronuclei was higher (P<0.05) for oocytes matured for 30 and 36 h (38.2 and 33.0%, respectively) than for those after IVM culture for only 24 h (18.5%). Total sperm penetration rate was highest (P<0.05) for oocytes that had been matured for 30 h (46.1%). After 30 h of IVM and 18 h of IVF culture, 66.3 and 24.8% of inseminated oocytes had cleaved and developed to the blastocyst stage, respectively. We concluded that IVM of feline oocytes for 30 h in TCM-199 resulted in optimal nuclear maturation and sperm penetration.     

In vitro growth, maturation, fertilization, and embryonic development of oocytes from porcine preantral follicles

This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.     

Effect of gonadotropins, steroids and culture media on bovine oocyte maturation in vitro

The present experiment was to investigate the effect of gonadotropins (LH and hCG), steroids (estradiol and progesterone) and culture media (TCM 199, Ham-F-12, BMOC-3 and modified KRB) on in vitro maturation of cumulus-enclosed bovine oocytes. Oocytes isolated from follicles of 相似文献   

In vitro maturation of caprine oocytes in different culture media

Oocytes were aspirated from caprine ovaries, washed and cultured in TCM-199 containing penicillin, streptomycin and 10, 15, or 20% of fetal bovine serum (FBS), estrous sheep serum (ESS) or estrous goat serum (EGS). After 24–26 h culture, oocytes were separated from cumulus and corona cells by hyaluronidase and by passing through a fine pipette, fixed in acetic alcohol, stained with aceto orcein and observed under a phase-contrast microscope for evidence of maturation. High maturation rates (74–94%) were observed in all concentrations of the three different sera examined. No significant difference was observed between different concentrations and among different sera. Almost no maturation (4%) was observed in the medium lacking protein supplement. In conclusion, these sera with the concentrations examined can be substituted for one another for in vitro maturation of caprine oocytes.     

Nuclear staining and culture requirements for in vitro maturation of domestic bitch oocytes

Oocyte nuclear staining and culture requirements for in vitro maturation (IVM) in the bitch have yet to be fully investigated. In the first part of this study we investigated 7 methods for labeling nuclear material (573 oocytes). The most favorable method involved fixation plus aceto-orcein staining and light microscopy. The influence of serum supplementation of the culture medium for IVM was then investigated (1292 oocytes). Culture was performed in media supplemented with no serum or with 5, 10 and 20% fetal calf serum (FCS) and 0.3 or 4% bovine serum albumin (BSA). Identifiable nuclear material was either a germinal vesicle (GV) or GV breakdown (GVBD). After 48 h in medium plus 0, 5, 10 or 20% FCS and 0.3 or 4% BSA, the percentage of oocytes matured to GVBD was 13, 9, 15, 23, 36 and 40%, and the percentage matured to metaphase I/anaphase I/metaphase II was 4, 12, 24, 14, 36 and 13%, respectively. After 96 h, maturation to GVBD was 31, 14, 21, 11, 50 and 38%, and to metaphase I/anaphase I/metaphase II it was 6, 5, 3, 19, 15 and 9%, respectively. Within the limits of this study, BSA or high concentrations of FCS appear to be optimal for bitch oocyte maturation in vitro.     

In vitro grown sheep preantral follicles yield oocytes with normal nuclear-epigenetic maturation

Background

Assisted reproductive technologies allow to utilize a limited number of fully grown oocytes despite the presence in the ovary of a large pool of meiotically incompetent gametes potentially able to produce live births. In vitro folliculogenesis could be useful to recruit these oocytes by promoting their growth and differentiation.

Methodology/Principal Findings

In vitro folliculogenesis was performed starting from sheep preantral (PA) follicles to evaluate oocyte nuclear/epigenetic maturation. Chromatin configuration, quantification of global DNA methylation, and epigenetic remodelling enzymes were evaluated with immunocytochemistry, telomere elongation was assessed with the Q-FISH technique, while the DNA methylation status at the DMRs of maternally IGF2R and BEGAIN, and paternally H19 methylated imprinted genes was determined by bisulfite sequencing and COBRA. Specifically, 70% of PA underwent early antrum (EA) differentiation and supported in culture oocyte global DNA methylation, telomere elongation, TERT and Dnmt3a redistribution thus mimicking the physiological events that involve the oocyte during the transition from secondary to tertiary follicle. Dnmt1 anticipated cytoplasmic translocation in in vitro grown oocytes did not impair global and single gene DNA methylation. Indeed, the in vitro grown oocytes acquired a methylation profile of IGF2R and BEGAIN compatible with the follicle/oocyte stage reached, and maintained an unmethylated status of H19. In addition, the percentage of oocytes displaying a condensed chromatin configuration resulted lower in in vitro grown oocytes, however, their ability to undergo meiosis and early embryo development after IVF and parthenogenetic activation was similar to that recorded in EA follicle in vivo grown oocytes.

Conclusions/Significance

In conclusion, the in vitro folliculogenesis was able to support the intracellular/nuclear mechanisms leading the oocytes to acquire a meiotic and developmental competence. Thus, the in vitro culture may increase the availability of fertilizable oocytes in sheep, and become an in vitro translational model to investigate the mechanisms governing nuclear/epigenetic oocyte maturation.     

In vitro maturation and fertilization of equine oocytes recovered during the breeding season

The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with frozen-thawed semen separated by swim-up and treated with heparin was carried out to determine the effects on fertilization of 1) increasing sperm concentrations (1x 10(6), 5 x 10(6) and 1 x 10(7)sperm cells/ml), 2) IVM medium supplementation with EMS or ECS and 3) partial cumulus mass removal before insemination. Forty-nine percent of the collected oocytes (335 683 ) showed a compact cumulus and homogeneous ooplasm and thus were selected for culture. In Experiment 1, high nuclear maturation rates were observed in both EMS (82%,32 39 ) and ECS (87.5%,56 64 ) groups, with no statistically significant difference. In Experiment 2, the percentage of normal fertilization (2 polar bodies, 2 pronuclei and sperm tail) was similar for all 3 tested sperm concentrations (12.9%,4 31 ; 15.2%,9 59 and 15.5%,9 58 ). No advantage in using the homologous serum in IVM medium was noted in terms of fertilization (12.2%, 5 41 with EMS vs 12.9%, 4 31 for ECS). However, significantly higher fertilization rates were obtained after partial cumulus removal compared with that of oocytes fertilized with a whole cumulus (32.6%, 14 43 vs 12.2%, 5 41 ; P < 0.05). The incidence of polyspermic fertilization was low under all culture conditions (0 to 2.4%). In a replicate in which the oocytes fertilized after the cumulus removal were further cultured for 72 h two embryos, one at the 2-cell stage and the other at the 4-cell stage, could be obtained. These results indicate that, in the horse, the cumulus can be partially removed to increase the fertilization of compact-cumulus oocytes recovered during the breeding season using frozen-thawed, heparin-treated semen.     

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

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

Embryo development,oocyte morphology,and kinetics of meiotic maturation in bovine oocytes exposed to 6-dimethylaminopurine prior to in vitro maturation

The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 ± 12% of the oocytes were at the germinal vesicle stage compared to 97 ± 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 ± 18% were at MII stage, compared with 93 ± 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 ± 8% vs 89 ± 7%; P < 0.01), oocyte activation higher (11 ± 5% vs 2 ± 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 ± 7% vs 4 ± 4%; P > 0.05), compared with the control group. Cleavage was lower (61 ± 13% vs 81 ± 6%; P < 0.001), as well as day 8 blastocyst formation (17 ± 7% vs 36 ± 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development. Mol. Reprod. Dev. 50:334–344, 1998. © 1998 Wiley-Liss, Inc.     

In vitro maturation of domestic dog oocytes cultured in advanced preantral and early antral follicles

Initial studies in our laboratory demonstrated that a large proportion of domestic dog advanced preantral (APAN) and early antral (EAN) follicles contained grown oocytes that had acquired the dense cytoplasmic lipid characteristic of preovulatory oocytes. The objective of this study was to assess nuclear maturation of those oocytes after in vitro culture. Both APAN and EAN follicles (152 to 886 microns in diameter) were isolated from ovaries by treatment with collagenase and DNase. The follicles were cultured in Dulbecco's Modified Eagle's medium/nutrient mixture F-12 Ham culture medium supplemented with 20% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 1% (v/v) antibiotic-antimycotic, 1 microgram FSH/ml, 10 IU hCG/ml and 1 microgram estradiol/ml. Within each group (APAN or EAN), control follicles were not cultured (0 h), and 2 to 12 follicles per well were incubated under a humidified atmosphere of 5% CO2 in air at 37 degrees C for 24, 48 or 72 h. After 24 h of culture, significantly more (5.3%, 20/374; P < 0.05) oocytes from APAN follicles reached the metaphase I to metaphase II stages (MI to MII) than the percentage of control follicles observed at 0 h (0.9%, 3/318). Continued culture resulted in a further increase (P < 0.05) in the percentage of oocytes reaching MI to MII by 48 h (11.5%, 47/407), which remained unchanged at 72 h (9.9%, 40/404). The percentage of oocytes from EAN follicles reaching MI to MII did not significantly increase after 24 h of culture. However, there was an increase (P < 0.05) by 48 h of culture (8.7%, 11/126), which remained unchanged at 72 h (7.5%, 8/106). These results show that dog oocytes cultured within advanced preantral and early antral follicles in vitro are competent to resume meiosis to the metaphase stage.     

Nuclear maturation of ovine oocytes in cultured preantral follicles

Small (150–250 μm in diameter) and large (251–400 μm in diameter) preantral follicles (PFs) in sheep were cultured for 6 days in four different concentrations of transforming growth factor-alpha (TGF-), epidermal growth factor (EGF), FSH and LH. Proportions of follicles exhibiting growth, antrum formation and increase in follicular and oocyte diameter were the initial indicators of development. The ability of the oocytes isolated from these cultured follicles to mature to metaphase II (MII), after 24 h culture in a known in vitro maturation medium was the final criterion of success. TGF- 2.5 ng ml⁻¹, EGF 50 ng ml⁻¹ and FSH 1 and 2 μg ml⁻¹ supported good initial growth of the PFs. Thirty and seventeen percent of the oocytes from the large PFs cultured in TGF- 2.5 ng ml⁻¹ and FSH 2 μg ml⁻¹ respectively, matured to the MII stage. These proportions for oocytes from small PFs were 11 and 6%, respectively. Oocytes from follicles cultured in EGF did not mature to the MII stage. LH at all concentrations tested and TGF-, EGF and FSH above 5, 50 ng ml⁻¹ and 2 μg ml⁻¹, respectively, induced degeneration of the PFs. It was concluded that (i) TGF- 2.5 ng ml⁻¹ supports development of large PFs in sheep to obtain meiotically competent oocytes, (ii) PFs > 250 μm in initial diameter develop better in vitro, and (iii) in vitro development of sheep PFs could be obtained independent of gonadotropin stimulation.     

Induction and inhibition of in vitro oocyte maturation and production of steroids in fish follicles by forskolin

The effects of forskolin (FK) on in vitro oocyte maturation and production of steroids were examined in Oryzias latipes. When oocytes within preovulatory follicles were preincubated in the presence of FK for 2-10 hr, they matured normally after additional incubation for 10-20 hr in plain culture medium. Naked (follicle cell-free) oocytes did not mature under these conditions. FK stimulated dose-dependent production of steroids (estradiol-17 beta, E2, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta-diOHprog) and cAMP in follicle (granulosa) cells. On the other hand, exposure to FK within 2 hr after 17 alpha,20 beta-diOH prog stimulation caused reversible inhibition of gonadotropin (PMS)- or 17 alpha,20 beta-diOH prog-induced maturation of the intrafollicular oocytes in vitro. FK also significantly inhibited the 17 alpha,20 beta-diOHprog-induced maturation of naked oocytes, suggesting the existence of adenylate cyclase in fish oocytes. These data indicate that in Oryzias latipes, FK induces oocyte maturation by stimulating follicular production of maturation-inducing steroid (MIS), probably 17 alpha,20 beta-diOH prog, via an increase in cAMP, and that it may inhibit oocyte maturation by increasing ooplasmic cAMP and some inhibitory interaction between the granulosa cells and the oocyte through intercellular communication.     

Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro

Effects of gonadotropins on the maturation of isolated oocytes and production of progesterone by porcine ovarian follicles from gonadotropin treated gilts have been studied in vitro. The addition of gonadotropins (2 I. U./ml, PMSG, HGC or 2 mg/ml FSH) to the culture medium resulted in increasing the number (84 - 90 %) of isolated oocytes which reached metaphase II. Expansion of the whole cumulus mass was observed only in media containing PMSG, whereas FSH or HCG alone did not cause these marked changes in the cumulus cells. Denudation of the eggs prior to culture gave no significant differences in the maturation rates between oocytes cultured in media with or without gonadotropins. In vitro maturation of follicle-enclosed oocytes took place only in HCG treated animals. Removing the ovary at 15 or 60 minutes after intravenous HCG administration induced oocyte maturation only in 22% and 17% respectively. A sharp increase in the number of oocytes which resume meiosis during follicle culture was observed 4 hours after HCG injection (84 %) and all of the oocytes of the gilts ovariectomized at 8 hours after HCG injection matured during the culture period. The progesterone production of isolated follicles from control gilts (only PMSG injected) increased slowly during a 96-hour culture period (from 48 to 240 ng progesterone/follicle), whereas the secretion of progesterone was drastically increased after a 15 minute interval between HCG injection and ovariectomy (from 42 to 950 ng progesterone/follicle). Follicles removed 24 hours after HCG injection showed a further increase in steroid production (2000 ng progesterone/follicle) and consistently secreted large amounts of progesterone during the culture period.     

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.