Oocyte maturation in humans: the role of gonadotropins and growth factors*

Transcription

1 FERTILITY AND STERILITY Vol. 6, No.1, July 1993 Copyright e 1993 The American Fertility Society Printed on acid-free paper in V. S. A. Oocyte maturation in humans: the role of gonadotropins and growth factors* Emilio Gomez, Ph.D. Juan J. Tarin, Ph.D. Antonio Pellicer, M.D.t Instituto Valenciano de lnfertilidad, and Department of Pediatrics, Obstetrics and Gynecology, Valencia University School of Medicine, Valencia, Spain Objective: To determine the effect of FSH/LH in vivo and epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in vitro on human oocyte maturation. Design: Oocyte-cumulus complexes were harvested from three different groups of patients: [1] unstimulated ovaries from women undergoing surgery; [2] multifollicular development achieved with a combination of FSH and LH in the absence of an ovulatory dose of hcg; and [3] oocyte-cumulus complexes retrieved after appropriate ovarian stimulation with FSH/LH and hcg for IVF purposes. Setting, Patients: In vitro fertilization program and patients undergoing surgery for benign disorders at the Instituto Valenciano de Infertilidad, Valencia, Spain. Interventions: Oocyte-cumulus complexes from unstimulated ovaries collected at surgery by follicular puncture and washing. Oocyte-cumulus complexes from stimulated cycles obtained by ultrasound-guided transvaginal aspiration. Oocyte-cumulus complexes cultured in vitro in the absence or presence of different concentrations of EGF and IGF-1. Main Outcome Measure: Germinal vesicle breakdown and metaphase-ii stage after 24 and 48 hours. Results: Comparison of the spontaneous resumption of meiosis and metaphase II oocytes among groups showed significant differences between unstimulated and stimulated ovaries after 24 and 48 hours in culture. Administration of hcg accelerated the percentage of maturation by 24 hours. Further incubation of unstimulated oocyte-cumulus complexes with EGF and IGF-I significantly increased the percentage of metaphase-ii oocytes after 24 and 48 hours in culture. Conclusions: Epidermal growth factor and IGF-I are able to augment spontaneous maturation in immature human oocytes. Because spontaneous maturation is mainly observed when follicles have been exposed to pharmacological doses of hmg, it is suggested that increasing FSH levels within the follicle is coincident with the generation of a positive signal necessary to complete oocyte maturation in humans. This signal may be linked to the dynamics of growth factors within the follicle itself. Fertil Steril 1993;6:4-6 Key Words: Human oocytes, oocyte maturation, epidermal growth factor, insulin-like growth factor I In humans, as well as in many other mammalian species, the first meiosis of the oocyte is initiated during fetal life and is arrested at the diplotene Received December 29, 1992: revised and accepted March 29, * Supported by Direcci6n General de Investigaci6n Cientifica y Tecnol6gica PM9-68 and Fondo de Investigaciones Sanitarias de Ia Seguridad Social , Madrid, Spain. t Reprint requests: Antonio Pellicer, M.D., Instituto Valenciano de Infertilidad, Guardia Civil, 23, Valencia-462, Spain. stage of the prophase before birth. In 1935, Pincus and Enzman (1) observed that mammalian oocytes spontaneously undergo resumption of meiosis when removed from the follicular environment. This process is commonly known as oocyte maturation. Since then, many investigators have searched for putative intraovarian regulators of this process. Specific molecules that have been implicated in this meiosis-arresting action include cyclic adenosine monophosphate (2) and purines (3, 4). In addition, evidence accumulated in mammals 4 Gomez et al. Oocyte maturation in humans

2 suggests the existence of a positive signal that stimulates oocyte maturation; Downs et al. ( 5) found that induction of maturation in cumulus-enclosed mouse oocytes by hormones was greater than the spontaneous resumption of meiosis observed in denuded oocytes. In rodents, spontaneous oocyte maturation is achieved in >95% of oocytes removed from their follicular environment in gonadotropin-primed as well as unprimed animals (6). In humans, however, spontaneous maturation in vitro is achieved only in 3% to 5% of oocytes (7-9) from non-stimulated ovaries, whereas almost 9% of the oocytes from hmg/hcg-treated women are metaphase-ii oocytes in IVF programs. These data suggest that, at least in humans, a positive signal may be generated by the gonadotropic stimulus within the follicle that induces the resumption of meiosis. Information as to whether hmg alone is sufficient to provide this signal or if the concurrence ofhcg is absolutely necessary is lacking. Several growth factors (GFs) have been related to the process of oocyte maturation in mammals. Dekel and Sherizly (1) showed that epidermal growth factor (EGF) induces maturation of follicle-enclosed rat oocytes. Pellicer et al. (11) demonstrated that EG F induces germinal vesicle breakdown (GVBD) faster than FSH and hcg in the same rat model. Downs (12) has provided evidence that EGF induces GVBD and cumulus cell expansion in oocyte-cumulus complexes maintained at arrested meiosis by hypoxanthine. The effect of EGF is more evident than for other GFs and hormones tested (12). The implications ofegf on human oocyte maturation have not yet been established, although immunodetectable EG F levels are found in follicular fluid (FF) (13), and maturation from the immature stage has been observed in a small population of human oocytes retrieved for IVF purposes (14). Insulin-like growth factor I (IGF-I) stimulates oocyte maturation in xenopus oocytes ( 15). A possible relationship between IGF-I and human oocyte maturation remains to be determined because high levels of IGF-I have been found in the cumulus oophorus in the rat (16), and immunoreactive IGF-I has been localized in several studies in the thecainterstitial compartment as well as in the cumulus cells surrounding the oocyte (17). Based on the above information, the purpose of this study has been to analyze possible factors associated with human oocyte maturation in vitro. Our studies confirm previous observations in which it was shown that spontaneous maturation in vitro occurs in humans to a lesser extent than in other mammals. Treatment of patients with gonadotropins or addition of GFs in vitro override this problem, suggesting that a positive signal is necessary in the process of oocyte maturation in humans. MATERIALS AND METHODS Oocytes from Spontaneous Cycles Oocytes were obtained from 22 women undergoing gynecological surgery for a number of benign disorders; mean age was 3.7 ± 2.7 years, and informed consent was obtained from all of them after appropriate information was provided. All patients had spontaneous and regular menstruations, and none was under any medication that could interfere with the ovarian cycle. Women were scheduled for surgery on days 7 to 1 of the menstrual cycle, and all visible follicles ranging from 5 to 1 mm in diameter were punctured for isolation of the oocyte-cumulus complexes. Two different methods were employed to obtain oocytes. First, women in whom an ovarian wedge resection and/ or ovariectomy was indicated. The surgical pieces were transferred to the laboratory in sterile conditions and the follicles punctured with an 18-gauge needle adapted to a syringe containing phosphate-buffered solution (PBS) (GIBCO Co., Paisley, Scotland). The follicles were washed several times with PBS and the contents observed under the dissecting microscope. When the oocyte-cumulus complexes was not evident, the follicle was dissected with the aid of fine forceps and scissors, the follicle wall scraped with 3-gauge needles, and the entire inside of the follicle washed several times until the oocyte-cumulus complexes appeared. Second, women in whom partial or total ovarian mass resection was not indicated. In these patients, all visible follicles were gently aspirated and washed a couple of times, employing 18-gauge needles with PBS. Only oocytecumulus complexes composed of an oocyte surrounded by several layers of cumulus cells were considered for the study. Those denuded in the process of oocyte retrieval or with signs of degeneration were discarded. All oocyte-cumulus complexes obtained from a given patient were randomly transferred to the media tested in each experiment. Oocyte-cumulus complexes were cultured in 5 #LL B2 medium (BIO Merieux, Marcy-L'Etoile, France) that contains albumin as the only source of serum. In a series of experiments, EGF (Sigma Chemical Co., St. Louis, MO) was diluted in B2 medium and added at final concentrations of 2 and 1 ng/ml (.33 and 1.65 nmoljl). In another series of experiments, the effect of IGF-I on oocyte maturation in Vol. 6, No.1, July 1993 Gomez et al. Oocyte maturation in humans 41

3 vitro was tested. Insulin-like growth factor I (Bachem Co., Torrance, CA) was diluted in B2 medium and added to a final concentration of 1 ng/ml (13.1 nmoljl). A concentration of 2 ng/ml EGF was chosen based on the physiological levels found in FF (13). Supraphysiological concentrations were also selected for the experiments with EGF; IGF-I was only tested at supraphysiological concentrations because these experiments were performed after EGF analysis. Twenty-four hours later, the oocytes were denuded by repeated shearing using hand-drawn pipettes. Then GVBD as well as the extrusion of the first polar body was recorded. Those oocytes that did not reach the metaphase-ii stage after 24 hours were cultured for an additional 24 hours under the same conditions as when the cultures were started. Oocytes From Stimulated Cycles Eighteen women were included in our IVF program because of tubal infertility; all were spontaneously ovulatory, as determined by endometrial biopsies and serum P levels in the luteal phase. Pituitary desensitization was achieved by administration of 1 mg/d SC leuprolide acetate (LA, Procrin; Abbott S.A., Madrid, Spain) starting in the luteal phase of the previous menstrual cycle. Serum E 2 levels< 6 pg/ml (.22 nmoljl) and negative vaginal ultrasonographic scans were used to define ovarian quiescence. Days 1 and 2 of ovarian stimulation, two ampules per day hmg (Pergonal; Serono Laboratories, Madrid, Spain) were administered together with two ampules pure FSH (Fertinorm; Serono Laboratories). Days 3, 4, and 5 of ovarian stimulation, three ampules per day hmg were administered to each patient. Beginning on day 6, hmg was administered on an individual basis according to the serum E 2 and transvaginal ovarian ultrasound scans. The criteria for hcg administration (1, IU, Profasi; Serono Laboratories) were the presence of two or more follicles ~ 1.9 em in greatest diameter and serum E 2 levels > pg/ml (2.94 nmoljl). Those patients (n = 4) at high risk of developing a hyperstimulation syndrome based on the presence of multiple small ovarian follicles and very high E 2 levels were counseled to cancel the cycle. Two different sources of oocytes were obtained for this study. First, in women who underwent a regular IVF cycle, LA and hmg injection were discontinued the day of hcg administration. Oocyte retrieval was scheduled 36 to 38 hours after hcg administration. The follicular aspirates were decanted in plastic Petri dishes, the oocyte-cumulus complexes identified, and the nuclear status ascertained by the cumulus spreading technique described by Veeck (18). All the oocytes were inseminated 4 to 6 hours after retrieval regardless of the maturational status. Seventeen to 2 hours later, the extrusion of the first polar body was evaluated after appropriate removal of the cumulus cells. Second, women who were canceled and therefore stopped all medication were asked to enter the study. Only four volunteers had transvaginal asp iration of all visible follicles. Oocytes in these circumstances (after hmg stimulation in the absence of the final dose of hcg) are very difficult to obtain because it has been demonstrated that oocyte recovery rates improve with longer hcg-to-recovery intervals and that the chance of obtaining oocytes in the absence of hcg administration is very low (19). The oocytes obtained from these follicles are at the germinal vesicle stage (2) and were processed as the oocyte-cumulus complexes retrieved from unstimulated ovaries. The study was approved by the Ethical Committee of the Instituto Valenciano de Infertilidad. All patients entering the study gave their consent to have their oocytes studied. The oocytes from patients being canceled because of the risk of hyperstimulation were not inseminated; the oocytes from IVF patients did not suffer additional manipulation because they were just observed for nuclear maturation and subsequently inseminated. No hormone was added to these oocytes. Statistical Analysis For statistical comparison among groups, x 2 test was applied. A P value <.5 was considered as statistically significant. RESULTS Figure 1 shows percentage of GVBD and metaphase II after 24 and 48 hours in culture of three different groups of oocytes. First, those (n = 24) obtained from nonstimulated ovaries. A total of 33.3% of the oocytes resumed meiosis during the first 24 hours; the percentage of GVBD was 75% after 48 hours in culture. Metaphase II was observed in % and 16.7%, after 24 and48 hours incubation, respectively. Second, the small population of oocytes (n = 8) retrieved from canceled patients because of the risk of ovarian hyperstimulation showed a significant (P <.5) increase in the percentage of GVBD (87.5%) as compared with controls after 24 hours' incubation. No significant differences were observed in the percentage of G VBD 42 Gomez et al. Oocyte maturation in humans

4 1 =! 6.c a. 4 i il Control Hyperstlmulatlon IVF n:24 n=b n=93 Figure 1 Spontaneous rates of GVBD and metaphase II observed in oocytes obtained in different clinical situations: oocytes matured in vitro from unstimulated cycles (n = 24); oocytes retrieved after stimulation with hmg in the absence of the ovulatory dose of hcg (n = 8); oocytes harvested for IVF purposes (n = 93). *P <.5; **P <.1; ***P <.1. between EGF-treated and control oocytes (1% versus 77.3%) were not significant. Similarly, pharmacological concentrations of EGF (1 ng/ml) also significantly (P <.1) induced GVBD after 24 hours' incubation (81.9%, n = 33). However, after 48 hours' incubation the percentage of GVBD (96.8%) was not significantly different from the control group. The percentage of metaphase II oocytes after incubation with EG F was also evaluated. Doses of2 (12.5%) and 1 (12.9%) ng/ml EGF induced a borderline significant (P =.7) increase in the percentage of metaphase II oocytes as compared with zero found in the controls after 24 hours in culture. Further incubation to a total period of 48 hours displayed a significant (P <.1) increase in the percentage of metaphase II oocytes using 1 ng/ml EGF (54.8%) as compared with the controls (19.2%). No significant differences with the control group were observed when oocyte-cumulus complexes were incubated with 2 ng/ml EGF (5.%). Figure 3 shows the results of adding 1 ng/ml IGF-1 to the culture medium of oocyte-cumulus complexes obtained from nonstimulated ovaries. Insulin -like growth factor I induced a significant (P <.1) increase in GVBD after 24 hours (87.5%, n = 16) as compared with the controls (31.8%, n = 22). ThepercentageofGVBD (1.%) after48 1 (1%) after 48 hours' incubation. The percentage of metaphase II in those oocytes was significantly (P <.1) higher than in the controls 24 (5%) and 48 hours (87.5%) after retrieval. Third, oocytes (n = 93) obtained after hmg-hcg stimulation for IVF were observed only for 24 hours because they were obtained 36 hours after hcg administration and subsequently inseminated. In these conditions, oocytes showed a significantly higher percentage of GVBD than did the controls (P <.1). No significant differences were found in the percentage of GVBD between patients who received or not hcg. Similarly, the percentage of metaphase II oocytes in the group of IVF oocytes after 24 hours was significantly (P <.1) higher than in the remaining two groups of oocytes. Figure 2 shows the effects of culturing the oocyte-cumulus complexes obtained from nonstimulated ovaries with EGF. Epidermal growth factor (2 ng/ml) significantly induced GVBD at a higher percentage than the controls after 24 hours' incubation (87.5%, n = 16, versus 3.8%, n = 26, P <.1). After 48 hours' incubation, differences c ID > (:J il- =!.c a. I il- 2 [EGF] ngtml Figure 2 Rates of GVBD and metaphase II of human oocytes cultured in the presence of different concentrations of mouse EGF. *P <.1; **P <.1. Vol. 6, No. 1, July 1993 Gomez et al. Oocyte maturation in humans 43

5 c Ill 1 6 > "'# = I.c 6 a. I 4 '#. 2 1 [IGF-1] ng/ml n 22 no:16 Figure 3 Rates of G VBD and metaphase II of human oocytes cultured in the presence and absence of human IGF-1. *P <.1. hours' incubation was not significantly higher than the value obtained in the control group (77.4%). Metaphase II oocytes were increased after 24 (12.5%) and48 (37.5%) hours of incubation as compared with the controls (% and 18.2%, respectively). However, none of these percentages reached statistical significance because of the low number of oocytes analyzed. DISCUSSION The results of the present study confirm previous observations that showed that spontaneous oocyte maturation occurs at a rate ranging from 3% to 5% (7-9), which is far from what has been described in primed and unprimed rats (6) and other mammalian species. We found a rate of GVBD in unstimulated oocytes of 7% to % after 48 hours in culture. However, <2% reached metaphase II. When women were treated with hmg (a combination of FSH and LH) alone, or hmg plus hcg to induce ovulation, oocyte maturation occurred to an extent comparable with animal observations. These experiments show that middle-size follicles in unstimulated cycles are unable to complete nuclear maturation. However, addition of GFs in vitro or short treatment ( <8 days) with hmg in vivo are sufficient to allow maturation to proceed. When analyzing experiments involving human oocytes, one has to appreciate that, in contrast to animal studies, the availability of human oocytes is never ideal because of technical and ethical constraints. This difficulty may always limit interpretation of the results, and any conclusion drawn must be taken with caution. Several problems may be considered in the present study. First, the source of unstimulated human oocytes was follicles 5 to 1 mm in diameter on days 7 to 1 of the menstrual cycle. There is no doubt that at this time of the cycle there is only one healthy follicle. Thus, it seems that three fourths of the oocytes collected from unstimulated ovaries had some minor degree of atresia. The question raised here is how this atresia relates to the process of GVBD. Gougeon and Testart (21) have shown that oocytes from follicles in early atresia have the same dynamics of GVBD as healthy oocytes. Therefore, we believe that this methodological constraint did not affect our observations. The second problem in explaining our results is the fact that oocytes derived from follicles of different sizes. In rodents, follicle and oocyte sizes have been related to the meiotic competence of the oocyte (22), and therefore a higher rate of GVBD in stimulated follicles may be simply explained by a greater size of these follicles as compared with unstimulated ovaries. However, Tsuji et al. (8) showed that when comparing GVBD dynamics in the follicular phase of medium- and large-size human follicles, the rate of G VBD was similar in both groups. Thus, we do not believe that the differences found in the rate of GVBD between control and hmgand/or hcg-stimulated cycles are the consequence of different follicular sizes but rather a direct effect of the exogenous gonadotropins. The third concern that deserves consideration is the fact that oocytes were denuded after 24 hours in culture. Even assuming that the effects described after 48 hours in culture are the consequence of mechanical denudation and not of hormonal action, there were significant effects after 24 hours in culture that can be only explained by the effect of hormones. Hence, assuming that there may be some methodological problems with the use of human tissue that in our opinion did not seriously affect our observations, we propose that in humans, both a positive and a negative signal controls oocyte maturation. The positive signal is predominant over the negative and is probably triggered when a certain FSH threshold level is reached within the follicle. This signal may be coincident with the appearance of LH receptors in the granulosa cells because the block in metaphase I or anaphase I has been related 44 Gomez et al. Oocyte maturation in humans

6 to this phenomenon (23). Therefore, unstimulated small- or medium-size follicles do not generate this signal, and the oocytes do not mature in vitro when removed from their environment. Once the positive signal is present in the follicle, maturation does not proceed because of the coexistence of a negative signal. In fact, when follicles and oocytes from stimulated ovaries have been fixed and analyzed before the LH surge, most of the oocytes were found to be at the germinal vesicle stage (2). The LH peak at midcycle will eliminate this negative stimulus, allowing the positive signal to act. If cumulus-enclosed oocytes are removed from the follicle once the positive signal has already been generated in the follicle, oocyte maturation will take place regardless of the action of LH/hCG because the negative stimulus may disappear when the oocyte-cumulus complexes are removed from the follicular milieu. This hypothesis needs further confirmation but agrees with the observations of the present study and previous suggestions of a meiosis-inducing substance in humans (24). Resumption of meiosis is observed in mouse cumulus-enclosed oocytes treated with FSH and EGF to a higher extent than in denuded oocytes (5), suggesting the existence of a positive signal also in rodents. Similarly, treatment of cumulus-enclosed human oocytes with EGF and IGF-I in vitro increased the rate of G VBD and metaphase II oocytes in our study. Thus, in humans as in mice, the intrafollicular environment induced by FSH may be able to generate the protein synthesis involved in oocyte maturation, and GF peptides seem to be very closely related to this process. Alternatively, however, our observations do not exclude the possibility that both GFs induce metabolic uncoupling between the cumulus and the oocyte. This possibility needs to be further explored because it is difficult to understand how two GFs that function via different receptor systems have the same biological effect. The results of the present study also show that metaphase II can be reasonably reached in human oocytes from unstimulated ovaries. This observation might have potential clinical applications. First, oocyte donation programs are widely established, the lack of donated oocytes being the main problem for reproductive endocrinologists treating these patients. Our observations and previous observations (9, 14) provide evidence that oocytes from women undergoing surgery may be a potential source of donated oocytes. In fact, Cha et al. (9) demonstrated that these oocytes are able to fertilize and cleave normally in vitro. They have described the first term pregnancy after implantation of embryos derived from immature oocytes (9). Second, in young women subjected to treatment with cytotoxic agents, the possibility exists of freezing the ovaries rather than isolated oocytes. In fact, cryopreservation of the entire tissue is more successful than freezing of germ cells. In these exceptional cases, the ovaries can be removed, frozen, and subsequently thawed in small wedge sections. The oocytes from each section may be matured in vitro and fertilized. Experiments on similar procedures in animals have been successful to date (25). REFERENCES 1. Pincus G, Enzman EV. The comparative behaviour of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. J Exp Med 1935;62: Eppig JJ, Downs SM. Chemical signals that regulate mammalian oocyte maturation. Biol Reprod 1984;3: Downs SM, Coleman DL, Ward-Bailey PF, Eppig JJ. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid. Proc Natl Acad Sci USA 1985;82: Miller JGO, Behrman HR. Oocyte maturation is inhibited by adenosine in the presence of follicle-stimulating hormone. Biol Reprod 1986;35: Downs SM, Daniel SAJ, Eppig JJ. Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic origin. J Exp Zool1988;245: Vander hyden BC, Armstrong DT. Effects of gonadotropins and granulosa cell secretions on the maturation and fertilization of rat oocytes in vitro. Mol Rep rod Dev 199;26: Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 1965;28: Tsuji K, Sowa M, Nakano R. Relationship between human oocyte maturation and different follicular sizes. Biol Reprod 1985;32: ChaKY, KooJJ, KoJJ,ChoiDH, HanSY, Yoon TK. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991;55: Dekel N, Sherizly I. Epidermal growth factor induces maturation of rat follicle-enclosed oocytes. Endocrinology 1985;116: Pellicer A, Gestone-Parmer T, Stoane JM, Behrman HR. Desensitization to follicle-stimulating hormone in cumulus cells is coincident with hormone induction of oocyte maturation in the rat follicle. Mol Cell Endocrinol1989;64: Downs SM. Specificity of epidermal growth factor action on maturation of the murine oocyte and cumulus oophorus in vitro. Biol Reprod 1989;41: Westergaard LG, Andersen CY. Epidermal growth factor (EGF) in human preovulatory follicles. Hum Reprod 1989;4: Das K, Stout LE, Hensleigh HC, Tagatz GE, Phipps WR, Leung BS. Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes. Fertil Steril 1991;55: Janicot M, Lane D. Activation of glucose uptake by insulin and insulin-like growth factor I in xenopus oocytes. Proc Natl Acad Sci USA 1989;86: Vol. 6, No.1, July 1993 Gomez et al. 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7 16. Oliver JE, Aitman TJ, Powell JF, Wilson CA, Clayton RN. Insulin-like growth factor-i gene expression in the rat ovary is confined to the granulosa cells of developing follicles. Endocrinology 1989;124: Balboni GC, Vannelli GB, Barni T, Orlando C, Serio M. Transferrin and somatomedin C receptors in the human ovary follicles. Fertil Steril 1987;48: Veeck LL. Oocyte assessment and biological performance. Ann NY Acad Sci 1988;541: Templeton AA, Van Look P, Angell RE, Aitken RJ, Lumsden MA, Baird DT. Oocyte recovery and fertilization rates in women at various times after the administration of hcg. J Reprod Fertil1986;76: Bomsel-Helmreich, Huyen LVN, Durand-Gasselin I, Salat-Baroux J, Antoine J-M. Mature and immature oocytes in large and medium follicles after clomiphene citrate and human menopausal gonadotropin stimulation without human chorionic gonadotropin. Fertil Steril 1987;48: Gougeon A, Testart J. Germinal vesicle breakdown in oocytes of human atretic follicles during the menstrual cycle. J Reprod Fertil1986;78: Eppig JJ, Schroeder AC, O'Brien MJ. Developmental capacity of mouse oocytes matured in vitro: effects of gonadotrophic stimulation, follicular origin and oocyte size. J Reprod Fertil 1992;95: Erickson GF, Ryan KJ. Spontaneous maturation of oocytes isolated from ovaries of immature hypophysectomized mice. J Exp Zoo! 1976;195: Westergaard L, Byskov AG, Andersen CY, Grinsted J, MeN a tty KP. Is resumption of meiosis in the human preovulatory oocyte triggered by a meiosis-inducing substance (MIS) in the follicular fluid? Fertil Steri11984;41: Carroll J, Whittingham DG, Wood MW, Telfer E, Gosden RG. Extraovarian production of mature viable mouse oocytes from frozen primary follicles. J Reprod Fertil 199;9: Gomez et al. Oocyte maturation in humans