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MAMMALIAN OOCYTE MATURATION: MECHANISMS FOR REGULATION AND PROSPECTS FOR PRACTICAL APPLICATION OF IN VITRO TECHNOLOGY John J. Eppig In 193S, Pincus and Enzmann observed that germinal vesicle (GV) stage oocytes isolated from rabbit follicles underwent spontaneous germinal vesicle breakdown (GVB) and emitted a polar body when cultured in medium without the gonadotropin hormones that promote follicular maturation and ovulation. They suggested that the ovarian follicle exerts a meiosis-arresting influence on the oocyte until the preowlatory surge of gonadotropins. This observation and hypothesis began an era that has used culture systems for mammalian oocytes to resolve the mechanisms that govern oocyte maturation. In addition to providing clues to the molecules and pathways that regulate oocyte maturation, the culture systems may provide the technology for resolving problems of human infertility and for expanding populations of rare and endangered species. This paper will review some current concepts of the regulatory systems that govern mammalian oocyte development, evaluate the prospects for the practical utilization of this information, and suggest some of the future directions for research. The Ovarian Follicle as the Support System for Oocyte Development In Viva After a period of mitotic proliferation in the fetus, oogonia enter meiosis, but the germ cells now called oocytes progress only as far as the diplotene stage where they are arrested until shortly before ovulation. Diplotene oocytes develop a close association with somatic cells that are probably progenitors of granulosa cells and form a primordial follicle. It is likely that oocytes that do not become associated with these pregranulosa cells degenerate. Until an oocyte is recruited into the pool of growing oocytes, the pregranulosa cells remain in a single, often flattened, layer around the oocyte. But when the oocyte is recruited, by unknown mechanisms, into the pool of growing oocytes, granulosa cell proliferation occurs concurrently with the increase in the size of the oocyte. Both growing and nongrowing oocytes appear to be coupled to their companion somatic cells by membrane specializations called gap junctions, which allow the passage of low molecular weight molecules to pass from one cell to another (Anderson and Albertini, 1976~. Gap junctional communication between granulosa cells and the oocyte is essential for oocyte growth, since they are a conduit for nutritional and regulatory substances into the oocyte (Eppig, 1977, 1979; Brower and Schultz, 1982; Herlands and Schultz, 1984; Buccione, et al,, 1987~. Although gap junctional communication can be 176
established between oocytes and other somatic cell types in vitro, oocyte growth occurs only when oocytes communicate with granulosa calls (Buccione et al., 1987~. Some aspects of oocyte development, nevertheless, are independent of oocyte growth or association with granulosa cells. Normally, as oocytes approach completion of their growth phase, they become competent of undergoing spontaneous maturation when liberated from their follicles and cultured (Szybec, 1972; Sorensen and Wasserman, 19761. The developmental program governing the acquisition of meiotic competence, however, is triggered at the onset of oocyte growth, but proceeds in the absence of continued oocyte growth or granulosa cells (Canipari et al., 19847. Once meiotic competence is achieved , granulosa cel is , via gap junctional communication, help to maintain the oocyte in meiotic arrest (Eppig and Downs, 1987~. Because cumulus granulosa cells are coupled by gap junctions to the oocyte and to the mural granulosa cells which make up the follicle wall, the follicle may be a functional syncytium allowing the movement of low molecular weight molecules throughout the follicle by diffusion. In theory, therefore, production or mobilization of low molecular weight regulatory substances in mural granulosa cells in response to an agonist could result in the diffusion of these substances to the oacyte, which may itself be unable to produce or mobilize them. These substances could participate in the maintenance of meiotic arrest, the induction of maturation, or both . Identification of Molecules and Pathways that Participate in the Maintenance of Meiotic Arrest Cyclic Adenosine Monophosphate (cAMP). In the ovarian follicles of most studied mammal fan species, cAMP probably participates in the maintenance of oocyte meiotic arrest. Support for this conclusion is based on the following observations. Membrane permeable analogs of cANP, such as dibutyryl cAMP (dbcAMP), and cAMP phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), maintain meiotic arrest in vitro (Cho et al., 1974; Wassaman et al., 1976; Magnusson and Hillensjo, 1977~. Microinjection of an inhibitor of cAMP-dependent protein kinase (PRI) induces GVB in oocytes cultured in medium containing dLcAMP or IBMX (Bornslaeger et al., 1986~. Conversely , microinj ection of the active catalytic subunit of cAMP-dependent protein kinase maintains meiotic arrest (Bornslaeger et al., 1986~. Finally, a decrease in the cAMP content of mouse (Schultz et al ., 1983 ); and rat (Racowsky, 1984 ~ oocytes appears to precede GVB, but such a decrease is not observed in the oocytes of hamsters (Racowsky, 1985a; Hubbard, 1986), sheep (Moor and Heslop, 1981), or pigs (Racowsky, 1985b). Oocytes of several mammalian species appear able to produce their own cAMP because they possess an adenylate cyclase system (Urner et al., 1983; Ekholm et al., 1984; Bornslaeger and Schultz, 1985a; Racowsky, 1985ab; Ruyt et al., 1988~. It is not clear, however, that the oocytes of all of these species can produce levels of cAMP adequate for the maintenance of Idiotic arrest. If not, the cAMP molecule is small enough to 177 -
traverse gap junctions and, theoretically therefore, should be able to enter the oocyte from the cumulus/mural granulosa cell syncytium. Thus far, however' attempts to demonstrate such movement (Racowsky, 1984, 1985b; Bornsleager and Schultz, 1985b) have resulted in ambiguous conclusions because it is not known whether increases in the concentrations of cAMP in oocytes after elevating levels of cAMP in cumulus cells are the result of the transfer of cAMP from the cumulus cells, or the generation of substances in the cumulus cells that increase production of cAMP, or decrease loss of cAMP, in the oocyte. Oocyte Maturation Inhibitor (OMI) Preparations of follicular fluid from several species prolong the maintenance of the GV stage in cultured oocytes (Tsafriri et a1., 1982~. Evidence has been presented that the active meiosis-arresting fraction of porcine follicular fluid is a low molecular weight peptide and has been referred to as OMI (Tsafriri et al., 19827. Confirmation of the existence of OMI awaits purification to homogeneity. Crude preparations of OMI, however, apparently act via cumulus cells, and the meiosis-arresting action is reversed by treatment with luteinizing hormone (LH) (Tsafriri et al., 1982~. Mullerian Inhibiting Substance (MIS) MIS, also known as anti-Mullerian hormone (AMH), has been identified in bovine follicular fluid and evidence from one group suggests that partially purified MIS has transient meiosis-arresting activity in rat, but not mouse oocytes (Takahashi et al., 1986~. Recently, however, it has been demonstrated that immunopurified, biologically active MIS does not maintain meiotic arrest in rat oocytes (Tsafriri et al., 1988~. It was suggested, therefore, that the meiosis-arresting action of he partially purified preparation of MIS could be that of a contaminant (Tsafriri et al., 19887. These studies emphasize the importance of a complete purification of biological preparations before clear identification and characterization of active factors can be possible. Purines Hypoxanthine was found in preparations of porcine follicular fluid (Downs et al ., 1985 ~ . This purine accounted for most of the meiosis-arresting activity of porcine follicular fluid on mouse oocytes. Concentrations of hypoxanthine in biological fluids can be increased substantially by a variety of pathological conditions, including anoxia. Since the porcine foil icular fluid used for analysis was not collected under conditions that would prevent the artifactual generation of hypoxanthine, the concentration of purines in mouse follicular fluid was assessed when collection was made under more controlled conditions (Eppig et al., 1985~. For example, the conversion of adenosine to hypoxanthine was prevented by treatment with an inhibitor of adenosine deaminase; nonetheless, millimalar concentrations of hypaxanthine were found. In addition, murine follicular fluid was found to contain substantial amounts of adenosine. Although it is still possible that the concentration of hypaxanthine in mouse follicular fluid is not as high as reported because of rapid intrafollicular changes, it should be kept in mind that the ovarian follicle is not vascularized central to the thecal layers and a low oxygen concentration may be a natural condition in situ. In addition, - 178 -
hypoxanthine is the predominant purine found in rapidly frozen intact ovaries (Eppig, unpublished results). When assessed at concentrations estimated to be present in murine follicular fluid, hypoxanthine maintained most mouse oocytes in meiotic arrest. Adenosine had only a transient meiosis-arresting action, but augmented the stable inhibitory effect of hypoxanthine (Eppig et al., 1985~. The meiosis-arresting action of hypoxanthine andadenosine was fully reversible. Moveover, oocytes that were maintained in the GV stage by these purines for 24 hours and then matured in medium without purines could be fertilized and underwent embryonic development (Downs et al., 1986a). Inhibitors of inosine monophosphate (IMP) dehydrogenase induced GVB in oocytes that had been maintained in meiotic arrest by hypoxanthine in vitro. The IMP-dehydrogenase inhibitors, however, did not induce GVB in oocytes arrested in the GO stage by guanosine, the most potent of the purines tested for meiosis-arresting activity (Downs et al., 1986b). These results indicate that the meiosis-arresting action of hypoxanthine in vitro may be mediated through the production of guanyl compounds. Likewise, IMP dehydrogenase inhibitors induced GVB upon injection into mice primed for 24 hours with PMSG. One of these inhibitors, bredinin, induced the maturation of almost all the meiotically competent ovarian oocytes within 6 hours of injection (Downs and Eppig, 1987). It is not likely that the G1lB-inducing action of IMP dehydrogenase inhibitors was mediated by gonadotropins since other manifestations of gonadotropin action such as cumulus expansion, thinning of the apical wall of the follicle, or cessation of DNA synthesis in the granulosa cells were not observed after injection of the inhibitors (Downs and Eppig, 1987). These results support the idea that, in mice, the formation of guanyl compounds via the IMP dehydrogenase pathway is essential for the maintenance of meiotic arrest in vivo. Because of the evidence indicating the importance of cAMP in maintaininq meiotic arrest in mouse oocytes, the hypothesis that purines, particularly hypoxanthine and adenosine, may maintain meiotic arrest by promoting meiosis-blocking levels of cAMP in oocytes was tested. The maintenance of meiotic arrest was correlated with elevated cAMP levels in cumulus cell-enclosed oocytes cultured in medium containing hypoxanthine or hypoxanthine plus adenosine . In addition, microinj ection of oocytes with an inhibitor of cAMP-dependent protein kinase induced GVB in oocytes cultured in medium containing hypaxanthine ~ Downs et al ., 19 8 9 ~ . These results show that hypaxanthine arrests mouse oocytes in the GV stage, at least in part, by maintaining meiosis-blocking levels of cAMP in the oocytes. Hypaxanthine, and also guanosine and adenosine, inhibited cAMP phosphodiesterase activity in ly~ates of mouse oocytes and augmented the cAMP-elevating action of follicle-stimulating hormone (FSH) in intact oocyte-cumulus cell complexes. These result. suggest that one way that hypaxanthine may maintain meiosis-blocking levels of cAMP is by preventing hydrolysis of cAMP. It is likely, though, that purines promote the elevation of cAMP by other mechanisms as well. - 179
Future research on the maintenance of meiotic arrest must focus on the identif ication of specif ic molecules in the cAMP-dependent pathway in oocytes. Moreover, ~ greater understanding is needed regarding the role of purines, such as hypoxanthine, adenosine, and GHP in meiotic arrest, and how the metabolism of these purines is regulated. It is likely that several different interconnecting pathways participate in the maintenance of meiotic arrest and that the relative contribution of these pathways may vary from species to species. The interactions of various factors such as gonadotropins, steroid hormones, and growth factors in the regulation of these pathways will be dif f icult to study, but their resolution is essential for a basic understanding of the mechanisms that maintain meiotic arrest. Induction of Oocyte Maturation Oocyte maturation in viva is induced in ovulatory follicles by the preovulatory surge of LH. The mechanism by which LH promotes maturation, however, is not well understood. LH could stimulate the granulosa cells to some action that would deprive the oocyte of maturation-arresting substances. For example, LH could terminate the production of meiosis-arresting substances or promote the catabolism of these substances, or it could disrupt the gap junctional system that delivers the meiosis arresting substances to the oocyte. The concentration of cAMP in the intact follicle does not decrease after stimulation with hCG (Schultz et al., 1983~. Nor is there a decrease in the concentration of hypoxanthine or adenosine in murine follicular fluid before GVB (Eppig et al., 1985~. It is clear, therefore, that a decrease in the concentration of these substances in the whole follicle is not the mechanism of hCG-induced oocyte maturation. Moreover, the gap junctional communication between the oocyte and its companion cumulus cells does not decrease before G~B in vivo (Moor et al., 1981; Eppig, 1982; Salustri and Siracusa, 1983~. It is possible that there are differences between the movement of the molecular markers used to measure gap junctional communication and the movement of meiosis-arresting substances, but if they are analogous, then it can be concluded that a disruption in the communication between cumulus cells and the oocyte is not the mechanism of LH-induced oocyte maturation. It has been suggested that LH induces a reduction in the communication between the mural granulosa cells and cumulus cells and tbereby reduces the movement of meiosis-arresting substances to the oocyte-cumulus cell complex (Larsen et al., 19873. If cAMP is the meiosis-arresting substance that moves from the follicle wall to the oocyte-cumulus cell complex to maintain meiotic arrest, then a decrease in the cAMP content of the intact complex should precede GVB, or commitment to undergo GVB. Such a decrease in the cAMP content of the intact oocyte-cumulus cell complex was not detected after injecting mice with human chorionic gonadotropin (hCG) (Shultz et al., 1983; Eppig and Downs, 19883. These results, however, do not eliminate the possibility that the passage of other meiosis-arresting substances from the follicle wall to the oocyte-cumulus cell complex could be reduced. 180 -
After mice are injected with hCG, the cAMP content of the intact oocyte-cumulus cell complexes increases (Shultz et al., 1983) even though the gap junctional communication between the oocyte and the cumulus cells, which should allow the transfer of cAMP, is not reduced (Eppig and Downs, 1988~. One explanation for this observation is that the oocytes become stimulated to secrete or hydrolyze their cAMP by a signal produced by the granulosa/cumulus cells in response to LH/hCG. Alternatively,-the cumulus cells may have been stimulated to selectively reduce the transfer of cAMP, and not markers of gap junctional communication, to the oocytes. There is evidence for a positive, maturation-inducing signal produced by granulosa cells in response to gonadotropin or epidermal growth factor (EGF) (Eppig and Downs, 1987; Downs et al., 1988~. This signal overcomes the ef feet of meiosis-arresting substances. GOB is induced in cumulus cell-enclosed oocytes by either FSH or EGF even in the continual presence of hypoxanthine, dbcAMP, or IBMX. The frequency at which these agonists induced GVB is greater than the frequency of GVB promoted by simply denuding the oocytes in medium containing the meiosis-arresting substance, thus indicating that the agonists do not function merely by promoting the. uncoupling of the cumulus cells from-the oocyte. The identification of maturation-inducing signals is a major area for future research. Moreover, the possible physiological role, if any, of growth factors such as EGF in the regulation of oocyte maturation needs to be resolved. Similarities Between Mechanisms that Govern Meiotic and Mitotic Cel 1 · · ~ Division ~ There are several lines of evidence suggesting that mechanisms that govern the induction of oocyte maturation are similar to the mechanisms involved in the induction of mitotic cell division. First, an autocatalytic maturation-promoting factor (MPF) is produced in frog oocytes in response to progesterone (Reynhout and Smith, 1974), in starfish oocytes in response to 1-methyladenine (Kishimoto and Kanatani, 1976), and in maturing mammalian oocytes (Sorenson et.al., 1974~. A similar factor is also present in somatic cells as they enter M phase of the cell cycle (Kishimoto et al., 1982~. Moreover, it has recently been shown that MPF purified from frog oocytes contains the product of a homolog of a mitosis control gene, cdc2+, of yeast (Dunphy et al., 1988; Gautier et al., 1988~. Second, mRNA- coding for the clam protein cyclin A, a protein whose level rises and falls during both meiotic divisions of the oocyte and during the cell cycle of early embryogenesis, induces meiotic- maturation upon injection into frog oocytes (Swenson et al., 1986~. Third, an increase in free intracellular calium appears to be an essential part of the mechanisms that promote somatic cell division (Poenie et al., 1985; Hafner and Petzelt, 1987 ; Iwigg et al., 1988) . Likewise, calcium ionophores, which increase free intracellular calcium by uptake, induce the maturation of mammalian oocytes maintained in meiotic arrest with dUcAMP or IBMX (Powers and Paleos, 1982; Racowsky, 19861. Moreover, inhibitors of calmodulin prevent the spontaneous maturation of murine oocytes, but do not prevent a decline in oocyte cAMP (Bornslaeger et al., 1984~. This observation supports the idea that intracellular calcium plays an important role in the initiation of GVB and functions in a - 181
pathway distinct from, but perhaps parallel to, the pathway activated by a decline in cAMP levels. Increases in the concentration of free calcium in oocytes could result from either the mobilization of calcium from intracellular stores or the uptake of external calcium. One could speculate that the maturation-inducing signal communicated from granulosa cells to the oocyte could be a factor(~) that promotes an increase in free calcium in the oocyte, by internal mobilization or uptake, or the signal could be calcium itself. It will be important to investigate these parallels between the control mechanism of mitotic and meiotic cell division further. Murine Oocyte Growth and Development in Culture Murine oocytes grow and acquire competence to undergo GVB in culture (Eppig, 19771. Oocyte growth is dependent upon metabolic coupling to granulosa cells, but the acquisition of competence to undergo GVB is not (Canipari et al. , 1984~ . Therefore, the developmental program governing the acquisition of GVB competence is independent of oocyte growth. Moreover, the acquisition of GVB competence does not appear to require gonadotropins or steroid hormones (Eppig, 19771. In the rat, however, ESH and estradiol increase the frequency at which GVB competence is acquired (Bar-Am) et al., 19837. For many years it was thought that oocytes that had matured in vitro were defective because they could not be fertilized and undergo embryonic development. The main reason for failure was thought to be the inability of ova matured in vitro to promote the development of the male pronucleus, but the failure was probably due to inadeguate culture systems because it was later demonstrated that cumulus cell-enclosed murine oocytes that mature spontaneously in vitro are competent of fertilization and embryonic development at frequencies equivalent to oocytes that mature in response to superovulatory regimen in viva (Schroeder and Eppig, 1984~. Subsequently, normal development after maturation in vitro has also been reported for oocytes form some other species such as the sheep {Staigmiller and Moor, 1984 ~ . Much remains to be done, however, to resolve the optimal conditions for oocyte maturation for each species that will promote maximal developmental potential. Murine oocytes indicate that oocytes sequentially develop competence for maturation, then fertilization and cleavage to the two-cel1 stage, and then development to blastocyst (Eppig and Schroeder, 1989~. The competence for each of these developmental capacities is acquired in vitro and does not require the presence of gonadotropins. Oocytes isolated in mid-growth phase from 12 day old mice, have been grown , matured , fertil ized, and cultured to the 2-cell stage in vitro, and developed to term after transfer to the oviducts of pseudopregnant foster mothers. Continued progress in the development of the technology for oogenesis in 182 -
vitro is dependent upon basic studies on oocyte nutrition and metabolism, and on the action of hormones, growth factors, and other factors that control the differentiation and function of the functionally integrated oocyte-granulosa cell complex in viva. Prospects for the Practical Utilization of Oocyte Culture Systems Expansion of Populations of Rare or Agriculturally Important Mammals Very few primary oocytes that are present in the ovary are ovulated and made available for fertilization in mammals. For example, it has been estimated that there are more than 150,000 primordial follicles in the bovine ovary at birth, but not more than 3 DO of these will ever be ovulated normally (Erickson, 19663. Techniques of superovulation could increase the reproductive capacity of ~ cow only about tenfold even i f it could be superovulated yearly during its reproductive lifespan (Seidel, 1981). This general rule probably holds true for most mammals. There is, therefore, a tremendous pool of oocytes having the potential for development and, therefore, expanding populations if the oocytes could be rescued from their normal fate of degeneration. One approach to such a rescue is to isolate the oocytes from their natural environment and grow them in vitro and, as described above, significant progress has been made to achieving this using mice as a model system. This technology would be applicable to the expansion of populations of agriculturally important animals such as those that have been genetically engineered for desirable characteristics, of important genetically engineered experimental animals, or of endangered species. Our culture system for murine oocyte maturation in vitro rescues degenerating oocytes from deceased mice. Significant necrotic changes are readily apparent in the oocytes of mice dead for six hours at ambient temperatures but these changes are reversed during oocyte maturation in vitro. The eggs derived from the rescued oocytes can be fertilized and undergo normal development. Thus, it may be possible to salvage the oocytes of rare or agriculturally important animals even after unforeseen death. Eggs derived from murine ova that mature in vitro can also be frozen and show almost normal capacity for fertilization and development after thawing. Thus valuable ova can be presented after development in vitro if appropriate sperm are not immediately available. Genetic Engineering. Jenkins and Copeland (1985) have reported that the germ line of SWR/J-RF/J hybrid mice become infected with ecotropic retroviru~es during oogenesis. Moreover, Panthier et. al (1988) have produced tranagenic mice by inoculation of newborn SWR/J females with an ecotropic murine leukemia virus. These findings, along with the development of oocyte culture systems, could present a unique opportunity for the introduction of genes into the germline. Protocols could be - 183
developed to infect oocytes with retroviruses carrying specific genes that would become integrated into the germ line, introducing a significant advancement in the technology for producing transgenic mice. Preliminary studies have produced one mouse with integrated retrovirus after infecting Skytel during maturation in vitro. Perhaps greater success will be achieved in experiments wherein oocytes are infected during more prolonged development or maintenance in culture. Clinical Applications In vitro fertilization ~ I~F) has become a ~list, c clinical resolution to a variety of infertility problems in both women and men. Nevertheless, the success rate could be increased by a radical change in this protocol to util ize oocyte maturation in vitro rather than the protocols of hormonal stimulation currently in use. The stipulatory protocols which promote follicular development and maturation of oocytes in multiple follicles in humans probably produces eggs with a reduced developmental capacity. Moreover, these protocols probably do not promote optimal development of the uterine epithelium for the implantation of the embryo. An alternative to the hyperstimulatory techniques is oocyte maturation in vitro. Our studies with mice indicate that ova with full developmental capacity can be recovered even from degenerating follicles. Thus, it may be possible to recover immature oocytes form several antral follicles, excluding the dominant preovulatory follicle, and mature them in culture while the preovulatory follicle prepares the uterus normally for implantation. Current ultrasonic techniques for follicular visualization and oocyte aspiration do not allow sufficient resolution of the follicles for this procedure, but this is an engineering problem with the ultrasound equipment and could be resolved with appropriate incentive. Current protocols for human I~F and embryo culture are not based on exhaustive experimentation to define optimal culture conditions for human ova or embryos. Ethical problems have hindered progress in this area. One of these problems involves the source of ova for the studies. However, if immature oocytes could be recovered from ovaries removed for various clinical reasons and matured in culture with full developmental potential, the ethical problems associated with the source of ova for embryo research would be reduced. Clinical realization of maturation in vitro as an antecedent to IVF will require basic studies to resolve likely differences in conditions for oocyte maturation that impart full developmental capacity between human and mouse oocytes. It would probably be advantageous and appropriate to carry out initial studies using non-human primate oocytes. Moreover, continued basic studies on oocyte metabolism and its regulation by companion somatic cells, hormones, meiotic regulatory substances and growth factors using rodent systems is essential. Mouse ova were used in the pioneering studies on IVF that have produced the clinical successes we now see every day. Certainly, experiments with mouse oocytes will continue to be indicators of what is possible with other species. - 184
Acknowledgments Research in my laboratory is supported by grants HD20575, HD21970 and ED23839 from the National Institutes of Health. The contents of this paper do not necessarily represent the official views of that agency. My most sincere-thanks to Roberto Buccione, Allan Schroeder and Barbara V~nderhyden for their helpful suggestions for this paper. - 185
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