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MOLECULAR EVENTS PRE- AND POST-FERTILIZATION OF MOUSE EGGS: OOCYTE MATURATION, EGG ACTIVATION, AND POLYSPERMY BLOCK R. M. Schultz,. S. Kurasawa, Y. Endo, and G. S. Kopf INTRODUCTION Prior to the fusion of the sperm with the egg, the egg undergoes a series of complex biological processes that allow it to mature and be fertilized. In turn, fertilization initiates a complex series of events teemed egg activation. One aspect of egg activation results in cell cleavage and further development. Another aspect of egg activation prevents polyspermy, which leads to aberrant development. This brief review will first discuss events during oogenesis and oocyte maturation that are involved in production of a fertilizable female gamete. Events comprising the fertilization-induced block to polyspermy will then be addressed. The discussion will be mainly restricted to the mouse, since this is the best characterized system for processes involved in mammalian oocyte maturation and fertilization. ACQUISITION OF MEIOTIC COMPETENCE During the period of oocyte growth, which takes about 14 days, mouse oocytes that are arrested in the first meiotic prophase grow from about 15 Am to 80 Am in diameter. Acquisition of meiotic competence is correlated with a specific stage of oocyte growth. In mice, oocytes that are obtained from juvenile mice less than 15 days of age are less than 60 Am in diameter and will not resume meiosis, i.e., undergo meiotic maturation, when placed in a suitable culture medium (Sorensen and Wassarman, 1976). One of the earliest morphological manifestations of meiotic maturation is breakdown of the nuclear membrane, which is called the germinal vesicle. Subsequent to germinal vesicle breakdown (GVBD), a spindle forms. Separation of homologous chromosomes then occurs, with emission of the first polar body and arrest at metaphase II. The frequency at which growing oocytes can resume meiosis increases with increasing diameter, which is a linear function of the age of the donor juvenile mice (Sorensen and Wassarman, 1976). Stage-specific differences in the spectrum of synthesized polypeptides are correlated with acquisition of meiotic competence (Schultz et al., 1979a). These changes are likely to underlie, in part, the biochemical basis for the acquisition of meiotic competence. Nucleate fragments obtained from fully-grown, meiotically competent oocytes that are less than 60 Am in diameter undergo GVBD, emit the first polar body and arrest at metaphase II (Balakier and Czolowska, 1 977 ; Schultz et al ., 1 97 8 ) . Thus, it is likely that the quality of the cytoplasm, and not the amount of cytoplasm, is involved in acquisition of meiotic competence. Oocyte growth per se may not be involved in acquisition of meiotic competence, since acquisition of meiotic competence can be dissociated from oocyte growth (Canipari et al., 1984). Oocytes obtained from juvenile mice about 15 days of age are about 6 0 em in diameter. oocytes obtained from juvenile mice 10 days of age are less than 60 em in 262

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diameter and meiotically incompetent. When there meiotically incompetent oocytes obtained from juvenile mice 10 days of age are cultured for 5 days in a medium that does not support oocyte growth but does support oocyte viability a significant fraction of the oocytes undergo GVBD. Results from a series of similar experiments indicated that a constant amount of combined time that totals 15 days of in Vito growth or in vitro culture is necessary for acquisition of meiotic con etence. To provide a tighter correlation between the changes in the proteins synthesized during oocyte growth and the acquisition of meiotic competence, it should be demonstrated that the changes in protein synthesis that occur during oocyte growth also occur under the conditions of in vi tro culture that do not sustain oocyte growth but do foster meiotic competence. OOCYTE MATURATION Oocytes undergo meiotic maturation, which culminates in the production of an egg; eggs, not oocytes, are capable of being fertilized and giving rise to normal development. Although oocytes that have not reached and arrested at metaphase II can be penetrated by sperm, such oocytes do not develop normally and very quickly degenerate. The subsequent section will discuss briefly some molecular aspects of oocyte maturation. 1. Role ot cAME and protein-pho~phQrylaLlQn The follicle exerts an inhibitory influence on oocyte maturation, since oocytes present in preovulatory ant ral follicles do not resume meiosis, whereas liberation of these oocytes from their follicles results in resumption and completion of meiotic maturation (Pincus and Enzmann, 1935). A substantial body of evidence implicates cAMP in the maintenance of meiotic arrest, although a number of molecules are likely to participate in this process (See Schultz, 1988 and reference therein for a more complete review of this area.). In vitro maturation is reversibly inhibited by membrane permeable c AMP analogs, (e.g., dibutyryl cAMP;(dbcAMP), or inhibitors of cyclic nucleotide phosphodiesterase (PDE), (e.g., 3-isobutyl-1-methyl xanthine;(I8MX) (Cho et al., 1974; Bornslaeger et al., 1984); the corresponding cGMP analogs do not inhibit maturation in vitro. In addition, treatment of oocytes with either the activator of adenylate cyclase, forskolin, or microinjected cAMP inhibits GVBD (Schultz et al., 1983). The best documented mode of action of cAMP in eukaryotes is to activate a cAMP-dependent protein kinase (protein kinase A, PK-A). Accordingly, it was proposed that cAMP is involved in maintenance of meiotic arrest by activating PK-A. This enzyme phosphorylates, either directly or indirectly, a protein fs) X, which is capable of promoting GVBD (Bornslaeger et al., 1986a). The phosphorylated form, XP, is inactive. Resumption of meiosis in vitro is proposed to occur by a decrease in cAMP, which would result in a decrease in PK-A activity. Consequently, a protein phosphatase would shift the equilibrium between XP and X to the dephosphorylated form of X and GVBD would ensue. Consistent with this model is that microinjection of oocytes, which are incubated in medium containing a concentration of dbcAMP that inhibits maturation, with protein kinase inhibitor (PKI) undergo GVBD - 263

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(Bornslaeger et al., 1986a); PKI inhibits PK-A by combining with the free catalytic subunit (C). This result in anticipated, since inactivation of C would result in the conversion of XP to X, and hence maturation would resume. In addition, microinjection of oocytes,.which are incubated in a medium that supports oocyte maturation, with the catalytic subunit of PK-A results in inhibiting GVBD. Presumably the excess of C..relative to regulatory subunit maker C essentially a cAMP- independent protein kinase. C continues to phosphorylate X and to keep it in its inactive phosphorylated form and accordingly maturation is inhibited. A decrease in oocyte clamp does in fact occur prior to GVBD during maturation in vitro (Schultz et al., 1983; Vivarelli et al., 1983). This decrease may be causally related to GVBD, since PDE inhibitors, which inhibit GVBD, inhibit this maturation-associated decrease in oocyte cAMP that occurs during a period of time in which the oocytes become committed to resume meiosis (Schultz et al., 1983). Commitment is experimentally defined an follows: After a given period of time in culture, oocytes possessing an intact germinal vesicle are transferred to medium containing either dbcAMP or IBMX and then scored for GVBD at later times. If an oocyte resumes meiosis it is termed "committed". Moreover, microinjection of oocytes, which are incubated in medium containing a concentration of IBMX that inhibits maturation, with purified phosphodiesterase undergo GVBD (Bornslaeger et al., 1986a) . Presumably, even though the exogenous microinjected PDE is inhibited by >85% by the IBMX, the excess amount of PDE activity is sufficient to hydrolyze oocyte cAMP. This decrease in cAMP would then occur and GVBD would ensue in the presence of IBMX. A maturation-associated set of changes in protein phosphorylation occurs during the commitment period (Bornslaeger et al., 1986a) and may be causally related to GVBD. A basic phosphoprotein of Mr 60,000 undergoes an apparent dephosphorylation and a set of phosphoproteins of Mr 24,000, 29,000, and 36,000 exhibit an apparent increase in phosphorylation. These same changes occur in oocytes incubated in medium containing dbcAMP and microinjected with PKI, which induces GVBD. In addition, activators of the calcium, phospholipid- dependent protein kinase, protein kinase C (PK-C) and antagonists of calmodulin, which could regulate a calmodulin-modulated protein kinase, inhibit maturation and the maturation-associated set of changes in protein phosphorylation (Bornslaeger et al., 1986b; Bornslaeger et al., 1984). These agents do not inhibit the maturation-a~sociated decrease in.cAMP and microinjection of oocytes incubated in these agents with PKI does not result in GVBD. Thus, the mode of action of there compounds may be distal to that of cAMP. These changes in protein phosphorylation do not occur in meiotically incompetent oocytes, but do occur in the lO% of oocytes 60 Am in diameter that can undergo GVBD. Injection of meiotically incompetent oocytes with an amount of PKI sufficient to induce maturation in fully-grown oocytes does not result in GVBD in these incompetent oocytes but does elicit the decrease in phosphorylation of the Mr 60,000 phosphoprotein. The increase in apparent phosphorylation of phosphoproteins of Mr 24,000, 29,000 and 36,000 is not observed. It is likely that meiotically competent oocytes lack the ability to phosphorylate these phosphoproteins, wince low levels of phosphorylation of these phosphoproteins are detected. Thus, dephosphorylation of the 60,000 Mr protein is not sufficient to induce GVBD and meiotic - 264

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incompetence may entail deficiencies in the ability of the oocytes to execute phosphorylations that occur downstream to the dephosphorylation of this protein (Bornslaeger et al., l9B8). Future work may provide additional insights regarding the proteins involved in this phonphorylation cascade. 2. .~.ti ~ ~ - Li ILL -ear An activity central to oocyte maturation and subsequent cell cycles is that of Maturation Promoting Factor (MPF)(Masui and Clarke, 1979 and references therein; ~ishimoto, 1988 and references therein). MPF in more aptly described an an M phase promoting factor that is involved in the G2 to M transition of the cell cycle (Gerhart et al., 1984). It is directly implicated in chromosome condensation and breakdown of the nuclear membrane and its activity oscillates during the cell cycle. MPF activity shown no evidence for species specificity, since MPF obtained from one species will induce nuclear membrane breakdown when injected into cells of distantly related species (Masui and Clarke, 1979, Kishimoto et al., 1984.;;Sorensen et al., 1985) . Until recently, MPF has defied purification, due to its instability and cumbersome biological assay (Gerhart et al., 1984). The development of methods for stabilizing the activity and in vitro assays that support nuclear membrane breakdown have led to MPF's recent purification from Xenopus oocytes (Lohka et al., 1988). The purified protein possesses protein kinase activity, and is itself phosphorylated; it is not known whether phosphorylation regulates its activity. Results of experiments using extracts that contain MPF activity suggest that phosphorylation of MPE, which may be capable of autophosphorylation, may generate the active form of MPF (Cyert and and Kirschner; 1988). Such a mechanism also explains the self-amplification properties of MPF. Interestingly, the Xenopus MPF is a CDC2 homologue (Dunphy et al., 1988; Gautier et al., 1988). The CDC2 protein is directly involved in the yeast cell cycle. The protein has an associated protein kinase activity and is itself likely to be regulated by phosphorylation; the level of phosphorylation increases as cells undergo a G2 to M transition (Simanis, V. and Nurse, 19861. Thus, MPF may be an intrinsic component of the cell cycle. Mouse oocytes appear to possess an MPF-like activity. Fusion of a meiotically competent oocyte that has undergone GVBD with a meiotically incompetent oocyte results in breakdown of the germinal vesicle of the incompetent oocyte (Balakier, 1978). Injection of cytoplasm from mouse oocytes that have undergone GVBD into Xenopus (Sorensen et al., 1985) or Asterina Pectinifera oocytes (Kish~moto et al., 1984) induces maturation; cytoplasm of mouse oocytes inhibited from maturing does possess MPF activity as deduced by its inability to induce GRAD in the recipient oocytes. Mouse oocyte MPF activity oscillates during maturation (Hash~moto and Kish~moto, 1988), as is the care in other systems (Gerhart et al., 19 8 4 ) . In a series of elegant experiments, cytoplasm f ram oocytes at dif ferent stages of maturation was in jected into Asterina Pectiniera oocytes, which were subsequently scored for GUBD . In this manner, the amount of MPF activity was quantified and shown to increase Subsequent to GVBD . It reaches a peak at metapha~e I, decreases during polar body emission, and then increases again as the oocyte arrests at metaphase II. Inhibiting protein synthesis does not affect the initial appearance of MPF after GVBD, which occurs in the absence of protein synthesis. 265 ~

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MPF generation does not require nuclear contents, since fusion of anucleate fragments of fully-grown oocytes with interphase blastomeres derived from 2-cell mouse embryo n results in almost ~ ediate chromosome condensation in the interphase nuclei (Balakier and Czolowska, 1977) ; the anucleate fragments were used at a time when the nucleate fragments had undergone GVBD. Meiotically incompetent mouse oocytes also possess an "anti-MPF" activity. Fusion of a meiotically competent oocyte with an intact GV with a meiotically incompetent oocyte preserves the integrity of each nucleus, which resides in the common cytoplasm (Fulka et al., 1985). The loss of this anti-MPF activity may be required for acquisition of meiotic competence, and further characterization of this activity is required. 3. The nucleus of the fertilizing sperm is highly condensed due to chromatin-~ssociated basic proteins called protamines, which contain high amounts of cysteine (Yanagimachi, 19 88 and references therein). These cysteine residues are oxidized during sperm maturation and their reduction during fertilization is necessary for the sperm nucleus to decondense in the egg cytoplasm and thus be transformed into the male pronucleus. A pivotal role for glutathione in the process of sperm nuclear Recondensation is likely, since glutathione is the major biological thiol reducing agent and treatment of eggs with diamide, which is an antioxidant of glutathione, reversibly inhibits the Recondensation of mucroinjected hamster sperm nuclei (Perreault et al., 1984). In addition, treatment of mice in viva with an inhibitor of glutathione synthesis, L-buthionine-S,R-sulfoximine (BSO) inhibits sperm Recondensation of mouse sperm nuclei following in vitro fertilization - (Calvin et al., 1986). The inability of the sperm nucleus to Recondense is apparently due to inadequate amounts of reducing power present in the egg, since disulfide-poor sperm nuclei Recondense when microinjected into either GV intact oocytes or eggs (Perreault et al., 1984; Zirkin et al., 1985). The ability of sped nuclei to Recondense in egg cytoplasm is dependent on the maturational state of the oocyte (Perreault et al., 1988). Sperm mucroinjection experiments indicate that Recondensing II arrested eggs but in barely Correlated with these dif ferences activity is maximal in metaphase detectable in GV intact oocytes. ~orre~acea worn enese adherences in these decondensing potentials is a maturation-associated increase in the amount of glutathione. Inhibiting this maturation-associated increase in glutathione content with BSO inhibits the ability of the matured oocyte to Recondense microinjected sperm nuclei. Sperm nuclei in a GV-intact oocyte will not deconden~e, whereas sperm nuclei in an oocyte that has undergone GVBD will Recondense. Moreover, since dithiothreitol-treated hamster sperm nuclei will Recondense in the cytoplasm of GV-intact oocytes (Perreault, et al., 1984), the contents of the GV may be necessary for the development of this Recondensing activity. The interaction of the nucleoplasm with the cytoplasm may result in generating the sperm recondensing activity by providing the cytoplasm with (1) the activity (2) a "co-factor'' necessary for activity, (3) an activity that '"activates" the sperm- 266 -

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deconden~ing activity, or ( 4 ) an activity that inhibits a sperm- decondensing inhibitory activity . Last, it should be noted that GVBD apparently required for development of cytoplasmic activities that control male and female pronuclei formation (Balakier and Tarkowski, 1980; Yanag~machi, 1988 and references therein). FERTILIZATION AND EGG ACTIVATION IS Although fertilization of mouse eggs induces a small increase in the absolute rate of protein synthesis (Schultz et al., 1979b), it initiates a dramatic series of changes in the pattern of protein synthesis (Schultz et al., 1979b; Cullen et al., 1980). Although the one-cell embryo supports a low level of transcription (Clegg and Piko, 1982), transcription is not necessary for these changes in protein synthesis, since they occur in either physically enucleated zygotes or zygotes incubated in the presence of a-amanitin (Petzoldt et al., 1980; Braude et al., 1979). Most of these changes are due to po~t- tran~lational modifications of existing proteins (Van Blerkom, 1981) or recruitment of maternal ~ A (Cascio and Wassarman, 1982); In addition, there is a small subset of changes that appear autonomous of fertilization and are apparently initiated by oocyte maturation (Howlett and Bolton, 1985). Both the fertilization-induced and fertilization- independent changes in protein synthesis are likely involved, at least in part, in the onset of DNA synthesis in the pronuclei, cleavage to the 2-cell stage, and transition from a nonproliferative to a proliferative state with the concomitant conversion of a meiotic cell cycle to a mutotic one. What controls these events is not known, but perturbing protein phosphorylation during the first cell cycle can inhibit both cleavage to the two-cell embryo, as well as activation of transcription of the zygotic genome, which also occurs in the two-cell embryo (Poueymurou and Schultz, 1987). This result is consistent with protein phosphorylation being a major type of protein modification theta occurs during the first cell cycle (Van Blerkom, 1981). 1. Block to Polyspermy A major response of the egg to the fertilizing sperm is the block to polyspermy. All species apparently have mechanisms to block polyspermy. For example, sea urchins possess a fast electrical block that operates at the level of the plasma membrane (Jaffe and Gould, 1983 and references therein). Although mouse eggs generate a plasma membrane block to polyspermy (Wolf, 1978; Stewart-Savage, and Baiter, 1988 for hamster), there is no evidence to support the existence of a fast electrical block to polyspermy at the level of the plasma membrane (Jaffe et al., 1983). The major mechanism for the polyspermy block in mice appears to be an egg-induced modification of the zone pellucids (ZP). The ZP is an extracellular coat that surrounds the oocyte and is responsible for ~pecie^-specific binding of sperm and induction of the acrosome reaction (Wasserman, 1987, 1988 and references therein). It Should be noted, however, that rabbits do not possess a zone block to polyspermy; their primary block apparently resides at the plasma membrane (Yanag~machi, 1988). Moreover, dispermic mouse eggs can be restored to the monospermic condition by spend loss due to cytoplasmic blebbing (Yu, and Wolf, 1981); this would constitute a third line of defense against polyspermy. The mechanism of this sperm loss is poorly understood, ~ 267

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although mucrofilaments are likely involved, since the process is inhibited by cytochalasin B (Yu, and Wolf, 1981). The mouse egg's zone pellucida is composed of three sulfated glycoproteins called ZP1,. ZP2,. and ZP3 (Wasserman, 1988 and references therein that pertain to structural and functional aspects; Bleil and Wassarman, 1980a; Shimizu et al., 1983). ZP1, which has an apparent molecular weight of about 200,000 daltons (Bleil and Wassarman, 1980a), is a clime r connected by intermolecular disulfide bonds and is likely to serve as a cross-linker responsible for maintaining the three dimensional structure of the. zone pellucida (Greve and Wa~sa~man, 1985). ZP2 isolated from oocyte~ or unfertilized eggs has an apparent molecular weight of 120,000 daltons, which in observed under either non-reducing or reducing conditions of gel electrophoresis (Bleil and Wassanman, 1980a). Fertilization results in the modification of ZP2 to a form called ZP2f. Under nonreducing conditions, ZP2f has an apparent molecular weight of 120,000, whereas under reducing conditions it has an apparent molecular weight of 90,000 daltons (Bleil et al., 1981). This modification is a proteolytic cleavage that results in two fragments held together by disulfide bonds. Since ZP2 can bind to acrosome reacted sperm, whereas ZP2f cannot, ZP2 may mediate sperm binding subsequent to the acrosome reaction (Bleil and Wassarman, 1986). ZP3 has an apparent molecular weight of 83,000 daltons and the O-linked carbohydrate portion accounts for the sperm receptor activity of ZP3, which is lost after fertilization (Bleil and Wassanman, 1980b; Florman et al., 1984). ZP3 also possesses all of the sperm acrosome reaction- inducing activity of the ZP, and can do so to the same extent as that induced by the ionophore A23187 (Bleil and Wassarman, 1983). Based on the aforementioned properties of the mouse zone and that only acrosome-intact sperm bind to the zone -acrosome-reacted sperm do not bind- the following sequence of events has been proposed for sperm- zona interactions and fertilization of mouse eggs. Acrosome-intact sperm bind to the zone pelf ucida (Saling et al., 1979a; Florman and Storey, 1982); binding is species-specific and mediated by-ZP3. Sperm bound to ZP3 then undergo the acrosome reaction, which is also mediated by ZP3. ZP2 mediates the binding of acrosome-reacted sperm (Bleil and Wassarman, 1986), which then penetrate the zone pellucida and gain access to the perivitelline space. The acrosome-reacted sperm then bind to and fuse with the egg's plasma membrane. In response to fertilization, the egg undergoes the "cortical granule reaction" (Szollsi, 1967; Barros and Yanag~machi, 1971, 1972; Wolf and Hamada, 1977). Cortical granules subjacent to the plasma membrane are thought to fuse with the plasma membrane and release into the perivitelline space enzymes that convert ZP2 to ZP2f and modify ZP3 such that it no longer possesses either sperm receptor activity or the ability to induce an acrosome reaction. Thus, acrosome-intact sperm can no longer bind to the zone and acrosome-reacted sperm bound to the zone can no longer interact with and penetrate the zone, since these sperm do not interact with ZP2f (Bleil and Wassarman, 1986). This series of events is proposed to comprise the zone reaction or the zone block to polyspermy. A membrane block to polyspermy, however, also develops with time. Although much is known about the cortical granule reaction in lower species and how the contents of the granules modify the extracellular coats surrounding eggs in these species, little is known 258 -

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about the mammalian cortical granule reaction (Gulyas, 1980). This is in large part due to the difficulty in obtaining large amounts of biological material from mammalian species. In turn, this has prevented generation of molecular markers for mammalian cortical granules. Moreover, until recently, the only way to assess accurately the status and distribution of cortical granules was by electron microscopy, which is a tome consuming process and difficult to quantify easily. Polyphosphatidylinositol turnover, which has been implicated as a response of the egg to fertilization in lower species (Turner et al., 1984, 1986; Whitaker and I ovine, 1984; Swann and Whitaker, 1986), generates two second messengers, i.e., an 1, 2-diacylglycerol, which activates the calcium- and phospholipid-dependent protein kinase, protein kinase C (PK-C), and inositol-1,4, 5-tri~phosphate (IP3) , which releases calcium from intracellular stores (Berridge, 1984; Berridge and Irvine, 1984) . Protein pho~phorylation(~) catalyzed by PK-C is implicated in regulating exocytotic processes (Nishizuka, 1984; Takai et al., 1984). Since the cortical granule reaction in mammalian eggs involves an exocytotic process, the role of mouse egg PK-C in the early events of the fertilization profess was examined (Endo et al., 1987 b, c ) . Treatment of eggs with biologically active phorbol diesters or a diacylglycerol, compounds that activate PK-C, inhibits both sperm penetration and fertilization (Endo et al., 1987c). Biologically inactive phorbol diesters, which do not activate PK-C, do not inhibit either sperm penetration or fertilization. This inhibition is due to an egg-induced modification of the zone pelf ucida, such that ZP2 is converted to ZP2f, while ZP3 retains its sperm receptor activity. This latter observation accounts for the finding that sperm binding is not reduced in eggs treated with PK-C activators. The inhibition of fertilization is due to the inability of ZP3 to induce a complete acrosome reaction, which was determined by using an assay that monitors of the progression of the acrosome reaction. The progression of capacitated, acrosome-intact sperm to acrosome- reacted sperm can be monitored by changes in staining patterns using-the fluorescent probe chlortetracycline (Saling and Storey, 1979b). Three major fluorescent staining patterns have been characterized with this assay. The B-pattern of capacitated sperm is correlated with acrosome intact sperm, as assessed by transmission electron microscopy (Flonman and Storey, 1982). The S-pattern represents an intermediate stage and appears prior to completion of the acrosome reaction. This pattern correlates with loss of the ability of sperm to maintain a transmembrane pH gradient (Lee and Storey, 1985). The AR-pattern corresponds to acrosome-reacted sperm, as determined by transmission electron microscopy (Saling et al., 1979b). ZP3 isolated from untreated eggs possesses the ability to induce the B to S to AR transitions. In contrast, ZP3 isolated from eggs treated with PK-C activators can induce the B to S transition, but not the S to AR transition. Accordingly, sperm treated with ZP3 isolated from these eggs treated with PK-C activators accumulate in the S pattern. Although previous studies indicated that ZP3 isolated from 2- cell embryos does not induce the acrosome reaction, the methods used in these studies assayed an end point, i.ee, the completion of the acrosome reaction, and would not detect intermediates in this process (Bleil and Wassarman, 1983). When tested with the chlortetracycline assay, sperm 269 -

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incubated with ZP3 isolated from 2-cell embryos do not even undergo the B to S transition. Sperm arrested in the S pattern by zonae isolated from PK-C activator-treated eggs can be induced to undergo the S to AR transition by treatment with either ionophore A 23187 or solubilized zonae from untreated eggs; solubilized zonae from 2-cell embryos do not induce this transition (Kligman, Storey, and Kopf, unpublished results). Thus, the S pattern in which the sperm accumulate by treatment with ZP3 isolated from PK-C activator-treated eggs represents an intermediate stage of the acro~ome reaction that can be induced to undergo subsequent steps and complete the acrosome reaction. ~ i , ~ . These studies demonstrate that treatment of eggs- with activators of protein kinase C results in an egg-induced modification of the zone such that there is a dissociation of the spe,`~-receptor activity from the acro~ome reaction-inducing activity of- ZP3. In contrast, fertilization results in the loss of both-the sperm receptor and acrosome reaction-inducing activities of ZP3. Presumably, differences in ZP3 obtained from untreated and phorbol diester-treated eggs reflect those portions of the molecules that participate in the acrosome reaction. Studies examining biochemical differences in ZP3 obtained from untreated and phorbol diester-treated eggs, therefore, should. facilitate analysis of those portions of ZP3 that are involved in inducing the acrosome reaction. The use of zonae from phorbol diester- t rested eggs should also facilitate studies on the mechanism(s) of the acrosome reaction, since it is now possible to use these zonae to study independently the B to S transition from the S to AR transition. Such an experimental system may be of great value in determining the biochemical correlates of the S pattern, which has characteristics of an intermediate stage prior to the completion- of the acrosome reaction. The effects of IP3 microinjected into mouse eggs have also been examined with respect to its effect on egg activation, zone modifications, and sperm receptor activities of the zone (unpublished results ) . A low percentage ( 15% ) of eggs microin jected with IP3 at a final concentration of 4 AM become activated, as evidenced by second polar body emission within 1.5 h. In contrast, -eggs injected with the vehicle do not activate. Zonae from the IP3-microinjected eggs that activate always show the loss of BP2 and its conversion to ZP2f. Although IP3-injected eggs do not activate, 85% of these eggs reveal a conversion of ZP2 to ZP2f. In about 70% of these cases the conversion is total and in the other 30% about 50% of the ZE2 is modified. Of the vehicle injected eggs, about 30% reveal a modification of ZP2, which is usually only partially modified. The half-maximal concentration of IP3 necessary to elicit the change in ZP3 is about 5 no, and this corresponds well to that necessary to induce the cortical granule-snediated elevation of the fertilization envelope in sea urchin eggs, as well as calcium release f ram intracellular stores in other systems (Whitaker and Irvine, 1984; Swann and Whitaker, 198 6) . Extracellular calcium is not required for mouse eggs injected with IP3 to display the ZP2 modification, and this is consistent with release of calcium from intracellular stores in the egg. Microin jection of either I(1,4)P2, I(2,4,5)P3, or I(1,3,4)P3, each of which does not release intracellular calcium, to a final concentration of 4 AM fails to induce a modification of ZP2. Moreover, microinjection of inositol 1, 3, 4,5-tetrakisphosphate, which is implicated in regulating 270 -

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calcium channels in the plasma membrane of eggs of lower species (Irvine and Moor, 1986, 1987),.does.not induce a conversion of ZP2 to ZP2f. IP3 in jection of mouse eggs may also modify ZP3, since IP3- in jected eggs bind fewer sperm than vehicle in jected eggs . The lower extent of binding could represent the inability of acrosome-reacted bound sperm to establish a secondary binding with ZP2, for the following reason: If ZP2 mediates the binding of acrosome-reacted sperm and ZP2f cannot interact with acrosome-reacted sperm, then although the sperm can bind to IP3-injected eggs and undergo the acrosome reaction, they cannot establish the secondary binding with ZP2, which has been modif fed to ZP2f. Thus, the bound sperm will dissociate. This explanation for the lower level of binding is made less likely since the sperm used in these experiments are treated with pertussis toxin, which prevents the spear` from undergoing the acrosome reaction (Endo et al., 1987a). Thus, their interaction with ZP2 is prevented and their interaction with the zone is restricted to ZP3. The lower level of sperm binding to IP3-injected eggs is therefore likely to be due to a reduced level of ZP3 sperm receptor activity. - Fertilization is associated with a characteristic set of changes in the pattern of protein synthesis. Although IP3 injected eggs display a modification in ZP2, they do not reveal the changes in the pattern of protein synthesis associated with fertilization. This is consistent with the very low level of egg activation, as assessed by pronuclear formation. Thus, although IP3 can apparently bring about an egg-induced modification of the zone, it does not elicit a full egg activation response, but rather a subprogram of events that occurs during egg activation. The mechanism of IP3-induced modifications of the zone is likely to be via an IP3-stimulated release of intracellular calcium, which somehow is involved in cortical granule exocytosis. Periodic increases in intracellular free calcium concentration occur following fertilization in hamster eggs (Miyazaki et al., 1986)~. Injection of IP3 to a final concentration of 80 nM induces a transient increase in intracellular free calcium that spreads over the entire egg within a second (Miyazaki, 1988). In addition, IP3 also stimulates a hyperpolarization of the membrane potential. These changes are very similar to those that normally occur after fertilization. G proteins are a family of guanine nucleotide binding proteins that are activated by GTP and serve to couple various extracellular signals to their intercellular effectors, which can be involved in generation of second messengers (Oilman, 1987 and references therein; Neer and Clapham, 1988, and references therein). Generation of an 1,2- diacylglycerol and IP3 is believed to be mediated by a G protein stimulated phospholipase C, and in sea urchins, fertilization is . correlated with a rapid turnover of phosphatidylinositol bisphosphate (Turner et al. , 1984). In addition, activation of G.proteins is believed to result in cortical granule exocytosis in sea.urchins (Turner et al., 1986). If mammalian sperm initiate a signal transduction sequence that is mediated by-a G protein, a cascade of fertilization-induced events should be triggered by microinjected GTP. Consistent with this hypothesis is the observation that microinjection of GTPgS into hamster - 271 ~

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oocytes also triggers transient increases in intracellular free calcium and hyperpolarization of the membrane potential, and GDPbS inhibited this GTPgS-induced response (Myasaki, 1988). Although these data are consistent with a fertilization-induced, G protein coupled, PK-C/IP3 mediated stimulation of a cortical granule reaction, which in turn effects the modifications of the zone that constitute the zone block to polyspermy, experiments have not yet correlated the biological and biochemical changes with changes in the number of cortical granules in response to there agents. [ens culinaris agglutinin is a lectin that apparently stains cortical granules, and accordingly provides a convenient marker to monitor the cortical granule reaction (Cherr et al., 1988). Other lectins may also provide potential markers for cortical granules (Lee et al., 1988). Results of recent experiments indicate that fertilized or ionophore-activated eggs have dramatically reduced numbers of there lectin staining granules (Ducibella, personal communication). Moreover, eggs treated with PK-C activators have a partially reduced number of these granules (Ducibella, Kopf, and Schultz, unpublished observations). Future studies are required to ascertain effects of IP3 and GTP on the release of these granules, as well BS characterizing the cortical granules with respect to their enzymatic contents. The development of micro-assay procedures will facilitate these studies, which may reveal the types of enzymes involved in the modification(s) of both ZP2 and ZP3. In addition, the development of cortical granule probes may be used to study cortical granule biogenesis and to reveal if there is a heterogeneity in the cortical granule population. Electron microscopy studies reveal the existence of light and dark staining populations of cortical granules in mouse eggs (Nicosia et al., 1977). This may reflect granules at different stages of packaging their internal contents, or could ref. lect heterogeneity of mature granules . Such heterogeneity exists in lower species. For example, in sea urchins, although the cortical granules appear homogeneous in the transmission electron microscope, only about 20% of them contain a cortical granule antigen, as determined by immunoelectronmicroscopy (Anstrom et al., 1988). The partial modification of ZP3 coupled with the partial reduction in the number of LCA-staining granules in response to PK-C activators is consistent with cortical granule heterogeneity in mammalian eggs, but further studies are clearly required to support this conjecture. UNRESOLVED QUESTIONS AND FUTURE DIRECTIONS Little is known regarding the molecular basis for the acquisition of meiotic competence. In the future, subtraction hybridization of cDNA libraries generated from meiotically competent and incompetent oocytes may allow the cloning of cDNAs specific to meiotically competent oocytes. Analysis of such clones may provide insights regarding how oocytes develop and differentiate. This may be of extreme importance, since an understanding of factors involved in acquisition of meiotic competence may lead to improved systems that support oocyte growth and acquisition of meiotic competence in vitro. Although protein phosphorylation is implicated in regulating Asiatic maturation, we know very little concerning the sequence of events that comprise meiotic maturation and the proteins involved in thin process. Meiotic maturation entails a G2 to M transition in the 279 -

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cell cycle, and therefore is a problem in cell cycle regulation. Specific cellular oncogenes, some of which are protein kineses, are Implicated in cell cycle regulation in other systems. Interestingly, recent studies show changes in the temporal patterns of expression of specific oncogenes, e.g., c-mos, which is a serine/threonine protein kinase, during oocyte growth and maturation (Propst et al., 1988). Future studies addressing functional aspects of these gene products, coupled with the recent purification and identification of maturation promoting factor as a homolog of the fission yeast cell cycle control protein encoded by the cdc2+ gene will undoubtedly shed light on the process of meiotic maturation at the molecular level and define more clearly the role of protein phosphorylation in regulating this process. Again, an understanding of the molecular mechanisms of the process of meiotic maturation may lead to Improvements in culture systems that support maturation in vitro. Not all oocytes that mature into eggs during maturation are capable of being fertilized and giving rise to normal development. This type of maturation is teemed 'tcytopla~mic" maturation. An example of cytoplasmic maturation was discussed above, namely, the acquired ability of the egg during maturation to Recondense the sperm nucleus. We still do not understand at the molecular level what constitutes "cytoplasmic" maturation and its regulation. A major problem in medically assisted conception is to identify eggs that are capable of being fertilized and giving rise to normal development. Although matured eggs may appear similar on morphological grounds, they are likely to possess profound differences with respect to their state of cytoplasmic maturation. These differences may compromise their ability to be fertilized and develop. The sequence of events from sperm fusion with the egg's plasma membrane to egg activation and the zone polyspermy block is still not well defined. Although it is likely that this process is mediated by a G protein(s), the biochemical nature of the sperm receptor on the egg's plasma membrane, the molecular identity of the G protein(s) involved, and the coupling mechanism of the putative receptor with the G protein(s) and the consequence of this interaction are still unknown. For example, does fertilization of mammalian eggs result in activation of a phospholipase C with the subsequent production of diacylglycerol and IP3? Analysis at the biochemical and molecular levels should provide basic and essential information regarding these issues. Such an understanding may lead to refinement of conditions that foster fertilization. The molecular basis for the mammalian cortical granule reaction in response to fertilization is still unknown. Although studier in lower species and initial studies in the mouse and hamster implicate calcium, much more work is required to understand this exocytotic event at the molecular level. The biochemical composition of mammalian cortical granules is still undefined and how the contents of these granules modify the zone to elicit the zone block to polyspe,~`y remains to be determined. Moreover, the actual biochemical changes that occur in the zone proteins following egg activation needs to be elucidated. Work directed at isolating and characterizing mammalian cortical granules will help resolve many of these issues. In addition, studies that focus on first determining the biochemical identity of determinants on ZP3 and ZP2 that are involved in sperm binding and the acrosome reaction will pave the way for subsequent studies that address the nature of the

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changes in these proteins that result in loss of these biological activities associated with these zone proteins. Results of such studies should establish the biochemical basis for the block to polyspenmy. Such knowledge may result in in vitro fertilization culture systems in which the incidence of polyspenmy is further reduced. ACKNOWLEDGMENTS Research performed by the authors was supported by grants from the- National Institutes of Health (HD 180604 to R.M.S. and 19096 to G.S.K. and HD 22732 to G.S.K. and R.M.S.) and grants from the Mellon Foundation (G.S.K.) and the University Research Fund (R.M.S.). S.K. was supported by the Rockefeller Foundation. G.S.K. and R.M.S. would like to thank Philip Hugo for assistance with some of the experiments described above and Jeff Bleil for stimulating discussions about the role of ZP2 in sperm binding. - 274

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