TOM DUCIBELLA, 2 ' 3 SHIGEAKI KURASAWA, 4 PAUL DUFFY, 3 GREGORY S. KOPF, 4 and RICHARD M. SCHULTZ 5

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1 BIOLOGY OF REPRODUCTION 48, (1993) Regulation of the Polyspermy Block in the Mouse Egg: Maturation-Dependent Differences in Cortical Granule Exocytosis and Zona Pellucida Modifications Induced by Inositol 1,4,5-Trisphosphate and an Activator of Protein Kinase C' TOM DUCIBELLA, 2 ' 3 SHIGEAKI KURASAWA, 4 PAUL DUFFY, 3 GREGORY S. KOPF, 4 and RICHARD M. SCHULTZ 5 Department of Obstetrics and Gynecology, 3 Division of Reproductive Endocrinology New England Medical Center, and Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 2111 Division of Reproductive Biology, 4 Department of Obstetrics and Gynecology, School of Medicine and Department of Biology, 5 University of Pennsylvania, Philadelphia, Pennsylvania 1914 ABSTRACT Germinal vesicle (GV)-intact fully grown mouse oocytes do not undergo cortical granule (CG) exocytosis in response to A23187 treatment, whereas metaphase II (MII)-arrested eggs do. This differential response may reflect the development of the ability of the egg to undergo CG exocytosis, which is responsible for the biochemical modification of the glycoprotein ZP2 in the zona pellucida. Accordingly, we compared in these two stages the ability of 12-O-tetradecanoyl phorbol 13-acetate (TPA) or inositol 1,4,5-trisphosphate (IP3) to promote CG exocytosis and/or the ZP2 to ZP2f conversion; these agents are known to stimulate early events of mouse egg activation. TPA (1 ng/ml) treatment for 6 and 12 min resulted in a 25% and 52% CG loss in GV-intact oocytes and a 38% and 76% loss in MII eggs, respectively; fertilization resulted in a CG loss of -7-8%. Although a similar extent of ZP2 to ZP2f conversion was observed in oocytes and eggs after a 12-min TPA treatment (-7-8% ), a greater extent of conversion was observed in oocytes after a 6-min treatment (8% for oocytes, 5% for eggs). Microinjection of IP 3 (final concentration 1 tm) into MII eggs resulted in an extent of ZP2 conversion similar to that observed following fertilization, whereas little conversion occurred in GV-intact oocytes similarly injected. These results indicate that a protein kinase C sensitivity develops prior to meiotic maturation, whereas responsiveness to IP 3 develops after maturation has resumed. We propose that the regulatory mechanism involving an IP,-mediated calcium release is deficient in GV-stage oocytes. INTRODUCTION Fertilization in many species causes the release of egg cortical granules (CGs) whose contents modify the egg's extracellular coat, which is called the zona pellucida (ZP) in mammals. In the mouse, these changes in the ZP constitute a block to polyspermy ([1] and references therein). The mouse ZP is composed of three glycoproteins, ZP1, ZP2, and ZP3. ZP3, which binds acrosome-intact sperm and induces the acrosome reaction of those bound sperm, is modified to a form called ZP3f after fertilization and loses both of these biological properties. ZP2, which binds acrosome-reacted sperm, is modified to a form called ZP2f after fertilization [2] and no longer binds to acrosome-reacted sperm. The modification of ZP2 to ZP2f is most likely due to the release of a CG-associated protease and is detectable by a change in electrophoretic mobility; the ZP3 to ZP3f conversion does not result in a detectable change in electrophoretic mobility. These changes result in the inability of sperm to penetrate the ZP and constitute the ZP block to polyspermy. Accepted January 18, Received April 4, 'This research was supported by grants from the NIH (HD to G.S.K. and RM.S., HD 6274 to G.S.K, HD to R.M.S., and HD to T.D.). S.K was supported by the Rockefeller Foundation. 2Correspondence: Tom Ducibella, New England Medical Center, Box 61, 75 Washington St., Boston, MA FAX: (617) The ability of eggs to undergo CG exocytosis is critical for the establishment of the zona block to polyspermy. Fertilization of germinal vesicle (GV)-intact oocytes is frequently associated with polyspermy, which may be due to the inability to undergo CG exocytosis and mount a zona block (see references in [31). Consistent with this observation is that ionophore A23187 treatment of metaphase II (MII) mouse eggs results in a mean CG loss of 71% and the ZP modifications [3-5]; the extents of these changes are similar to those that normally occur in response to sperm. In contrast, A23187 treatment of fully grown GV-intact mouse oocytes does not result in a detectable loss of CGs [3, 4]. Treatment of metaphase I (MI) eggs with A23187, however, results in a mean CG loss of 28% [3]. These results suggest that meiotic maturation is accompanied by the development of competence to undergo CG exocytosis. Moreover, these observations emphasize the importance of ascertaining when oocytes become capable of undergoing CG exocytosis for timing insemination during in vitro fertilization (IVF) procedures as well as understanding the temporal and spatial development of pathways.involved in stimulating the cortical reaction. This acquisition of competence to undergo CG exocytosis could be due to either direct mechanisms (e.g., changes in the properties of the CGs to undergo exocytosis) or indirect mechanisms (e.g., development of competent signal transduction pathways required for CG exocytosis). In the mouse egg, G-protein activation, as well as second messen Downloaded from on 7 July 218

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3 REGULATION AND MATURATION OF THE BLOCK TO POLYSPERMY 1253 gers produced by the hydrolysis of polyphosphoinositides-inositol 1,4,5 trisphosphate (IP 3 ) and diacylglycerol-can induce the fertilization-associated modifications of the ZP [6-9]. These modifications are most likely due to CG exocytosis because IP 3 results in the release of intracellular calcium, which is a well-known activator of CG release in eggs [1], and 'protein kinase C (PKC) activation results in exocytosis in other cell types. CG exocytosis is therefore likely to be mediated by signal transduction mechanisms known to regulate exocytosis in other cell types (reviewed in [11]). The purpose of the present study was to determine whether there is a failure in signaling mechanisms that could account for the incompetence of GV-intact oocytes to undergo CG exocytosis in response to ionophore. The experiments examined whether maturation-associated changes occur in the ability of oocytes and eggs to respond to an activator of PKC or to microinjected IP 3 by undergoing CG exocytosis and/or the ZP2 to ZP2f conversion. We report that treatment of either GV-intact oocytes or MII eggs with a biologically active phorbol diester resulted in CG release and a conversion of ZP2 to ZP2f; the extent of this conversion was similar to that observed following fertilization. Although MII eggs microinjected with IP 3 displayed a ZP2 conversion to an extent similar to that observed following fertilization, GV-intact oocytes similarly microinjected were refractory and showed little, if any, ZP2 conversion. MATERIALS AND METHODS Collection and Culture of Oocytes Fully grown GV-intact oocytes and ovulated MII eggs were obtained from either CD-1 (Charles River Breeding Labs., Wilmington, MA) or CF-1 (Harlan Indust., Indianapolis, IN) mice primed with gonadotropins, and the cumulus was removed as previously described [12]. Eggs and oocytes were collected in Earle's Balanced Salt Solution (without bicarbonate) containing 25 mm HEPES buffer, ph 7.3, and.3% polyvinylpyrrolidone (PVP) [3] and cultured in the same salt solution with bicarbonate (25 mm), pyruvate (1 plg/ml), gentamicin (1,ug/ml), and.3% PVP (37 C, 5% CO 2 ) during treatments. GV breakdown was inhibited by including.2 mm 3-isobutyl-l-methyl xanthine (IBMX) in the culture medium [13]. Treatment of Oocytes and Eggs with Phorbol Diesters Oocyte and egg treatments were conducted with the PKC agonist 12-O-tetradecanoyl phorbol 13-acetate (TPA; Sigma FIG. 1. Representative fluorescence micrographs of LCA-stained CGs after phorbol diester treatments. All micrographs were taken of the cortex and do not include CGs below the cortex due to the depth of field of the 1x, N.A. 1.4 objective. GV-intact oocytes after 6 min in 4-PDD (A) or TPA (B). MII eggs after 6 min in 4a-PDD (C) or TPA (D). The phorbol diester concentration was 1 ng/ml. In MII eggs, the CG-free domain appears in the lower left corner in (C) and (D). Bar = 1 pim. Chemical Co., St. Louis, MO) or the biologically inactive phorbol diester 4a-phorbol 12,13-didecanoate (4a-PDD; Sigma). Treatment with 4a-PDD resulted in no loss of CGs (data not shown) or ZP2 conversion [7] when treated oocytes or eggs were compared to untreated oocytes or eggs. These compounds were dissolved in dimethylsulfoxide (1 mg/ml), aliquoted for a single use, stored at -25 C, thawed just prior to use, and vortexed immediately upon dilution into culture medium. For CG studies, CD-1 or CF-1 GV-intact oocytes and MII eggs were collected, combined into a single group, and then split into two equivalent groups containing both GV-intact oocytes and MII eggs. One group was treated with TPA and the other with 4a-PDD. In subsequent staining procedures described below, GV-intact oocytes and MII eggs in each group were co-incubated in the same solution at each step of the procedure. GV-intact oocytes and MII eggs were distinguished by nuclear staining. For those experiments analyzing the ZP2 to ZP2f conversion, CF-1 GV-intact oocytes and MII eggs were separated prior to ZP isolation (see below). Staining and Quantification of Cortical Granules Cortical granules were stained with biotin-conjugated Lens culinaris agglutinin (LCA) [14], developed with Texas Red streptavidin as described previously [3], and then visualized by fluorescence microscopy with a Nikon 1x objective (N.A. = 1.4). The CG density for each oocyte was computed by image analysis based on the same principles as manual counting described previously [3, 12]. Fluorescent 35-mm slide images were mounted on a slide box, captured by a Panasonic CD-5 camera with a 6-mm AF micro Nikon lens, and then transferred to an Apple Macintosh IIx computer with Image Analyst software (Automatix, Inc., Billerica, MA). An area of cortex, usually 6 pm 2, was analyzed from an Ektachrome 4 slide. In MII eggs, CG density determinations were made from the region within the large CG domain. The area of this domain was calculated (not shown) on the basis of its size relative to the area of the domain of a hemisphere (= 5% of the egg cortex) in the same egg. Chromatin patterns indicating the stage of meiotic maturation were visualized by co-staining with 1,ug/ml 4,6- diamidino-2-phenyl indole for 2-4 min. Calculation of Cortical Granule Loss For each time point, the CG densities and the corresponding CG-occupied areas in the cortex of control (4a- PDD) and TPA-treated GV-intact oocytes and MII eggs were determined. For each stage, the percentage of CG loss from the cortex was calculated by comparing the TPA group to the corresponding 4a-PDD group: % Loss = [1-(TPA')/ (4oa-PDD')] X 1, in which TPA' is the product of the CG density and CG-domain area in eggs treated with TPA, and 4ct-PDD' is the control value similarly calculated. At 1 ng/ Downloaded from on 7 July 218

4 1254 DUCIBELLA ET AL. 4 1 x 3 o E en _J 2 o * 1 8 m on 4 N o o o o ' 6' 12' I GV I 12' FIG. 2. Relationship between time of TPA treatment and loss of CGs from the cortex and ZP2 conversion. For the 12-min groups, GV-intact oocytes and MII eggs were removed from phorbol diester-containing media after 6 min. At each time point, more than 25 GV-intact oocytes and MII eggs were co-incubated in the same well with TPA or 4a-PDD in CG experiments. The mean number of CGs lost from the cortex per GV-intact oocyte or MII egg was calculated from the percent CGs lost after treatment (see Fig. 3) as described in Materials and Methods. Thus, at zero time, the number (#) of CGs lost also would be zero. Data are expressed as the mean + SEM in this and subsequent figures. ml, the data for each time point represent at least two experiments and a total of at least 2 oocytes per experimental or control group. The number of CGs lost from the cortex was determined from the mean percentage of CG loss and mean total number of CGs in controls. GV-intact oocytes have a significantly higher mean total number of CGs than MII eggs [3, 12]. The mean percentages of CG release at different stages were compared by means of the pooled Student's t test. IP3 Microinjection IP 3 was microinjected to a final concentration of 1 M as previously described [6]. IBMX was included in the medium when GV-intact oocytes were microinjectioned. Quantification of the ZP2 to ZP2f Conversion ZP were isolated from GV-intact oocytes or MII eggs that were either treated with TPA (or 4ot-PDD) or microinjected with IP 3 as previously described [6]. The ZP were radioiodinated and processed for ZP2 to ZP2f conversion by twodimensional reduction electrophoresis as previously described [3], and the extent of the ZP2 conversion was quantified as previously described [15]. I ' 6' MII Time, min. FIG. 3. Relationship between time of TPA (1 ng/ml) treatment and percentage of loss of CGs from the cortex and ZP2 conversion. The percentage of CG loss was calculated as described in Figure 4; for each time point more than 2 MII eggs were analyzed. RESULTS Response of MII Eggs to Phorbol Diesters Initial experiments were conducted to determine whether TPA results in both the ZP2 conversion and CG loss, since, although this is assumed to be the case, this correlation has not been previously established, and furthermore, if such a CG loss was observed, to determine whether its extent was similar to that following fertilization. Eggs from CD-1 or CF-1 mice responded similarly to TPA treatment. Incubation of MII eggs for 6 min with 1 ng/ml of TPA resulted in significant (p <.1) decreases in the mean CG number per egg: CD-1, 25%; CF-1, 33%. In addition, a timedependent increase in both the loss of CGs from the cortex (Fig. 1, C and D) and the extent of the ZP2 conversion was observed (Fig. 2). At each of the two time points examined, the percentage of the ZP2 to ZP2f conversion was linearly related to the number of the CGs lost from the cortex. By 12 min, both the extent of the ZP2 conversion [2] and the loss of CGs [5] were similar to those in eggs observed following fertilization. TPA-induced CG loss was associated with a reduced CG density and/or smaller area of the CG-occupied domain (Fig. ID). The intermediate percentages of ZP2 conversion and CG loss at 6 min were not due to a sub-saturating concentration of TPA, since similar results were observed at 1 ng/ ml of TPA (Fig. 3). Downloaded from on 7 July 218

5 REGULATION AND MATURATION OF THE BLOCK TO POLYSPERMY x a) 1t: o E o () ( T T1 cm a. N o c- o (D C T 2 -I 2- GV MII bu' 1ZU' 1; U' I GV II MII I FIG. 5. The effect of microinjected IP 3 (1 LpM, final concentration) on the percentage of ZP2 to ZP2f conversion in GV-intact oocytes and MII eggs. For each bar, the experiment was performed three times; within each experimental group, at least 5 individual ZP were analyzed. Solid bars, vehicle-injected; open bars, IP 3 -injected. FIG. 4. Relationship between time of TPA treatment and percentage of loss of CGs from the cortex. For the 12-min groups, GV-intact oocytes and MII eggs were removed from phorbol diester-containing media after 6 min. For each bar, more than 25 cells were analyzed. The percentage of CG loss was calculated by comparing the mean number of CGs in the TPA-treated group to that in the 4a-PDD-treated control group at the same time point (see Materials and Methods). Unlike the number of CGs lost (Fig. 2), the percentage loss at each time point is different for GV-intact oocytes and MII eggs as explained in Results. Response of GV-lntact Oocytes to TPA To determine whether the development of competence to undergo CG release [3,4] is associated with a change in the responsiveness of the CGs to second messengers implicated in CG exocytosis, we examined the effect of TPA on both CG loss and the extent of the ZP2 conversion in GV-intact oocytes. Treatment of CD-1 or CF-1 GV-intact oocytes with 1 ng/ ml of TPA for 6 min resulted in a similar mean CG loss per oocyte, 38% and 42%, respectively; this loss was similar to that observed in MII eggs treated with TPA for 6 min. While this represents only a partial loss of CGs, the extent of the ZP2 conversion was similar to that observed following fertilization (Fig. 1, A and B, and 2). There was a further loss of oocyte CGs by 12 min (Fig. 2), and again this loss was similar to that observed in MII eggs treated with TPA. It should be noted that the limited CG loss in TPA-treated oocytes appeared to be global and did not result in the formation of a CG-free domain (Fig. 1, A and B). Moreover, there was no change in the area of the CG domain. Although the numbers of CGs lost from the cortex in GV-intact oocytes and MII eggs were similar after TPA treatment (Fig. 2), the percentage decrease in CG number in the cortex did not appear to be the same in the two treated groups. At both 6 and 12 min, the percentage of CG loss from the cortex appeared to be greater in MII eggs than GV-intact oocytes (Fig. 4). The higher percentage of CG loss in MII eggs than in GV-intact oocytes was offset by the smaller number of CGs per cell (in the cortex) in MII eggs; this resulted in the similar numbers of CGs lost per cell for both stages portrayed in Figure 2. Response of GV-Intact Oocytes and MII Eggs to IP 3 As mentioned above, treatment of eggs with A23187 results in both CG exocytosis and the ZP2 conversion, whereas A23187 treatment of GV-intact oocytes does not [3,4]. In order to examine the effect of a physiological regulator of intracellular calcium release, the effect of microinjected IP 3 on the ZP2 conversion was examined in both GV-intact oocytes and eggs. When eggs were microinjected with IP 3 at a final concentration of 1!zM, the extent of conversion of ZP2 was similar to that observed following fertilization (Fig. 5) [6]. In contrast, GV-intact oocytes microinjected under the same conditions did not display a ZP2 conversion (Fig. 5). DISCUSSION The development of the intracellular signaling pathways involved in egg activation is an important process that oc- Downloaded from on 7 July 218

6 1256 DUCIBELLA ET AL. curs during meiotic maturation. The development of such signaling pathways also has important ramifications for in vitro fertilization programs because eggs are often collected before the completion of maturation [16]. The present work extends previous studies [3, 4] by demonstrating that the inability of GV-intact oocytes to release CGs in response to A23187 is probably not due to an inability of CGs themselves to undergo release-tpa stimulates CG exocytosis and the ZP2 conversion-but rather to an immaturity in the Ca2+-dependent pathways that are involved in CG exocytosis. In comparison with MII mouse eggs, GV-intact oocytes are deficient in their ability to undergo CG exocytosis in response to microinjected IP 3. In contrast, both GV-intact oocytes and MII eggs are similarly competent to respond to TPA in terms of the extent of the ZP2 conversion and number of CGs released. These data suggest that the response to IP 3 is dependent on oocyte maturation, whereas the response to PKC activators is independent of maturation. Because IP 3 is a physiological mediator of intracellular calcium release in vertebrate eggs [17,18], the observed increased incidence of polyspermy in immature oocytes in both animal research and clinical IVF programs (see Discussion in [3]) may be due, at least in part, to their inability to release sufficient amounts of intracellular calcium in response to IP 3. Treatment of GV-intact oocytes with TPA results in a partial CG loss (25-5%) that is accompanied by the ZP2 to ZP2f conversion whose extent (> 8%) is similar to that observed following fertilization. Oocyte maturation in vitro in the absence of serum also results in a partial CG loss (3-4%), but the extent of the ZP2 to ZP2f conversion is less than that observed following fertilization [3]; serum contains a component(s) that does not inhibit CG release but does inhibit the ZP modifications [3]. A possible explanation for this difference may be related to the kinetics of CG release from the cortex. During oocyte maturation, a slow and continual release of CGs occurs over 5-1 h. In contrast, a similar extent of CG release from oocytes in response to TPA occurs between 1 and 2 h. Since the protease thought to mediate the ZP2 to ZP2f conversion is labile [5], a slow release of CGs may be insufficient to provide a critical concentration of active protease required to mediate a complete ZP2 to ZP2f conversion. In addition to the kinetics of CG release, differences in the spatial relationship between the oocyte/egg plasma membrane and the ZP may account for the higher extent of ZP2 conversion in GV-intact oocytes. In comparison to MII eggs, GV-intact oocytes have little or no perivitelline space; thus, the released CG contents from GV-intact oocytes would come into contact with the ZP more rapidly and at a higher concentration due to less dilution by perivitelline fluid. There are several possible explanations for the differential sensitivity of GV-intact oocytes and MII eggs to IP 3. The first possibility may be that IP 3 does not release sufficient intracellular calcium in GV-intact oocytes. Although exogenous IP 3 can release intracellular calcium in GV-intact mouse oocytes [19], there may be insufficient calcium to cause CG release. Consistent with this interpretation are the findings that A23187 fails to elicit exocytosis in GV-intact oocytes [3,4] and that a four-fold increase in the amount of internally stored calcium occurs between the GV and MII stages [2]. A second possibility may be related to maturation-associated changes in the spatial organization of the calcium stores that appear to be important in many secretory cells [21]. Although the GV-intact oocyte may have sufficient calcium stores, these stores may be inappropriately localized, e.g., not present in the oocyte cortex. Consistent with this possibility is the fact that a spatial and temporal increase occurs in putative calcium-containing organelles-smooth membrane and endoplasmic reticulum vesicles-in the cortex during oocyte maturation in several species [22-26]; these changes are associated with changes in the cytoskeleton [27]. Release of calcium by either IP 3 (this report; in the starfish [28]) or ionophore (in the mouse [3,4]; in the starfish [29]) from a spatially inappropriate store would account for the inability of these agents to stimulate CG exocytosis in GV-intact oocytes; this released calcium would be re-sequestered before it could act on regulatory systems that control CG exocytosis. This explanation may not apply to starfish oocytes and eggs because treatment with calcium-egta buffers, which should result in a uniform increase in intracellular calcium, results in only a partial CG exocytosis in oocytes but a complete exocytosis in eggs [29]. A third possibility is that an increase in the sensitivity of the IP 3 receptor occurs during oocyte maturation. For example, maximal IP 3 -stimulated CG exocytosis in starfish oocytes requires -1 3 higher IP 3 concentrations than those needed in eggs [28]. Phosphorylation of the brain IP 3 receptor by the camp-dependent protein kinase is associated with a decrease in its sensitivity to IP 3 [3]. The decreases in camp [31] and changes in protein phosphorylation [32] that accompany oocyte maturation in the mouse could result in a net dephosphorylation of the IP 3 receptor and hence an increased sensitivity to IP 3. The inability of A23187, however, to stimulate CG exocytosis in the GV-intact oocyte [3,4] minimizes this possibility, since this ionophore would be expected to release calcium from both IP 3 -dependent and -independent stores. A fourth possibility is that there are maturation-associated changes in regulatory components that modulate CG exocytosis. These changes would be independent of both spatial alterations in the calcium stores and the sensitivity of the calcium release mechanisms of those stores. The changes in the pattern of protein synthesis that occur during oocyte maturation [33] may provide the molecular basis for the "maturation" of the regulatory components involved in CG exocytosis. Downloaded from on 7 July 218

7 REGULATION AND MATURATION OF THE BLOCK TO POLYSPERMY 1257 In contrast to the differential sensitivity of CG exocytosis to IP 3 between GV-intact oocytes and MII eggs, no readily apparent differences in sensitivity are observed with TPA. PKC-catalyzed phosphorylation is implicated in CG exocytosis [7,8,34-36] and its effect on CG exocytosis appears to be distal to IP 3 -induced calcium rises [36]. The observation that TPA stimulates CG exocytosis in both oocytes and eggs indicates that the protein substrate(s) that are directly or indirectly regulated by PKC are present in the oocyte and can function to mediate CG exocytosis, and moreover, that the CGs present in the oocyte are competent to undergo CG exocytosis. At the concentration of TPA used in these and other studies, the PKC-induced CG exocytosis is unlikely to involve a rise in intercellular calcium [37, 38]. Taken together, the observation that IP 3 /A23187-mediated exocytosis is both calcium- and maturation-dependent, whereas PKC-stimulated exocytosis is calcium- and maturation-independent, suggests that maturation-associated acquisition of Ca2+-dependent regulatory mechanisms are proximal to the PKC step(s) involved in CG exocytosis. ACKNOWLEDGMENT The excellent technical support of Jan Prange in the CG studies is appreciated. REFERENCES 1. Wassarman PM. Profile of a mammalian sperm receptor. Development 199; 18: Bleil J, Beall CF, Wassarman P. Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zonapellucida glycoprotein, ZP2. Dev Biol 1981; 86: Ducibella T, Kurasawa S, Rangarajan S, Kopf GS, Schultz RM. Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with egg-induced modification of the zona pellucida. Dev Biol 199; 137: Ducibella T, Duffy P, Reindollar R, Su B. 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Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus /aevis. Cell Regulation 199 1: Ciapa B, Whitaker M. Two phases of inositol polyphosphate and diacylglycerol production at fertilization. FEBS Lett 1986; 195: Colonna R, Tatone C, Malgaroli A, Eusebi F, Mangia F. Effects of protein kinase C stimulation and free Ca 2+ rise in mammalian egg activation. Gamete Res 1989; 24: Downloaded from on 7 July 218