SNAP-25 is essential for cortical granule exocytosis in mouse eggs

Synaptosome-associated protein of 25 kDa (SNAP-25) has beenshown to play an important role inCa²⁺-dependent exocytosis inneurons and endocrine cells. During fertilization, sperm-egg fusioninduces cytosolic Ca²⁺mobilization and subsequentlyCa²⁺-dependent cortical granule(CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25is involved in this process. In this study, we determined theexpression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 weredetected in metaphase II (MII) mouse eggs by RT-PCR and immunoblotanalysis, respectively. Next, to determine the function of SNAP-25, weevaluated the change in CG exocytosis with a membrane dye,tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjectionof a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25in MII eggs. Sperm-induced CG exocytosis was significantly inhibited inthe BoNT/A-treated eggs. The inhibition was attenuated by coinjectionof SNAP-25. These results suggest that SNAP-25 may be involved inCa²⁺-dependent CG exocytosisduring fertilization in mouse eggs.

     

Translocation of the classic protein kinase C isoforms in porcine oocytes: implications of protein kinase C involvement in the regulation of nuclear activity and cortical granule exocytosis

Protein kinase C (PKC) is a family of Ser/Thr protein kinases categorized into three subfamilies: classical, novel, and atypical. The subcellular localization of classical PKCalpha, -betaI, and -gamma in the process of porcine oocyte maturation, fertilization, and parthenogenetic activation and their involvement in cortical granule (CG) exocytosis were investigated. The results of Western blot showed that PKCalpha, -betaI, and -gamma were expressed in the oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Confocal microscopy revealed that the three PKC isoforms were concentrated in the GV but evenly distributed in the cytoplasm of MII eggs. PKCalpha and -gamma were translocated to the plasma membrane soon after sperm penetration. cPKCs migrated into the pronucleus in fertilized eggs. Following treatment with a PKC activator, phorbol 12-myristate 13-acetate (PMA), CGs were released and PKCalpha and -gamma were translocated to the membrane. The CG exocytosis and PKC redistribution induced by PMA could be blocked by the PKC inhibitor staurosporine. Parthenogenetic stimulation with ionophore A23187 or electrical pulse also induced cPKC translocation and CG exocytosis. Eggs injected with PKCalpha isoform-specific antibody failed to undergo CG exocytosis after PMA treatment or fertilization. The results suggest that cPKCs, especially the alpha-isotype, regulate nuclear function and CG exocytosis in porcine eggs.     

Study of protein kinase C antagonists on cortical granule exocytosis and cell-cycle resumption in fertilized mouse eggs

Although pharmacological agonists of protein kinase C (PKC) stimulate some events of mammalian egg activation, including cortical granule (CG) exocytosis, it is not known if these events are dependent on PKC activation during the normal process of fertilization. In order to examine the potential role of PKC in CG exocytosis, this study investigated whether PKC agonists faithfully mimic CG release and whether PKC antagonists block fertilization-induced CG release in mature mouse eggs. Phorbol ester (TPA, 2.5 ng/ml) treatment resulted in an atypical pattern of CG release in which there was a greater net loss of CGs in the equatorial region of the egg than in the region opposite the spindle. This pattern also was in contrast to that during fertilization, in which CG release occurred randomly throughout the cortex. Fertilization experiments utilized two different PKC inhibitors, bisindolyl-maleimide (5 μM) and chelerytherine (0.8 μM), targeted to both the “conserved” substrate and ATP binding domains of PKC. Simultaneous use of both inhibitors at maximal concentrations (compatible with fertilization and above their IC₅₀S) resulted in no detectable inhibition of CG release in treated fertilized eggs compared to controls. In addition, no inhibition of anaphase onset was observed in treated fertilized eggs. Activity of the inhibitors was verified by demonstrating that they blocked the induction of CG loss by TPA. Moreover, 1 μM staurosporine, a potent but less specific antagonist of PKC, also did not block CG loss, whereas the metaphase-anaphase transition was temporarily inhibited. The results indicate that TPA does not faithfully mimic CG release in fertilized eggs, that a role for PKC in CG release at fertilization remains to be established, and that other calcium-dependent effectors may be involved in CG exocytosis. Mol Reprod Dev 46:216–226, 1997. © 1997 Wiley-Liss, Inc.     

Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation

The ability of parthenogenetically activated mouse eggs to establish a plasma membrane (PM) block to sperm penetration was studied. Zona-free eggs preloaded with Hoechst 33342 were activated by exposure to ethanol or OAG (1-oleoyl-2-acetyl-sn-glycerol) and inseminated after different periods. Eggs challenged with sperm at 30- or 60-min postactivation displayed a fertilization frequency significantly lower than that of control eggs. Conversely, when insemination was carried out at 120-min postactivation, the proportion of fertilized eggs was equivalent to that observed in the control group. Moreover, we report that when the eggs were induced to resume meiosis without any notable loss of CGs (egg exposure to OAG at 100 μM external Ca²⁺ or to heat shock), a normal ability to be penetrated was recorded at 30-min postactivation. Similar behaviour was exhibited by eggs that underwent a CG exocytosis close to that triggered by sperm in absence of nuclear activation (microinjection of inositol 1,4,5-trisphosphate into the egg at 1 μM cytosolic concentration). Present data support the conclusion that parthenogenetically activated mouse eggs are capable of a transitory PM block response that requires both CG exocytosis and meiosis resumption to occur. © 1994 Wiley-Liss, Inc.     

Phalloidin inhibits cortical granule exocytosis and ooplasmic segregation in loach eggs

Injections of phalloidin under the surface of loach eggs, followed by activation of the eggs in tap water, result in local inhibition of cortical granule (CG) exocytosis. Light and electron microscopy revealed that in the region where exocytosis is inhibited the thickness of the microfilamentous cortex (MC) separating CGs from the plasma membrane (PM) is increased significantly, and many CGs are detached and have moved away from the MC. Injections of phalloidin also inhibit ooplasmic segregation in fertilized eggs. The experiments suggest that in intact eggs the MC represents a physical barrier to CG exocytosis, and that interactions of the MC with the PM and CGs are crucial for the retention of CGs near the sites of fusion.     

Mechanistic studies of the plasma membrane block to polyspermy in mouse eggs

The mechanisms responsible for the plasma membrane associated block to polyspermy in mouse eggs were studied. Reinsemination experiments using zona-free eggs indicated that, after fertilization, the egg plasma membrane is altered such that sperm binding to the egg plasma membrane is blocked, except in the region of the second polar body. Activation of the egg with either ethanol or strontium chloride did not result in a block to polyspermic penetration, as artificially activated eggs displayed identical penetration levels as to nonactivated control eggs. The penetrability of activated eggs was not altered by the presence or absence of the zona pellucida during activation. Lectin staining for egg cortical granule material indicated that activation did cause cortical granule exocytosis; however, activated eggs remained penetrable. These data support the following conclusions: (1) an alteration in the ability of the egg plasma membrane to allow sperm adherence accounts for the block to polyspermy; (2) establishment of the plasma membrane block to polyspermy is sperm dependent, since artificial egg activation does not result in a block response; (3) the contents of the egg's cortical granules do not play a role in the establishment of the plasmalemma block response. © 1993 Wiley-Liss, Inc.     

蛋白激酶C在小鼠卵母细胞体外成熟和受精中的作用

蛋白激酶是一类重要的丝／苏氨酸蛋白激酶。本实验以小鼠为实验动物,研究了PKC在卵母细胞体外成熟、活化和受精中的可能作用,及两种PKC亚型在卵母细胞中的定位。PKC激活剂PMA可以阻止GV期卵母细胞在体外恢复减数分裂,该作用可被PKC抑制剂CalphostinC抵消,但不能被PLCγ抑制剂U73122或PKCδ专一性抑制剂Rottlerin所克服。Western印迹显示PKCα和βI在卵母细胞发育过程中恒量表达。激光共聚焦显微术研究发现,受精或受到活化刺激后PKCα转位到卵母细胞膜上,同时皮质颗粒排放,说明PKCα可能参与调节卵皮质反应。本实验首次在小鼠中研究了PLCγ与受精的关系,发现不存在PKC对PLCγ的正反馈调节。此外,本研究还对小鼠卵巢中对PKCα和βI进行了蛋白定位研究。     

Activation of protein kinase C induces cortical granule exocytosis in a Ca2+-independent manner, but not the resumption of cell cycle in porcine eggs

The effects of protein kinase C (PKC) activation on meiotic resumption and cortical granule (CG) exocytosis as well as its dependence on Ca²⁺ in porcine eggs matured in vitro were studied. Cortical granule release was judged by both confocal laser microscopy after the eggs were labeled with fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and electron microscopy. Meiotic resumption and pronuclear formation were observed after eggs were stained with acetic orcein. When eggs were treated with PKC activators, 1-oleyl-2-acetyl-glycerol (OAG) or phorbol 12-myristate 13-acetate (PMA), the pronuclear formation percentage was significantly lower than that of Ca²⁺ ionophore A23187-treated group, but not statistically different from that in negative control group (P > 0.05), and most of the eggs were still arrested at metaphase II stage, suggesting that PKC activation does not induce the resumption of meiosis and pronuclear formation. In contrast, PKC activation induced 89.1% to 100% of the eggs completely or partially released their CG in different groups, not statistically different from A23187-treated group, and this effect could be overcome by PKC inhibition. When the intracellular free Ca²⁺ was chelated with acetoxymethal ester form of 1,2-bis(0-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), and then treated with PMA or OAG in Ca²⁺-free medium, the proportions of eggs with CG release were 90.9% and 78.1%, respectively, not statistically different from the above-treated groups, suggesting that CG exocytosis induced by PKC activation is independent of Ca²⁺ rise. The results indicate that different events of porcine egg activation may be uncoupled from one another.     

Evidence for direct membrane retrieval following cortical granule exocytosis in Xenopus oocytes and eggs

Rapid exocytosis is typically followed by rapid resorption of exocytosed membrane; however, whether membrane retrieval occurs via indirect endocytosis of numerous small vesicles or direct resealing of the original, larger exocytotic vesicles is controversial. Here we show that cortical granule (CG) exocytosis in Xenopus oocytes and eggs is followed by rapid formation of endosomes as large as the CGs. Large endosomes are translucent, and their formation has the same developmental and pharmacological profile as CG exocytosis. Time course analyses show that large endosomes are not derived from small endosomes. Large endosome formation is triggered by stimuli that do not trigger increases in intracellular-free calcium and is insensitive to perturbation of microtubules by treatment with nocodazole. Perturbation of the f-actin cytoskeleton with latrunculin, however, sharply reduces large endosome formation. We conclude that CG membrane is directly retrieved in Xenopus oocytes and eggs and suggest that this retrieval is not directly dependent on an increase in intracellular-free calcium, but is dependent on the actin cytoskeleton.     

Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs

At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures.     

Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs

A cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca(2+) levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2(f) during formation of the new CGFD. Moreover, clamping intracellular Ca(2+) did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Incompetence of preovulatory mouse oocytes to undergo cortical granule exocytosis following induced calcium oscillations

Immature oocytes of many species are incompetent to undergo cortical granule (CG) exocytosis upon fertilization. In mouse eggs, CG exocytosis is dependent primarily on an inositol 1,4,5-trisphosphate (IP3)-mediated elevation of intracellular calcium ([Ca2+]i). While deficiencies upstream of [Ca2+]i release are known, this study examined whether downstream deficiencies also contribute to the incompetence of preovulatory mouse oocytes to release CGs. The experimental strategy was to bypass upstream deficiencies by inducing normal, fertilization-like [Ca2+]i oscillations in fully grown, germinal vesicle (GV) stage oocytes and determine if the extent of CG exocytosis was restored to levels observed in mature, metaphase II (MII)-stage eggs. Because IP3 does not stimulate a normal Ca2+ response in GV-stage oocytes, three alternate methods were used to induce oscillations: thimerosal treatment, electroporation, and sperm factor injection. Long-lasting oscillations from thimerosal treatment resulted in 64 and 10% mean CG release at the MII and GV stages, respectively (P < 0.001). Three electrical pulses induced mean [Ca2+]i elevations of approximately 730 and 650 nM in MII- and GV-stage oocytes, respectively, and 31% CG release in MII-stage eggs and 9% in GV-stage oocytes (P < 0.001). Sperm factor microinjection resulted in 86% CG release in MII-stage eggs, while similarly treated GV-stage oocytes exhibited < 1% CG release (P < 0.001). Taken together, these results demonstrate a deficiency downstream of [Ca2+]i release which is developmentally regulated in the 12 h prior to ovulation.     

Localization of cortical granule constituents before and after exocytosis in the hamster egg

Electrical activation of the hamster egg was used to study cortical granule constituents before and after exocytosis. The activated hamster eggs underwent cortical granule decondensation just prior to and at the time of exocytosis. Some of the cortical granules of aged, unactivated eggs underwent similar changes. FITC- and gold-conjugated Lens culinaris agglutinin (LCA) bound intensely to the surfaces of activated but not unactivated eggs. This labelling was associated with the microvilli. Permeabilized eggs exhibited discrete cortical labelling before activation, with a subsequent decrease following the cortical reaction. Gold-conjugated LCA specifically bound to cortical granules when incubated with thin sections. FITC-soybean trypsin inhibitor (SBTI) bound in discrete foci in the cortex of unactivated eggs. Following activation, cortical labelling by SBTI decreased. Aprotinin and benzamidine hydrochloride inhibited FITC-SBTI from binding to the egg cortex. Gold-avidin localization of biotin-SBTI in the electron microscope demonstrated that condensed cortical granules did not bind SBTI but decondensed or exocytosing granules did. This suggests that a cortical granule protease is exposed just prior to exocytosis. Activated eggs exhibited dramatic decreases in the number of hamster sperm penetrating the cytoplasm, suggesting that a plasma membrane block to polyspermy is temporally related to cortical granule exocytosis.     

Calcium ultrastructural localization in Xenopus laevis eggs following activation by pricking or by calcium ionophore A 23187

In the egg of Xenopus laevis a cortical network of smooth endoplasmic reticulum (SER) surrounds and interconnects each cortical granule (CG) (Campanella and Andreuccetti, '77). This network is a possible intracellular site of calcium storage to be called into action for CG exocytosis. In our experiments, Xenopus eggs, unfertilized or activated by pricking or by calcium ionophore A 23187, have been fixed in osmium-pyroantimonate for calcium localization. Our data show that deposits can be detected only in activated eggs. The calcium chelator edetate (EGTA) and x-ray microprobe analysis demonstrate that they contain calcium. Deposits are found on liposomes and on all intraovular cytomembranes, which therefore appear to be possible sites of calcium sequestration. In the case of ionophore-activated eggs, deposits are detectable independently of the presence of extracellular calcium. These data show that in Xenopus at activation an intracellular liberation of calcium occurs similar to that described in other species. Furthermore, the fact that antimony deposits are observed only after activation makes Xenopus eggs appropriate material in which to follow the temporal and spatial sequence of appearance of the deposits during the early stages of activation. Our results show that antimony deposits appear first in SER vesicles between the plasma membrane and CGs and then spread to the rest of the egg cytomembranes. These data corroborate our hypothesis that in Xenopus the cortical SER network is the first intracellular site where calcium is released at activation. The possible mechanism of calcium release and propagation along the egg cortex is discussed.     

Cortical granule exocytosis in hamster eggs requires microfilaments

Earlier work has demonstrated that hamster eggs that do not release a second polar body after fertilization in vitro lack a block to polyspermy (Stewart-Savage and Bavister, 1987: Gamete Res 18:333–338). Since polar body release requires microfilaments, the involvement of microfilaments in cortical granule exocytosis was examined. When hamster eggs were treated with cytochalsin B (CB) for 1 hr and then coincubated with sperm for 90 min, there was a dose-dependent increase in both the percentage of eggs with more than one sperm penetrating the zona pellucida and the mean number of sperm that penetrated the zona, with a maximum effect at 20 μg CB/ml (100% polypenetration, 3.0 ± 0.3 sperm/egg). Cytochalasin-treated eggs retained 85% of their cortical granules 55 min after insemination, as compared to unfertilized eggs. Longer time periods did not result in any further reduction. As seen with the scanning confocal microscope, an extensive microfilament network was present in the cortex of untreated eggs, with the cortical granules located within this cortical network. The cortical microfilament network was highly reduced in CB-treated eggs. When viewed with the electron microscope, the same number of cortical granules were located next to the plasma membrane in both cytochalasin-treated and untreated, unfertilized eggs. These data indicate that intact microfilaments are required for normal cortical granule exocytosis in the hamster egg, but the role of the microfilaments in exocytosis is unresolved. Mol. Reprod. Dev. 47:334–340, 1997. © 1997 Wiley-Liss, Inc.     

A monoclonal antibody that recognizes mammalian cortical granules and a 32-kilodalton protein in mouse eggs

The fertilization-induced exocytosis of egg cortical granules (CGs) is responsible for a block to polyspermy, crucial to the viability of many species. The contents of mammalian CGs have been an elusive target for analysis because of picogram quantities of CG proteins. By using media enriched in secreted CG contents from calcium ionophore-induced eggs as an immunogen, a monoclonal antibody was raised that immunolocalized to structures in the mouse egg cortex with all the hallmarks of CGs. These structures were the correct size, absent from the region over the metaphase II spindle, and greatly reduced after fertilization. Double-labeling experiments confirmed that the antibody recognized the same population of CGs as those recognized by Lens culinaris agglutinin. On Western blots, the antibody primarily recognized a 32-kDa protein (and secondarily one at approximately 25 kDa) in mouse eggs. Analysis of biotin-labeled secreted proteins from activated eggs confirmed that CGs release only a small number of major proteins (45, 34, 32, 28, and approximately 20 kDa by SDS-PAGE). We therefore propose that the 32-kDa protein identified by this antibody is likely to correspond to the 32-kDa protein released from activated eggs and that it may be involved in the block to polyspermy. These methods should make it possible to generate additional antibodies to study the structure of CG components as well as their roles in the polyspermy block and CG biogenesis.     

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.     

Mastoparan induces Ca2+-independent cortical granule exocytosis in sea urchin eggs

In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca(2+)-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca(2+) chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca(2+)-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca(2+)-independent late site of the signaling pathway that culminates in cortical granule discharge.