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Sea urchin eggs are surrounded by a network of extracellular matrix, consisting of the jelly coat (JC) and vitelline layer (VL). While the voluminous JC evokes acrosomal reaction in the approaching sperm, the tight VL ensheathing the plasma membrane of the subjacent microvilli is known to be the subcellular site where ‘sperm receptors’ reside. In this study, we have examined the roles of JC and VL at fertilization in a combinatorial approach utilizing two different pretreatments of the eggs: (i) incubation with dithiothreitol (DTT) in alkaline seawater to remove JC and VL, (ii) masking the egg extracellular matrix with a carbohydrate-binding protein concanavalin A (Con A). Surprisingly, the results showed that the DTT-denuded eggs still engulfed sperm at fertilization, even more effectively than intact eggs, as multiple sperm entered. On the other hand, Con A appeared to interfere with sperm entry in a dose-dependent manner and to delay the onset of the Ca2+ wave in intact eggs after the cortical Ca2+ release, representing sperm–egg fusion. This prolonged time lag in triggering the Ca2+ wave at fertilization was associated with compromised dynamics of the subplasmalemmal actin filaments in Con A-pretreated eggs. By using Alexa Fluor 633 Con A and BPA-C8-Cy3, respectively, we also report unprecedented fluorescent labeling of the egg JC and the spontaneous ‘acrosomal protrusion’ on the head of Paracentrotus lividus sperm diluted in natural seawater. Combined with electron microscopy observations of intact and denuded eggs, our results suggest that the glycoconjugate on the egg surface contributes to the fertilization signal transduction, affecting the Ca2+ wave via actin cytoskeletal changes and sperm entry.

In starfish oocytes, the hormone 1-methyladenine (1-MA) induces germinal vesicle breakdown (GVBD) through a signaling cascade involving PI3K, SGK, Cdc25, and Cdk1/cyclin via G-proteinβγ subunit. Following GVBD, fertilization triggers an intracellular calcium increase, leading to the formation of the fertilization envelope (FE) via cortical granule exocytosis. While transient calcium elevations are known to occur after 1-MA stimulation even without fertilization, the inability of these calcium elevations to induce cortical granule exocytosis and FE formation remained unexplained. In this study, we found that co-treatment with 1-MA and calcium ionophore A23187 prevents FE formation, revealing a transient period termed the “no FE phase” persisting for several minutes. After no FE phase, the oocytes regain full competence to form the FE. Furthermore, we identified that the GEF/Rac1 signaling cascade is activated during the no FE phase. Notably, constitutively active Rac1 expressed in oocytes reproduces this inhibition even in the absence of 1-MA stimulation. These findings suggest that the GEF/Rac1 cascade, triggered by 1-MA, initiates the no FE phase and plays a critical role in coordinating the progression of subsequent fertilization events.

Much of the scientific knowledge on oocyte maturation, fertilization, and embryonic development has come from the experiments using gametes of marine organisms that reproduce by external fertilization. In particular, echinoderm eggs have enabled the study of structural and biochemical changes related to meiotic maturation and fertilization owing to the abundant availability of large and transparent oocytes and eggs. Thus, in vitro studies of oocyte maturation and sperm-induced egg activation in starfish are carried out under experimental conditions that resemble those occurring in nature. During the maturation process, immature oocytes of starfish are released from the prophase of the first meiotic division, and acquire the competence to be fertilized through a highly programmed sequence of morphological and physiological changes at the oocyte surface. In addition, the changes in the cortical and nuclear regions are essential for normal and monospermic fertilization. This review summarizes the current state of research on the cortical actin cytoskeleton in mediating structural and physiological changes during oocyte maturation and sperm and egg activation in starfish and sea urchin. The common denominator in these studies with echinoderms is that exquisite rearrangements of the egg cortical actin filaments play pivotal roles in gamete interactions, Ca 2+ signaling, exocytosis of cortical granules, and control of monospermic fertilization. In this review, we also compare findings from studies using invertebrate eggs with what is known about the contributions made by the actin cytoskeleton in mammalian eggs. Since the cortical actin cytoskeleton affects microvillar morphology, movement, and positioning of organelles and vesicles, and the topography of the egg surface, these changes have impacts on the fertilization process, as has been suggested by recent morphological studies on starfish oocytes and eggs using scanning electron microscopy. Drawing the parallelism between vitelline layer of echinoderm eggs and the zona pellucida of mammalian eggs, we also discuss the importance of the egg surface in mediating monospermic fertilization. Graphical abstract

In sea urchin, the immediate contact of the acrosome-reacted sperm with the egg surface triggers a series of structural and ionic changes in the egg cortex. Within one minute after sperm fuses with the egg plasma membrane, the cell membrane potential changes with the concurrent increases in intracellular Ca2+ levels. The consequent exocytosis of the cortical granules induces separation of the vitelline layer from the egg plasma membrane. While these cortical changes are presumed to prevent the fusion of additional sperm, the subsequent late phase (between 1 and 4 min after fertilization) is characterized by reorganization of the egg cortex and microvilli (elongation) and by the metabolic shift to activate de novo protein and DNA syntheses. The latter biosynthetic events are crucial for embryonic development. Previous studies suggested that the early phase of fertilization was not a prerequisite for these changes in the second phase since the increase in the intracellular pH induced by the exposure of unfertilized sea urchin eggs to ammonia seawater could start metabolic egg activation in the absence of the cortical granule exocytosis. In the present study, we have demonstrated that the incubation of unfertilized eggs in ammonia seawater induced considerable elongations of microvilli (containing actin filaments) as a consequence of the intracellular pH increase, which increased the egg’s receptivity to sperm and made the eggs polyspermic at fertilization despite the elevation of the fertilization envelope (FE). These eggs also displayed compromised Ca2+ signals at fertilization, as the amplitude of the cortical flash was significantly reduced and the elevated intracellular Ca2+ level declined much faster. These results have also highlighted the importance of the increased internal pH in regulating Ca2+ signaling and the microvillar actin cytoskeleton during the late phase of the fertilization process.

In sea urchins, the sequence of the cellular and molecular events characterizing the fertilization process has been intensively studied. We have learned that to activate the egg, the fertilizing sperm must undergo morphological modifications (the acrosome reaction, AR) upon reaching the outer gelatinous layer enveloping the egg (egg jelly), which triggers the polymerization of F-actin on the sperm head to form the acrosomal process. The AR exposes bindin, an adhesive sperm protein essential for the species-specific interaction with the cognate receptor on the egg vitelline layer. To investigate the specific roles of the egg jelly and vitelline layer at fertilization of sea urchin eggs, Paracentrotus lividus eggs were incubated in acidic seawater, which removes the egg jelly, i.e., experimental conditions that should prevent the occurrence of the AR, and inseminated in the same medium. At variance with the prevailing view, our results have shown that these dejellied P. lividus eggs can still interact with sperm in acidic seawater, albeit with altered fertilization responses. In particular, the eggs deprived of the vitelline layer reacted with multiple sperm but with altered Ca2+ signals. The results have provided experimental evidence that the plasma membrane, and not the vitelline layer, is where the specific recognition between gametes occurs. The vitelline layer works in unfertilized eggs to prevent polyspermy.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs’ JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg’s VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8.

Abstract Background/Aims: Eggs of all animal species display intense cytoplasmic Ca2+ increases at fertilization. Previously, we reported that unfertilized eggs of Astropecten aranciacus exposed to an actin drug latrunculin A (LAT-A) exhibit similar Ca2+ waves and cortical flashes after 5-10 min time lag. Here, we have explored the molecular mechanisms underlying this unique phenomenon. Methods: Starfish eggs were pretreated with various agents such as other actin drugs or inhibitors of phospholipase C (PLC), and the changes of the intracellular Ca2+ levels were monitored by use of Calcium Green in the presence or absence of LAT-A. The concomitant changes of the actin cytoskeleton were visualized with fluorescent F-actin probes in confocal microscopy. Results: We have shown that the LAT-A-induced Ca2+ increases are related to the disassembly of actin flaments: i) not only LAT-A but also other agents depolymerizing F-actin (i.e. cytochalasin B and mycalolide B) induced similar Ca2+ increases, albeit with slightly lower efficiency; ii) drugs stabilizing F-actin (i.e. phalloidin and jasplakinolide) either blocked or significantly delayed the LAT-A-induced Ca2+ increases. Further studies utilizing pharmacological inhibitors of PLC (U-73122 and neomycin), dominant negative mutant of PLC-ɣ, specific sequestration of PIP2 (RFP-PH), InsP3 uncaging, and quantitation of endogenous InsP3 all indicated that LAT-A induces Ca2+ increases by stimulating PLC rather than sensitizing InsP3 receptors. In support of the idea, it bears emphasis that LAT-A timely increased intracellular contents of InsP3 with concomitant decrease of PIP2 levels in the plasma membrane. Conclusion: Taken together, our results suggest that suboolemmal actin filaments may serve as a scaffold for cell signaling and modulate the activity of the key enzyme involved in intracellular Ca2+ signaling.

While alkaloids often exert unique pharmacological effects on animal cells, exposure of sea urchin eggs to nicotine causes polyspermy at fertilization in a dose-dependent manner. Here, we studied molecular mechanisms underlying the phenomenon. Although nicotine is an agonist of ionotropic acetylcholine receptors, we found that nicotine-induced polyspermy was neither mimicked by acetylcholine and carbachol nor inhibited by specific antagonists of nicotinic acetylcholine receptors. Unlike acetylcholine and carbachol, nicotine uniquely induced drastic rearrangement of egg cortical microfilaments in a dose-dependent way. Such cytoskeletal changes appeared to render the eggs more receptive to sperm, as judged by the significant alleviation of polyspermy by latrunculin-A and mycalolide-B. In addition, our fluorimetric assay provided the first evidence that nicotine directly accelerates polymerization kinetics of G-actin and attenuates depolymerization of preassembled F-actin. Furthermore, nicotine inhibited cofilin-induced disassembly of F-actin. Unexpectedly, our results suggest that effects of nicotine can also be mediated in some non-cholinergic pathways.

In starfish, the addition of the hormone 1-methyladenine (1-MA) to immature oocytes (germinal vesicle, GV-stage) arrested at the prophase of the first meiotic division induces meiosis resumption (maturation), which makes the mature eggs able to respond to the sperm with a normal fertilization response. The optimal fertilizability achieved during the maturation process results from the exquisite structural reorganization of the actin cytoskeleton in the cortex and cytoplasm induced by the maturing hormone. In this report, we have investigated the influence of acidic and alkaline seawater on the structure of the cortical F-actin network of immature oocytes of the starfish (Astropecten aranciacus) and its dynamic changes upon insemination. The results have shown that the altered seawater pH strongly affected the sperm-induced Ca2+ response and the polyspermy rate. When immature starfish oocytes were stimulated with 1-MA in acidic or alkaline seawater, the maturation process displayed a strong dependency on pH in terms of the dynamic structural changes of the cortical F-actin. The resulting alteration of the actin cytoskeleton, in turn, affected the pattern of Ca2+ signals at fertilization and sperm penetration.