Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane,the other involving the follicle cells

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm–egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 μM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 μM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N-acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm–egg fusion. Mol. Reprod. Dev. 48:137-143, 1997. © 1997 Wiley-Liss, Inc.     

Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N- acetylglucosaminidase found in cortical granules was identified as beta- hexosaminidase B, the beta, beta homodimer. Inhibition of N- acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase- binding site, accounting for the block in sperm binding to the zona pellucida.     

Germ-cell warfare in ascidians: sperm from one species can interfere with the fertilization of a second species

Ascidians (invertebrate chordates) are very abundant in many marine subtidal areas. They often live in dense multispecies clumps; thus, interspecific competition for space may be intense. Although most noncolonial species are broadcast spawners, their eggs can be fertilized only by sperm of the same species (1). Multiple fertilization is lethal and all animals have evolved blocks to polyspermy. Ascidian eggs block polyspermy by enzymatic (2) and electrical mechanisms (3). Sperm bind to N-acetylglucosamine groups on the vitelline coat (4, 5, 6, 7). Follice cells surrounding the vitelline coat release N-acetylglucosaminidase during egg activation (8), preventing the binding of all sperm but a few (2). I show here that this interaction is not species-specific; sperm from one species can cause glycosidase release from follicle cells of a second species. Furthermore, once glycosidase release has been induced, the subsequent addition of sperm from the egg-producing species fails to fertilize a substantial proportion of these eggs. This leads to the hypothesis that sperm from one species of ascidian can interfere with fertilization of a second species. While intraspecific sperm competition has been well documented in several taxa (9, 10), this is the first record of sperm competition between species, or interspecific sperm competition.     

Glycolipid linkage of a polyspermy blocking glycosidase to the ascidian egg surface.

Ascidian eggs release N-acetylglucosaminidase rapidly into the seawater following fertilization. This glycosidase is detected seconds after fertilization, and histochemical tests suggest the cell surface as the prefertilization storage site (Lambert, C. C. (1989). Development 105, 415-420). Living eggs of Ascidia ceratodes, A. callosa, and A. paratropa all cleave a fluorogenic substrate in seawater. Following cell surface biotinylation and activation of the eggs, enzyme activity binds to streptavidin further substantiating the cell surface localization. The released glycosidase has a molecular weight of 180 kDa by size exclusion chromatography and exhibits bands at 62 and 70 kDa by SDS-PAGE, suggesting a possibly multimeric enzyme. The enzyme is released by a glycophosphatidylinositol-specific phospholipase C and HNO2 deamination, both of which are specific indicators of linkage to the cell surface via phosphatidylinositol. The enzyme from unfertilized eggs is quite hydrophobic in Triton X-114 phase partition experiments but becomes hydrophyllic after release by activation or deamination. All of these observations are consistent with the glycosidase being anchored to the cell surface via a GPI anchor that is cleaved at fertilization to yield the soluble form of the enzyme which helps protect the egg against polyspermy. We discuss the possible role of a cell surface PLC in this release.     

Independent Initiation of Calcium Dependent Glycosidase Release and Cortical Contractions during the Activation of Ascidian Eggs

During fertilization or ionophore induced activation, ascidian eggs rapidly release cell surface N-acetylglucosaminidase activity used in the block against polyspermy and undergo cortical contractions before they re-initiate meiosis. To better understand the activation process, we probed the relationship between these two processes in Ascidia ceratodes eggs by activating with different agents that increase intracellular Ca levels and under different ionic conditions. Glycosidase activity release was followed by the use of a fluorogenic substrate, and cortical contractions were followed by examining changes in cell shape with light microscopy. Ionomycin (2.7 μM) and thimerosal (1 mM) initiate glycosidase release and cortical contractions when administered in complete sea water (SW) but only the contractions in low Ca SW. Ryanodine (0.67 mM), known to raise free intracellular Ca in a number of cell types by release from the endoplasmic reticulum, causes glycosidase release but fails to initiate cortical contractions in complete SW. Thapsigargin (10 μM), which inhibits Ca dependent ATPase in the ER, causes glycosidase release but induces the contractions only about 50% of the time. These experiments show that, although glycosidase release normally precedes the ooplasmic shape changes that accompany the resumption of meiosis in ascidian eggs, they are not obligately coupled. That both processes can be induced by treatments known to raise intracellular Ca in other systems but under different conditions indicates that there may be a multiplicity of Ca requiring but functionally independent events during egg activation.     

Electrically mediated fast polyspermy block in eggs of the marine worm, Urechis caupo

Previous work has established that the polyspermy block in Urechis acts at the level of sperm-egg membrane fusion. (J. Exp. Zool. 196:105). Present results indicate that during the first 5--10 min after insemination the block is mediated by a positive shift in membrane potential (the fertilization potential) elicited by the penetrating sperm, since holding the membrane potential of the unfertilized egg positive by passing current reduces the probability of sperm entry, while progressively reducing the amplitude of the fertilization potential by decreasing external Na+ progressively enhances multiple sperm penetrations. Also, a normal fertilization potential is correlated with a polyspermy block even under conditions (pH 7) in which eggs do not develop. We have investigated the mechanism of the electrical polyspermy block by quantifying the relationship between sperm incorporation, membrane potential and ion fluxes. Results indicate that the polyspermy block is mediated by the electrial change per se and not by the associated fluxes of Na+, Ca++, and H+.     

Activation of follicle cell surface phospholipase by tyrosine kinase dependent pathway is an essential event in ascidian fertilization

Eggs of Ascidia ceratodes and Phallusia mammillata block polyspermy by releasing a phosphatidylinositol‐linked glycosidase from the follicle cell and egg surface that binds to and blocks all unoccupied sperm binding sites on the vitelline coat. Release of this glycosidase is thought to be under the control of a membrane‐bound phospholipase. To elucidate the mechanism of phospholipase activation, intact eggs and isolated follicle cells are activated by either sperm or the tyrosine kinase activator 9,10‐dimethyl‐1,2‐benzanthracene (DMBA). Both treatments caused release of comparable quantities of glycosidase activity, the earliest event following fertilization. A corresponding increase in phospholipase activity accompanied this glycosidase release. The tyrosine kinase inhibitor genistein blocked release by DMBA at concentrations as low as 1 μM, but had no effect on sperm‐induced release even when used up to 100 μM. Tyrphostin A23, another tyrosine kinase inhibitor, when used at 200 μM blocked glycosidase release and decreased phospholipase activity following both DMBA activation and fertilization. Western blot analysis probing for phosphotyrosine content of disrupted intact eggs with their follicle cells revealed the absence of a band in tyrphostin‐treated eggs corresponding to a 40 kDa protein that was present in both unfertilized and fertilized egg samples. Based on these results, we propose that phosphorylation of specific tyrosine residues is necessary for phospholipase activation and is sufficient to trigger subsequent glycosidase release. Mol. Reprod. Dev. 54:69–75, 1999. © 1999 Wiley‐Liss, Inc.     

Molecular basis of fertilization in the mouse

Recent studies of mouse fertilization have identified two complementary gamete receptors that mediate sperm-egg binding. Sperm surface β1,4-galactosyltransferase (GalTase) binds to specific oligosaccharides of the egg coat (zona pellucida) glycoprotein ZP3. Evidence suggests that these same molecules may stimulate the acrosome reaction in sperm. After the acrosome reaction, it is thought that sperm remain adherent to the zona by binding another glycoprotein, ZP2. The acrosome-reacted sperm releases hydrolytic enzymes, including acrosin and N-acetylglucosaminidase, enabling it to penetrate the zona pellucida. After the penetrating sperm binds to the egg membrane and activates development, N-acetylglucosaminidase is exocytosed from egg cortical granules and, as part of the zona block to polyspermy, globally removes the sperm GalTase binding site from ZP3 oligosaccharides.     

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat

In the initial stage of ascidian fertilization sequential sperm–egg coat interactions assure successful species-specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio-iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non-radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration-dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg-binding activity. Three biotinylated proteins (66kDa, 120kDa and 140kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120kDa protein possesses species-specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species-specific recognition in P. mammillata .     

An electrically mediated block to polyspermy in the primitive urodele Hynobius nebulosus and phylogenetic comparison with other amphibians

At fertilization, the egg of the primitive urodele, Hynobius nebulosus, produced a fertilization potential which rose from -12 to +47 mV. A similar activation potential was elicited by pricking with a needle, by applying A23187, or by electric shock. The potential change was mediated by an increased permeability to Cl-. Clamping the egg's membrane potential at +40 mV blocked fertilization, while clamping at +20 mV induced polyspermy. These results indicated the occurrence of an electrical polyspermy block, typical of anurans, but atypical of urodeles. Furthermore, Hynobius eggs fertilized by natural mating incorporated only one sperm nucleus, and experimentally polyspermic eggs underwent multipolar division. Accessory sperm did not degenerate in the egg cytoplasm, indicating lack of an intracellular polyspermy block. By comparison, fertilization of Bufo japonicus (anuran) was also voltage dependent, whereas that of Cynops pyrrhogaster (urodele) was voltage independent. Thus polyspermy prevention mechanisms in Hynobius closely resemble those of anuran amphibians and differ from those of higher urodeles.     

A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida

Past studies have suggested that mouse sperm surface galactosyltransferase may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the zona pellucida. In this paper, we examined further the role of sperm surface galactosyltransferase in mouse fertilization. Two reagents that specifically perturb sperm surface galactosyltransferase activity both inhibit sperm-zona binding. The presence of the milk protein alpha- lactalbumin specifically modifies the substrate specificity of sperm galactosyltransferase away from GlcNAc and towards glucose and simultaneously inhibits sperm binding to the zona pellucida. Similarly, UDP-dialdehyde inhibits sperm binding to the zona pellucida and sperm surface galactosyl-transferase activity to identical degrees. Of five other sperm enzymes assayed, four are unaffected by UDP-dialdehyde, and one is affected only slightly. Covalent linkage of UDP-dialdehyde to sperm dramatically inhibits binding to eggs, while treatment of eggs with UDP-dialdehyde has no effect on sperm binding. Heat-solubilized or pronase-digested zona pellucida inhibit sperm-zona binding, and they can be glycosylated by sperm with UDP-galactose. Sperm are also able to glycosylate intact zona pellucida with UDP-galactose. Thus, solubilized and intact zona pellucida act as substrates for sperm surface GlcNAc:galactosyltransferases. Finally, pretreatment of eggs with beta- N-acetylglucosaminidase inhibits sperm binding by up to 86%, while under identical conditions, pretreatment with beta-galactosidase increases sperm binding by 55%. These studies, in conjunction with those of the preceding paper dealing with surface galactosyltransferase changes during capacitation, directly suggest that galactosyltransferase is at least one of the components necessary for sperm binding to the zona pellucida.     

Fertilization acid release in Urechis eggs. II. The stoichiometry of Na+ uptake and H+ release

We tested the hypothesis that Na+ uptake and H+ release at fertilization of Urechis eggs might occur via a Na+:H+ exchange. Previous studies have shown that (1) Na+ uptake is proportional to the number of entering sperm in seawater with or without lowered Na+ and (2) H+ release is proportional to external pH. Therefore, to determine if Na+ uptake and H+ release are always proportional, we determined the effect of polyspermy on H+ release in natural and low Na+ seawater and the effect of external pH on Na+ uptake and release. Na+ uptake and H+ release do not covary in a manner consistent with a Na+:H+ exchange. H+ release under most conditions was manner consistent with a Na+:H+ exchange. H+ release under most conditions was independent of the number of sperm/egg and in low Na+ seawater was at most 53 +/- 16% of that in natural seawater. In contrast, Na+ uptake in low Na+ seawater can be more than in natural seawater (Jaffe et al., J. Gen. Physiol. 73, 469-492, 1979). In natural seawater Na+ uptake exceeded H+ release; at pH 7 Na+ uptake was 2 pmol/egg, but there was no H+ release. Since Na+ release did not increase at fertilization at pH 7, neither Na+:Na+ nor Na+:H+ exchange could account for the Na+ uptake. An alternate hypothesis is suggested: Na+ uptake is primarily via the channels responsible for the fertilization potential, while H+ release is by another route that is affected by the membrane potential during the fertilization potential.     

The fast block against polyspermy in fucoid algae is an electrical block

Fertilization potentials in Pelvetia fastigiata, Fucus vesiculosus, and Fucus ceranoides were studied to examine whether eggs of fucoid algae have an electrical block against polyspermy. The resting potential of eggs of all species was about -60 mV, depolarizing, respectively, to -24 +/- 5 mV (SD, n = 9) for 7.5 +/- 2.1 (n = 8) min, -26 +/- 5 (n = 9) mV for 6.4 +/- 2.3 (n = 9) min, and -24 +/- 6 (n = 5) mV for 6.7 +/- 1.9 (n = 4) min. The depolarization was slower, and the fertilization potential was about 10 mV more negative in eggs of both F. vesiculosus and Pelvetia fertilized in 45-mM Na+ ASW; many of these eggs were polyspermic. Steady current was passed through unfertilized eggs of F. vesiculosus prior to insemination to test the potential dependence of fertilization. Eggs (n = 10) bound sperm at all potentials tested (-45 to -23 mV), but fertilization was prevented if eggs were held at potentials more positive than -45 to -37 mV. Eggs underwent a second depolarization if artificially hyperpolarized to potentials more negative than -50 mV immediately after the rise of a normal fertilization potential. Thus, fucoid eggs have an electrical fast block against polyspermy. Only in F. ceranoides does the formation of the cell wall after fertilization appear to be fast enough (i.e., 3-6 min postfertilization versus at 10-15 min in F. vesiculosus and P. fastigiata) to replace the fertilization potential as a polyspermy block. Nonfertilizing fucoid sperm swim away from the egg surface by 1-3 min after rise of the fertilization potential. This suggests that there is another "intermediate block" against polyspermy.     

The ascidian egg envelope in fertilization: structural and molecular features

In this report, unpublished and recent findings concerning the structure and function of the ascidian egg coat are compiled in context with fertilization. In the initial stage of ascidian fertilization, sperm interact with a complex egg investment that consists of a layer of follicle cells attached to an acellular vitelline coat. Increasing evidence exists that ascidian sperm are activated at their encounter with the follicle cells. The molecular basis of sperm-follicle cell interactions is discussed in context with sperm binding, membrane proteins and sperm bound glycosidase. The model that suggests a block to polyspermy established by glycosidase released from the follicle cells on fertilization is evaluated and compared with assured facts. Although a number of questions remain to be answered, our recent findings that a cloned beta-hexosaminidase from P. mammillata binds exclusively to the follicle cells of unfertilized but not fertilized eggs, indicates that the follicle cells participate in the block to polyspermy. A dual function, mediating sperm activation and a block to polyspermy attributes to the ascidian follicle cells a key position in fertilization.     

An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis

In 27% DeBoer's saline (DBS), which yields maximum fertility rates, Xenopus eggs fertilized in vitro are monospermic, regardless of sperm concentration. One block to polyspermy (the “slow” block), described previously, occurs at the fertilization envelope that is elevated in response to the cortical reaction. This paper describes properties of an earlier, “fast” block at the plasma membrane and evaluates the functional significance of the two blocks at physiological sperm concentrations in natural mating conditions. Unfertilized eggs have a resting membrane potential of ?19 mV in 27% DBS. Fertilization triggers a rapid depolarization to +8 mV (the fertilization potential, FP); the potential remains positive for ca. 15 min. Activation of eggs with the ionophore, A23187, produces a slower but similar depolarization (the activation potential, AP). As in other amphibian eggs, the FP appears to result from a net efflux of Cl^?, since the peak of the FP (or the AP in ionophore-activated eggs) decreases as the concentration of chloride salts in the medium is increased. In 67% DBS no FP or AP is observed; eggs fertilized in 67% DBS become polyspermic and average 2 sperm entry sites per egg. In the 5–37 mM range, I^? and Br^?, but not F^?, are more effective than Cl^? in producing polyspermy. In 20 mM NaI the plasma membrane hyperpolarizes in response to sperm or ionophore; 100% levels of polyspermy and an average of 14 sperm entry sites per egg are observed. NaI does not inhibit or retard elevation of the fertilization envelope; the cortical reaction and fertilization envelope are normal in transmission electron micrographs. In 67% DBS, which also inhibits the fast block, the slow block was estimated to become functional 6–8 min after insemination. Eggs fertilized by natural mating in 20 mM NaI exhibit polyspermy levels of 50–90% and average 5 sperm entry sites per egg. Since eggs become polyspermic when fertilized by natural mating under conditions that inhibit the fast, but not the slow, block to polyspermy, we conclude that the fast block is essential to the prevention of polyspermy at the sperm concentrations normally encountered by the egg.     

Fertilization potential and polyspermy prevention in the egg of the nemertean, Cerebratulus lacteus

We investigated the electrical properties of the egg of the nemertean worm Cerebratulus, and found evidence that an electrically-mediated polyspermy block operates for a period of about 1 hr after fertilization. At fertilization, in natural or artificial sea water, the membrane potential shifts from its resting level of about -66 mV to a peak of about +43 mV, and in most cases remains greater than 0 mV for more than 1 hr. The average potential during the first 30 min is +22 +/- 8 mV (SD, n = 12). When the external Na+ concentration is reduced from 486 to 51 mM (choline substituted) the fertilization potential amplitude is reduced; the average potential during the first 30 min is -27 +/- 21 mV (SD, n = 5). Eggs inseminated in 51 mM Na+ sea water become polyspermic, indicating that polyspermy prevention depends on an electrically-mediated mechanism. The electrical block is required for about 60 min, since transfer to 51 mM Na+ sea water during this period results in polyspermy. During the first hour following fertilization, the egg is also developing a permanent, nonelectrical block; the degree of polyspermy which results upon transfer to low Na+ sea water decreases progressively with time. The permanent block appears to be at the level of the egg plasma membrane or glycocalyx, since the egg envelope is not a barrier to sperm penetration, nor does its removal induce polyspermy. Electron micrographs show no obvious changes in the morphology of the extracellular layers, plasma membrane or cortex of the egg after fertilization.     

Sperm surface proteases in ascidian fertilization.

Ascidian eggs are surrounded by a noncellular layer and two cellular layers, which are penetrated by sperm. Three sperm surface proteases are essential for fertilization of eggs from the stolidobranch ascidian Halocynthia: spermosin, acrosin, and the proteasome. In the phlebobranch Ciona, a chymotrypsin-like protease and the proteasome are essential in fertilization. Sperm from the phlebobranch ascidians Phallusia mammillata, Ascidia (=Phallusia) nigra, and Ascidia columbiana, all express spermosin, acrosin, and the proteasomal chymotrypsin activities on their surfaces. Chymostatin blocks cleavage in phlebobranchs, but inhibitors of spermosin and acrosin only delay it by several minutes. Protease inhibitors have little effect upon sperm binding in Phallusia but strongly affect the rate of sperm passage through the vitelline coat. Peptide substrates and inhibitors to spermosin and acrosin cause a significant decline in the number of eggs undergoing pre-meiotic contractions at 3 min after fertilization. Thus while chymotrypsin activity is essential for penetration of the vitelline coat, spermosin and acrosin both function to increase the rate of fertilization. A crucial step in the divergence of the phlebobranchs and stolidobranchs may have been the conversion of spermosin and acrosin to essential proteases in the stolidobranchs, or, perhaps, their essential function was lost in the evolution of phlebobranchs. Aplousobranch ascidians are all colonial with very small zooids. Sperm from Aplidium californicum, Aplidium solidum (Polyclinidae), and Distaplia occidentalis (Holozoidae) have acrosin and chymotrypsin activities but lack spermosin activity. This enzyme is also missing from sperm of colonial phlebobranch and stolidobranch ascidians, suggesting that spermosin is not necessary for small zooids with internal fertilization.     

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.     

Egg exudate-induced reduction of sperm lysin sensitivity in the vitelline coat after fertilization of Bufo japonicus and its participation in polyspermy block

The jellyless eggs of Bufo japonicus or those from which the vitelline coats (VCs) had been removed (denuded eggs) were electrically activated. The exudate that accompanied egg activation (AEX) was collected to study its role in preventing polyspermy. When dejellied (but VC intact) eggs were treated with AEX, the eggs lost not only fertilizability but also the sensitivity of their VCs to the sperm lysin. By contrast, denuded eggs treated with AEX were fertilizable; even activated eggs were highly fertilizable, provided they were deprived of their VCs and inseminated 30 min after activation. The loss of sensitivity to sperm lysin occurred in VCs 3-5 min after activation either in De Boer's or 1/20 De Boer's solution. The activity of AEX to reduce the sensitivity of VCs to sperm lysin was heat-sensitive and dependent on Ca2+, but it was not affected at all by the variety of protease inhibitors used. The activity was lost by the preincubation of AEX with fragmented VCs in the presence of Ca2+, suggesting Ca(2+)-dependent binding of AEX molecules to the VC at fertilization. Immunocytochemical studies employing anti-AEX rabbit serum showed that the pertinent antigens were localized in the cortical granules of unfertilized eggs and in both the inner surface of VCs and the perivitelline space of fertilized eggs. We conclude that the AEX-induced loss of lysin sensitivity in VCs and the deposition of cortical granule materials on the inner wall of VCs constitute a slow and permanent block to polyspermy.     

A sodium-dependent, fast block to polyspermy occurs in eggs of fucoid algae

More than 70% of Pelvetia fastigiata eggs and about 15% of Fucus distichus eggs become polyspermic when fertilized at natural sperm concentrations in a low-sodium (2.5 mM Na+, 450 mM N-methyl glucamine) artificial seawater. Natural levels of polyspermy are 1-3% for both species. Polyspermic eggs germinate and respond to photopolarization, but do not develop beyond an abnormal, "stumpy," four-cell stage. They die within 1-1.5 weeks. The sodium-dependent block is a fast block, and it is replaced by a second block (probably cell wall formation) no later than 9 min (Pelvetia) after eggs are shed. The sodium-dependent block in Pelvetia is very efficient; when external sodium is raised to only 47.5 mM, the level of polyspermy drops to about 25%. These results are compared with data on marine invertebrates in the context of factors such as the sperm/egg concentration at fertilization and natural, osmotic (salinity) stress.