Purification and characterization of an extracellular fragment of the sea urchin egg receptor for sperm

Fertilization in the sea urchin involves species-specific interaction between the ligand bindin on the surface of acrosome-reacted sperm and a receptor of high molecular weight on the surface of the egg. Efforts to understand this interaction and the resultant signal transduction events leading to egg activation have been limited because of the large size and extreme insolubility of the intact receptor on the egg surface. Earlier work suggested that an alternative strategy would be to isolate proteolytic fragments of the extracellular domain of this receptor. Consequently, we have treated S. purpuratus eggs with a specific protease, lysylendoproteinase C. This enzyme treatment abolished the ability of eggs to bind sperm and resulted in the release of proteolytic fragments that bound to sperm and showed inhibitory activity in a fertilization bioassay. One of these fragments, presumed to be a fragment of the extracellular domain of the receptor, was purified to homogeneity by gel filtration and anion exchange chromatography and shown to be a 70-kD glycosylated protein. Several lines of evidence support the contention that this fragment is derived from the receptor. First, the fragment inhibited fertilization species specifically. Second, species specific binding of the 70-kD glycoprotein to acrosome-reacted sperm was directly demonstrated by using 125I-labeled receptor fragment. Third, the fragment exhibited the same species specificity in binding to isolated bindin particles. Species specificity was abolished by Pronase digestion of the fragment. This observation supports the hypothesis that although binding is mediated by the carbohydrate moieties, species specificity is dependent on the polypeptide backbone. The availability of a structurally defined fragment of the receptor will facilitate further studies of the molecular basis of gamete interaction.     

Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata

The possibility that the surface of the egg of the sea urchin Arbacia punctulata contains a species-specific receptor for sperm has been investigated. The extent of fertilization of eggs of A. punctulata, which is proportional to the number of sperm, is unaffected by the presence of either eggs or membranes prepared from eggs of Strongylocentrotus purpuratus. In marked contrast, membranes prepared from eggs of A. punctulata quantitatively inhibit fertilization of A. punctulata eggs by A. punctulata sperm. Several lines of evidence indicate that this inhibition is due to the presence of a membrane-associated glycoprotein that binds to the sperm, thus preventing them from interacting with receptor on the surface of the eggs. First, eggs treated with trypsin are incapable of being fertilized, although they can be activated with the Ca2+ ionophore A23187. Moreover, membranes prepared from eggs pretreated with trypsin do not inhibit fertilization of eggs. Second, receptor isolated in soluble form from surface membranes binds to sperm and thus prevents them from fertilizing eggs; the inhibition by soluble receptor is species-specific. Third, the soluble receptor binds to concanavalin A-Sepharose. Fourth, eggs are incapable of being fertilized if they are pretreated with concanavalin A. The specificity of inhibition, and the affect of trypsin and concanavalin A on intact eggs, suggest that the receptor is a species-specific macromolecule located on the surface of the eggs. The sensitivity of the receptor to trypsin, and its ability to bind to concanavalin A, indicate that it is a glycoprotein.     

Isolation of a high molecular weight glycoconjugate derived from the surface of S purpuratus eggs that is implicated in sperm adhesion

Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin-like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4-B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin-mediated egg agglutination yet described.     

The sequence of the Arbacia punctulata bindin cDNA and implications for the structural basis of species-specific sperm adhesion and fertilization

Bindin is the major protein component of the acrosome granule of sea urchin sperm which mediates the species-specific adhesion of sperm to the egg surface during fertilization. Bindin isolated from both Arbacia punctulata and Strongylocentrotus purpuratus sperm demonstrate a distinct adhesive preference for eggs of the same species although a significant amount of cross-species reactivity is observed. Here we describe the isolation and sequence of A. punctulata bindin cDNA clones and a comparison of the predicted protein sequence with the sequence previously reported for S. purpuratus bindin (Gao et al., 1986, Proc. Natl. Acad. Sci., USA 83, 8634-8638). Bindins from these genera show substantial sequence similarity in both the mature bindin domain and the probindin precursor region. The most striking identity is a region of 42 conserved amino acids in the central part of the mature bindins. This conserved domain may be responsible for conserved functions of bindin. Regions flanking this conserved element on both the amino and carboxyl side are more highly divergent, suggesting that they are responsible for the species-specific properties of bindin. The mature A. punctulata sequence contains a putative transmembrane segment between residues 431 and 451 that is absent from S. purpuratus bindin. This structural element may account for the previous observation that isolated A. punctulata bindin uniquely forms multilamellar structures reminiscent of lipid bilayers and binds significant amounts of phospholipid and detergent. The structure of this hydrophobic segment also displays a number of similarities to viral fusion peptides.     

Isolation of a glycopeptide fraction from the surface of the sea urchin egg that inhibits sperm-egg binding and fertilization

The role of cell surface glycoproteins of the sea urchin egg in binding sperm has been examined by studying the biological activity of glycopeptides derived from these glycoproteins. Glycopeptides were produced from egg surface glycoproteins by Pronase digestion. After fractionation by gel filtration the glycopeptides were tested for their ability to inhibit the binding of sperm to eggs, presumably by competing with the egg surface glycoproteins for binding sites on the sperm. One glycopeptide fraction with an apparent molecular weight of approximately 6,000 was found to be a potent inhibitor of sperm-egg binding, as well as fertilization, even at nanomolar concentrations. This activity was heat stable and exerted its effect against the sperm and not the egg. Experiments with a radiolabeled form of the glycopeptide fraction directly demonstrated that at least one component of it bound to sperm. Specific binding of the radiolabeled glycopeptide occurred only to acrosome-reacted sperm. Because the isolated glycopeptide fraction has many of the characteristics that one would expect of a biologically active fragment of an egg surface receptor for sperm, these findings are consistent with the idea that one or more glycoconjugates on the surface of the egg are involved in sperm binding.     

Identification of the sea urchin egg receptor for sperm using an antiserum raised against a fragment of its extracellular domain

Sea urchin egg fertilization requires the species-specific interaction of molecules on the sperm and egg surfaces. Previously, we isolated an extracellular, 70-kD glycosylated fragment of the S. purpuratus egg receptor for sperm by treating the eggs with lysylendoproteinase C (Foltz, K. R., and W. J. Lennarz. 1990. J. Cell Biol. 111:2951-2959). To characterize the receptor further, we have generated a polyclonal antiserum (anti-70KL) against the purified 70-kD fragment. Anti-70KL was found to react with a single polypeptide of approximately 350 kD on Western blots, presumed to be the intact receptor, in an egg cell surface preparation. This polypeptide appeared to be tightly associated with the plasma membrane/vitelline layer complex, as it was released from these preparations only by detergent treatment. Immunofluorescence microscopy revealed that the receptor was distributed evenly over the egg surface. The anti-70KL was species specific both in its ability to recognize the egg surface protein and to inhibit sperm binding. Fab fragments generated from affinity-purified anti-70KL also bound to the egg surface and inhibited sperm binding in a concentration-dependent manner. Interestingly, treatment with Fabs caused a small percentage of eggs to undergo cortical granule exocytosis, even in the absence of external Ca2+. These results confirm earlier findings indicating that the receptor is a cell surface glycoprotein of high molecular weight that species specifically binds sperm. This antiserum provides a powerful tool for further investigation of gamete interactions and the structure of the sperm receptor.     

The sea urchin egg receptor for sperm: isolation and characterization of the intact, biologically active receptor

The species-specific binding of sea urchin sperm to the egg is mediated by an egg cell surface receptor. Although earlier studies have resulted in the cloning and sequencing of the receptor, structure/function studies require knowledge of the structure of the mature cell surface protein. In this study, we report the purification of this glycoprotein to homogeneity from a cell surface complex of Strongylocentrotus purpuratus eggs using lectin and ion exchange chromatography. Based on the yield of receptor it can be calculated that each egg contains approximately 1.25 x 10(6) receptor molecules on its surface. The receptor, which has an apparent M(r) of 350 kD, is a highly glycosylated transmembrane protein composed of approximately 70% carbohydrate. Because earlier studies on the partially purified receptor and on a pure, extracellular fragment of the receptor indicated that the carbohydrate chains were important in sperm binding, we undertook compositional analysis of the carbohydrate in the intact receptor. These analyses and lectin binding studies revealed that the oligosaccharide chains of the receptor are sulfated and that both N- and O-linked chains are present. Functional analyses revealed that the purified receptor retained biological activity; it inhibited fertilization in a species-specific and dose-dependent manner, and polystyrene beads coated with it bound to acrosome-reacted sperm in a species-specific manner. The availability of biochemical quantities of this novel cell recognition molecule opens new avenues to studying the interaction of complementary cell surface ligands in fertilization.     

Association of egg zona pellucida glycoprotein mZP3 with sperm protein sp56 during fertilization in mice.

Purified mouse sperm receptor, a zona pellucida glycoprotein called mZP3, binds to plasma membrane overlying acrosome-intact sperm heads (P.M. Wassarman, 1999, Cell 96, 175-183). Some evidence suggests that mZP3 binds to sp56, a protein reported to be associated peripherally with the plasma membrane of acrosome-intact sperm heads (J.D. Bleil and P.M. Wassarman, 1990, Proc. Natl. Acad. Sci., USA 87, 7215-7219; A. Cheng et al., 1994, J. Cell Biol. 125, 867-878). Here, we report that membrane vesicles prepared from acrosome-intact sperm contain sp56. When these vesicles are incubated with eggs they inhibit binding of sperm to eggs in vitro (ID50 approximately 50-100 microg protein/ml). On the other hand, a monoclonal antibody directed against sp56 relieves the inhibition of binding of sperm to eggs by membrane vesicles. As expected, incubation of intact sperm with the antibody directed against sp56 inhibits binding of the sperm to eggs. Results of immunoprecipitation of sperm extracts incubated with mZP3, by either a polyclonal antibody directed against mZP3 or a monoclonal antibody directed against sp56, suggest that mZP3 is specifically associated with sp56. Results of laser scanning confocal microscopy of fixed sperm probed with antibodies directed against either sp56 or a approximately 155 kDa acrosomal protein, suggest that the two proteins are present in the acrosome, but with different distributions. Furthermore, confocal images of sperm, fixed after exposure to purified mZP3 and probed with antibodies against mZP3 and sp56, reveal overlap between mZP3 and sp56 at the surface of the sperm head. The possible implications of these results are discussed in the context of mammalian fertilization.     

Purification of sea urchin sperm bindin by DEAE-cellulose chromatography

A procedure for purifying bindin from sperm of the sea urchin Strongylocentrotus purpuratus is presented in detail. The impure bindin, dissolved in 4 M urea, 50 mM sodium phosphate, pH 6.6, is adsorbed to DEAE-cellulose and eluted wit 4 M urea, 650 mM sodium phosphate, pH 6.6. The purified bindin is not contaminated with tubulin or histone HI. A precipitate of this DEAE-purified bindin, made by dialysis into Ca2+-free seawater and natural seawater, is a species-specific agglutinin of unfertilized eggs. This method of obtaining consistently pure preparations of bindin will aid in the analysis of its role in fertilization.     

Fate of the sea urchin egg receptor for sperm following fertilization

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH- terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi-associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane-bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane-bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.     

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 .     

Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.     

Species-specific sperm adhesion in sea urchins. A quantitative investigation of bindin- mediated egg agglutination

Bindin, a protein component of the acrosomal vesicle of sea urchin sperm, has been isolated from Arbacia punctulata and strongylocentrous purpuratus. Using this isolated bindin, we have devised a quantitative assay for bindin-mediated egg agglutination and compared the agglutination of bindin eggs from A. puntulata and S. purpuratus. Bindin- mediated agglutination is species –specific in both species, although a measurable degree of heterotypic interaction is observed. Homotypic bindin-egg interactions differ significantly from heterotypic interactions both in the extent of agglutination and the size of the resulting aggregates. We also provide direct evidence that bindin particles agglutinate eggs by adhering to the surfaces of adjacent eggs. Although the A. punctulata bindin preparation displays the same functional properties and consists of one major polypeptide of the same apparent molecular weight as S. purpuratus bindin, its morphology is very different. Unlike the spherical aggregates observed with S. purpuratus bindin, A punctulata bindin exists as lamellar vesicles and binds significant amounts of phospholipids and Triton X-100, suggesting that it may be tightly associated with the acrosomal membrane. Having defined a number of the basic parameters of bindin-mediated agglutination, we examined the effect of a number of saccharides and glycopeptides on bindin-mediated egg agglutination. Carbohydrate-containing components derived from the egg cell surface by proteolysis were found to inhibit bindin-mediated egg agglutination at low concentrations, but this inhibition is not species specific.     

Species-specific inhibition of fertilization by a peptide derived from the sperm protein bindin.

The sperm protein bindin is responsible for the species-specific adhesion of the sperm to the egg. The regions of the bindin molecule responsible for forming the contact between the sperm and the egg were investigated by measuring the ability of peptides representing various regions of the bindin sequence to inhibit fertilization. Twenty-four peptides were studied: 7 based on the Strongylocentrotus purpuratus bindin sequence, 11 based on the S. franciscanus bindin sequence, and 6 control peptides. Values for the concentration of peptide required to inhibit 50% of the productive sperm contacts (IC50) were extracted from experimental measurements of the extent of fertilization in the presence of various concentrations. of these peptides. The IC50 value averaged 220 microM for the control peptides. Active peptides representing certain specific subregions of the bindin sequence displayed IC50 values < 10% of the average value for control peptides, and the IC50 for the most potent of the peptides tested was only approximately 1% of the control peptide value (IC50 = 2.2 microM). Furthermore, we found that a peptide representing a particular region of the S. franciscanus bindin sequence that differs from the S. purpuratus bindin sequence inhibits fertilization species specifically. For the reaction of S. purpuratus sperm and eggs, the IC50 of this peptide was approximately 120 microM, whereas for the reaction of S. franciscanus sperm and eggs it was only 8.6 microM. These results demonstrate that a few specific regions of the bindin molecule are involved in the sperm-egg contact and that certain of these regions mediate the species specificity of the interaction in a sequence-specific manner.     

Solubilization and partial purification from mouse sperm membranes of the specific binding activity for 3-quinuclidinyl benzilate,a potent inhibitor of the zona pellucida-induced acrosome reaction

3-Quinuclidinyl benzilate (QNB), a potent antagonist of muscarinic acetylcholine receptors, has been demonstrated to inhibit specifically the zona pellucida (ZP)-inducud acrosome reaction (AR) in mouse sperm (Florman and Storey, 1982; Dev Biol 91:121–130). In this study we describe the solubilization and partial purification of the mouse sperm QNB binding activity which may represent a component of the putative receptor complex for ZP on the sperm plasma membrane. Sperm membranes were isolated from cell homogenates of washed, capacitated, epididymal mouse sperm. Scatchard plots of QNB binding to these membranes indicated a single class of binding sites with K_D = 7.2 nM and B_max = 8700 sites/cell. These binding characteristics are similar to those seen with QNB binding to whole cells (Florman and Storey, 1982, J Androl 3:157–164). Sperm membranes were solubilized using 1% digitonin/0.2% cholate, and the resultant detergent-soluble fraction possessed QNB binding activity similar to that of intact membranes. The detergent-soluble fraction maintained intact ZP receptor(s)–G protein coupling in that treatment of this fraction with either ZP or mastoparan resulted in a 35% or 65% increase in specific GTPγS binding, respectively. The solubilized membrane preparation was fractionated by gel permeation HPLC. A majority of specific QNB binding activity was confined to one HPLC fraction. Analysis of this fraction by SDS–PAGE revealed a complex of approximately 5 proteins unique to this fraction. The most prominent protein had a M_r of 72 kDa, which is within the M_r range for muscarinic receptors. A protein with M_r = 41 kDa was also present within this fraction. Subsequent pertussis toxin (PTX)-catalyzed ADP-ribosylation of this fraction revealed this protein to be the α subunit of the G_i class of G proteins. Although the QNB binding activity could not be positively identified, we propose that it is contained in one or more of the proteins unique to this fraction and that these proteins, including G_i, may act as part of a sperm receptor complex for the ZP. © 1994 Wiley-Liss, Inc.     

Interaction of the sperm adhesive protein, bindin, with phospholipid vesicles. I. Specific association of bindin with gel-phase phospholipid vesicles

Bindin is a 30,000-mol-wt protein of sea urchin sperm that is responsible for the specific adhesion of the sperm acrosomal process to the vitelline layer covering the egg plasma membrane during fertilization. Sulfated glycoconjugates are believed to be the egg surface receptors for bindin, but the mechanism by which bindin associates with the sperm acrosomal membrane is unknown. Here I report that bindin specifically associates with phospholipid vesicles in vitro. Interaction of the bindin polypeptide with liposomes was found to cause an increase in the density of the liposomes and induce the aggregation of the vesicles. A novel property of this association of bindin with membranes was that it required phospholipids in a gel phase. The interaction of bindin with liposomes was greatly reduced at temperatures above the phase transition temperature. The interaction of bindin with gel-phase vesicles appeared to be reversible, since the aggregated vesicles dissaggregated as the temperature was raised above the phase transition temperature. Association of bindin with the bilayer did not alter the accessibility of the polypeptide to cleavage by trypsin, which suggests that most of the polypeptide chain remains exposed at the surface of the membrane.     

Evidence for specific binding of uncapacitated boar spermatozoa to porcine zonae pellucidae in vitro

Washed ejaculated boar sperm and sperm from the cauda epididymis bind to the zona pellucida of fixed porcine eggs in large numbers. Sperm incubated in the presence of dextran sulfate (8 K daltons or 500 K daltons) or fucoidan and then washed no longer bind to eggs. Other acid carbohydrates (heparin, chondroitin sulfates, inositol hexasulfate, carboxymethylcellulose) fail to block sperm-egg binding even when added directly to sperm-egg suspensions. Seminal plasma and the seminal vesicle secretion contain basic proteins which bind tightly to sperm and bind reversibly to eggs preventing sperm from binding to eggs. When dextran sulfate or fucoidan are mixed with the vesicular secretion, from which seminal plasma basic proteins originate (Hunt et al., '83), the secretion loses the capacity to prevent sperm from binding to eggs; this suggests that seminal vesicle proteins can bind to the same site on zonae as do sperm and thus seminal plasma may modify sperm-egg interactions. Corpus and cauda epididymal sperm also bind in large numbers to the zona pellucida of isolated eggs but high concentrations of caput sperm, which exhibit high motility in the presence of caffeine, bind only in few numbers. Thus a component that enhances sperm-zona binding is apparently formed on the plasma membranes of uncapacitated sperm during passage through the epididymis. This finding, and an earlier observation that antibodies raised against uncapacitated sperm plasma membranes block sperm-egg binding in vivo (Peterson et al., '83) suggest that this component may be involved in sperm zona interaction in vivo.     

The Interactions of Sea Urchin Gametes During Fertilization

The interactions between sea urchin spermatozoa and ova duringfertilization usually exhibit a high degree of species specificity.Under natural conditions and reasonable gamete concentrations,most interspecific inseminations fail to yield zygotes. Macromoleculeson the external surfaces of the apposing gametes must surelybe responsible for successful gamete recognition, adhesion andfusion. Species specific recognition between surface componentsof sperm and egg could occur during at least three events comprisingthe fertilization process. The first event is the interactionof the sperm plasma membrane with the egg jelly coat. This inducesthe sperm acrosome reaction resulting in the exocytosis of the"bindin" -containing acrosome granule and also the extrusionof the acrosome process from the anterior tip of the sperm.The second event is the adhesion of the bindin-coated acrosomeprocess to glycoprotein "bindin receptors" on the external surfaceof the egg vitelline layer. The third event is the penetrationof the vitelline layer and the fusion of sperm and egg plasmamembranes. With the isolations of the component of egg jellywhich induces the acrosome reaction, sperm bindin from the acrosomevesicle and the egg surface bindin receptor from the vitellinelayer, there is hope of discovering the molecular basis of thismost interesting intercellular interaction which results inthe activation of embryonic development.     

Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin

Bindin is a gamete recognition protein of sea urchins that mediates species-specific attachment of sperm to an egg-surface receptor during fertilization. Sequences of bindin from closely related urchins show fixed species-specific differences. Within species, highly polymorphic bindin alleles result from point substitution, insertion/deletion, and recombination. Since speciation, positive selection favoring allelic variants has generated diversity in bindin polypeptides. Intraspecific bindin variation can be tolerated by the egg receptor, which suggests functional parallels between this system and other flexible recognition systems, including immune recognition. These results show that polymorphism in mate recognition loci required for rapid evolution of sexual isolation can arise within natural populations.      

Novel species-specific glycoprotein on the surface of Mytilus edulis and M. trossulus eggs

Protein–protein interactions play a central role in the gamete attraction, binding, and fusion stages of gamete interactions and fertilization for broadcast spawning species, such as marine mussels in the Mytilus edulis species complex. Although assortative gamete interaction has been implicated in the level of reproductive isolation among the three species in this complex, the molecular basis of these interactions has not been elucidated. Using mass spectrometry peptide sequencing, cDNA sequencing, and bioinformatics approaches, we have investigated species-level variation in the proteins expressed on the surface of mussel eggs. We herein describe an extracellular protein, MESP-1, from the surface of the eggs of M. edulis and M. trossulus that has a unique domain structure when compared to protein structures that have heretofore been identified. Given variation in the size of MESP-1 predicted from cDNA sequences versus those estimated from SDS-PAGE gels, we conclude this protein is subject to significant species-specific post-translation modifications. Further, bioinformatic analysis of the novel structure of MESP-1 suggests that this protein may be an integral membrane protein involved in sperm–egg fusion, and/or released to the vitelline envelope.