The Molecules of Mammalian Fertilization

Abstract

Fertilization proceeds as a well-choreographed cascade of cell–cell interactions between gametes of opposite mating types (68Yanagimachi, R. (1994). Mammalian fertilization. In The Physiology of Reproduction, E. Knobil and J.D. Neill, eds. (New York: Raven Press), pp. 189–317.Google Scholar, 62Wassarman P.M Towards a molecular mechanism for gamete adhesion and fusion during mammalian fertilization.Curr. Opin. Cell Biol. 1995; 7: 658-664Crossref PubMed Scopus (69) Google Scholar). This review focuses on mammalian fertilization (primarily in the mouse) and in particular on proteins involved in the cell–cell adhesion, fusion, and signaling events that ensue when sperm meets egg. It is noteworthy that although sperm and eggs are highly specialized, many of the mechanisms and some of the players in other cell adhesion and signaling reactions appear to be utilized in the dance of the gametes. Following their deposition and modification (“capacitation”) in the female reproductive tract (68Yanagimachi, R. (1994). Mammalian fertilization. In The Physiology of Reproduction, E. Knobil and J.D. Neill, eds. (New York: Raven Press), pp. 189–317.Google Scholar), mammalian sperm chart a tortuous path, and may even be lured, toward the egg (45Ralt D Goldenberg M Thompson D Dor J Mashiach S Garbers D.L Eisenbach M Sperm attraction to a follicular factor(s) correlates with human egg fertilizability.Proc. Natl. Acad. Sci. USA. 1991; 88: 2840-2844Crossref PubMed Scopus (153) Google Scholar, 57Vanderhaeghen P Schurmans S Vassart G Parmentier M Olfactory receptors are displayed on dog mature sperm cells.J. 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In mammals, sperm–egg interaction can be subdivided into six steps (Figure 1): acrosome-intact sperm first bind to the zona pellucida (ZP) (step 1), a thick extracellular matrix comprising the outer layer of the egg; interaction with the ZP signals sperm to undergo the acrosome reaction (step 2), a regulated exocytic event that is essential for subsequent steps; sperm penetrate the ZP (step 3) and, having done so, bind to (step 4) and fuse with (step 5) the egg plasma membrane; these latter events somehow “activate” the egg (step 6) to initiate zygotic development and to become refractory to the overtures of additional sperm. Thus, to deliver its haploid genome, a sperm must penetrate a first barrier imposed by a wall of cumulus cells, and then it must bind to and breach two barriers imposed by the egg proper, the ZP and the plasma membrane. Significant progress has been made in identifying proteins in both the egg ZP as well as in the sperm plasma membrane that mediate the first steps of sperm–egg interaction (Figure 2). Of the three major proteins in the mouse ZP (ZP1, ZP2, and ZP3), ZP3 has emerged as the molecule responsible for species-specific binding (63Wassarman P.M Litscher E.S Sperm–egg recognition mechanisms in mammals.Curr. Top. Dev. Biol. 1995; 30: 1-19Crossref PubMed Scopus (78) Google Scholar). As might be expected for a ZP molecule with important structural and functional roles, preventing expression of the mouse gene encoding ZP3 results in production of eggs lacking a ZP (19Gong X.H Dubois D.H Miller D.J Shur B.D Activation of a G protein complex by aggregation of β-1,4 galactosyltransferase on the surface of sperm.Science. 1995; 269: 1718-1721Crossref PubMed Scopus (149) Google Scholar, 34Liu C Litscher E.S Mortillo S Sakai Y Kinloch R.A Stewart C.L Wassarman P.M Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice.Proc. Natl. Acad. Sci. USA, in press. 1996; Google Scholar) and infertility in female mice (34Liu C Litscher E.S Mortillo S Sakai Y Kinloch R.A Stewart C.L Wassarman P.M Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice.Proc. Natl. Acad. Sci. USA, in press. 1996; Google Scholar). The ZPs in other mammalian species contain homologs to the mouse ZP proteins; in some, ZP3 homologs are implicated as the key adhesion molecules, whereas in others, ZP2 homologs may play a dominant role (56Topfer-Petersen E Mann K Calvete J.J Identification of porcine oocyte 55 kDa alpha and beta proteins within the zona pellucida glycoprotein families indicates that oocyte sperm receptor activity is associated with different zona pellucida proteins in different mammalian species.Biol. Chem. Hoppe Seyler. 1993; 374: 411-417Crossref PubMed Scopus (14) Google Scholar). Although O-linked oligosaccharides on ZP3 are strongly implicated as being responsible for both sperm binding and signaling for the acrosome reaction, the composition and structure of the essential carbohydrate moieties remain controversial. Compelling evidence supports roles for both a terminal Gal residue in α or β linkage (63Wassarman P.M Litscher E.S Sperm–egg recognition mechanisms in mammals.Curr. Top. Dev. Biol. 1995; 30: 1-19Crossref PubMed Scopus (78) Google Scholar) as well as for a terminal N-acetylglucosamine (GlcNAc) in β linkage (35Miller D.J Macek M.B Shur B.D Complementarity between sperm surface β-1,4-galactosyl-transferase and egg-coat ZP3 mediates sperm egg binding.Nature. 1992; 357: 589-593Crossref PubMed Scopus (399) Google Scholar). The finding, however, that mice deficient in the glycosyltransferase that amends terminal Gal in α linkage are fully fertile (54Thall A.D Maly P Lowe J.B Oocyte Gal alpha 1,3 Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse.J. Biol. Chem. 1995; 270: 21437-21440Crossref PubMed Scopus (313) Google Scholar) points to Gal in β linkage or GlcNAc (or both) as critical moieties. Moreover, even though in vitro experiments have failed to show more than a passive role for the ZP3 polypeptide, intact ZP3 exhibits more effective sperm binding activity than purified ZP3 glycopeptides, which themselves are more effective than simple O-linked oligosaccharides. Thus, it remains to be established if the ZP3 polypeptide plays a direct role in adhesion, or if it simply influences the “presentation” of the oligosaccharides (63Wassarman P.M Litscher E.S Sperm–egg recognition mechanisms in mammals.Curr. Top. Dev. Biol. 1995; 30: 1-19Crossref PubMed Scopus (78) Google Scholar). Having been identified as a key adhesion molecule on mouse eggs, ZP3 has become the lens through which many investigators have sought to identify sperm surface proteins responsible for ZP adhesion. Several features of ZP3 have helped to define criteria that sperm proteins should satisfy in order to fulfill this role: ZP adhesion molecules must be on the surface of live acrosome-intact sperm and they must bind to ZP3. ZP adhesion molecules should display species specificity in binding, and antibodies to them should interfere with adhesion. Moreover, reflecting the inactivation of ZP3 that accompanies the major block to polyspermy (63Wassarman P.M Litscher E.S Sperm–egg recognition mechanisms in mammals.Curr. Top. Dev. Biol. 1995; 30: 1-19Crossref PubMed Scopus (78) Google Scholar), candidate sperm adhesion molecules should not bind to ZP3 from fertilized eggs. Confirmation as a bona fide ZP adhesion molecule should involve a combination of gene disruption and gene transfer experiments. These criteria do not encompass the additional possibility that proteins involved in binding to the ZP may also be involved in triggering the acrosome reaction. As will be noted below, several candidate ZP adhesion molecules on sperm are proposed to fulfill both binding and signaling roles, as has been shown for adhesion/signaling molecules in other systems (24Hynes R.O Integrins versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8813) Google Scholar, 51Springer T.A Traffic signals for lymphocyte recirculation and leukocyte emigration the multistep paradigm.Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6277) Google Scholar, 12Clark E.A Brugge J.S Integrins and signal transduction pathways the road taken.Science. 1995; 268: 233-239Crossref PubMed Scopus (2758) Google Scholar). To date three proteins have emerged as candidate ZP3 adhesion molecules on acrosome-intact mouse sperm (Figure 2): a β-1,4 galactosyl transferase (GalTase) (35Miller D.J Macek M.B Shur B.D Complementarity between sperm surface β-1,4-galactosyl-transferase and egg-coat ZP3 mediates sperm egg binding.Nature. 1992; 357: 589-593Crossref PubMed Scopus (399) Google Scholar, 19Gong X.H Dubois D.H Miller D.J Shur B.D Activation of a G protein complex by aggregation of β-1,4 galactosyltransferase on the surface of sperm.Science. 1995; 269: 1718-1721Crossref PubMed Scopus (149) Google Scholar); a 56 kDa protein (4Bleil J.D Wassarman P.M Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking.Proc. Natl. Acad. Sci. USA. 1990; 87: 5563-5567Crossref PubMed Scopus (179) Google Scholar); and a tyrosine-phosphorylated protein, p95 (29Leyton L Saling P 95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding.Cell. 1989; 57: 1123-1130Abstract Full Text PDF PubMed Scopus (265) Google Scholar). Sperm surface GalTase stands out as the candidate adhesion/signaling molecule with the longest history and most extensive experimental support. Although the biosynthetic Golgi form of this enzyme is better known, both somatic cells and sperm express a cell surface GalTase that functions as a receptor for extracellular oligosaccharide ligands with terminal GlcNAc (35Miller D.J Macek M.B Shur B.D Complementarity between sperm surface β-1,4-galactosyl-transferase and egg-coat ZP3 mediates sperm egg binding.Nature. 1992; 357: 589-593Crossref PubMed Scopus (399) Google Scholar). GalTase first was implicated in ZP adhesion more than 15 years ago when it was reported that mouse sperm carrying certain t haplotypes had increased fertilizing ability that correlated with increased levels of surface GalTase activity (49Shur B.D Bennett D A specific defect in galactosyltransferase regulation on sperm bearing mutant alleles of the T/t locus.Dev. Biol. 1979; 71: 243-259Crossref PubMed Scopus (71) Google Scholar). The evidence supporting roles for GalTase is as follows. Sperm GalTase is found on the surface of live sperm (19Gong X.H Dubois D.H Miller D.J Shur B.D Activation of a G protein complex by aggregation of β-1,4 galactosyltransferase on the surface of sperm.Science. 1995; 269: 1718-1721Crossref PubMed Scopus (149) Google Scholar) and interacts selectively with ZP3, as evidenced by its ability to transfer 3H-galactose only to ZP3 and not to the other mouse ZP proteins, ZP2 or ZP1 (35Miller D.J Macek M.B Shur B.D Complementarity between sperm surface β-1,4-galactosyl-transferase and egg-coat ZP3 mediates sperm egg binding.Nature. 1992; 357: 589-593Crossref PubMed Scopus (399) Google Scholar). Purified GalTase, inhibitors of GalTase, and antibodies to GalTase each block sperm–egg binding. (50Shur B.D Neely C.A Plasma membrane association, purification, and partial characterization of mouse sperm beta 1,4-galactosyltransferase.J. Biol. Chem. 1988; 263: 17706-17714Abstract Full Text PDF PubMed Google Scholar). Overexpression of sperm surface GalTase in transgenic mice leads to the production of sperm that bind more radiolabeled ZP3 and are hypersensitive to ZP-induced acrosome reactions. (69Youakim A Hathaway H.J Miller D.J Gong X Shur B.D Overexpressing sperm surface β1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm–egg interactions.J. Cell Biol. 1994; 126: 1573-1583Crossref PubMed Scopus (72) Google Scholar). Crosslinking of GalTase by anti-GalTase antibodies activates G proteins and induces the acrosome reaction (35Miller D.J Macek M.B Shur B.D Complementarity between sperm surface β-1,4-galactosyl-transferase and egg-coat ZP3 mediates sperm egg binding.Nature. 1992; 357: 589-593Crossref PubMed Scopus (399) Google Scholar, 19Gong X.H Dubois D.H Miller D.J Shur B.D Activation of a G protein complex by aggregation of β-1,4 galactosyltransferase on the surface of sperm.Science. 1995; 269: 1718-1721Crossref PubMed Scopus (149) Google Scholar). In addition, modifications of the GalTase binding site on ZP3 by an N-acetylglucosaminidase released from egg cortical granules upon egg activation can account for the absence of sperm binding activity of ZP3 on the fertilized egg and two-cell embryo. Correspondingly, inhibition of the cortical granule N-acetylglucosaminidase blocks the inactivation of sperm binding that normally accompanies fertilization (36Miller D.J Gong X Decker G Shur B.D Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.J. Cell. Biol. 1993; 123: 1431-1440Crossref PubMed Scopus (132) Google Scholar). Although the precise location of GalTase on the sperm head, the extent of the acrosome reaction induced by GalTase crosslinking (10Cardullo R.A Wolf D.E Distribution and dynamics of mouse sperm surface galactosyltransferase implications for mammalian fertilization.Biochemistry. 1995; 34: 10027-10035Crossref PubMed Scopus (25) Google Scholar), and the species specificity of GalTase remain to be clarified, collectively the findings cited above support a role for GalTase in sperm–egg binding and signaling. A second candidate ZP adhesion molecule, sp56, emerged as one of the few sperm proteins that is radiolabeled preferentially by a photoactivatable crosslinker covalently linked to ZP3 (4Bleil J.D Wassarman P.M Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking.Proc. Natl. Acad. Sci. USA. 1990; 87: 5563-5567Crossref PubMed Scopus (179) Google Scholar). sp56 also binds tightly to ZP3 affinity columns and to ZP3 oligosaccharides recognized by sperm, but not to ZP from fertilized eggs. The sp56 protein is localized to heads of fixed, acrosome-intact sperm, and purified sp56 blocks sperm–egg binding (4Bleil J.D Wassarman P.M Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking.Proc. Natl. Acad. Sci. USA. 1990; 87: 5563-5567Crossref PubMed Scopus (179) Google Scholar, 7Bookbinder L.H Cheng A Bleil J.D Tissue- and species-specific expression of sp56, a mouse sperm fertilization protein.Science. 1995; 269: 86-89Crossref PubMed Scopus (137) Google Scholar, 11Cheng A Le T Palacios M Bookbinder L.H Wassarman P.M Suzuki F Bleil J.D Sperm-egg recognition in the mouse characterization of sp56, a sperm protein having specific affinity for ZP3.J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (140) Google Scholar). Recent studies show that sp56 is a peripheral membrane protein unique to testis, that its mRNA is detectable only in spermatids and testicular spermatozoa, and that a highly related sp56 homolog is present in hamster but not in guinea pig or human. Analysis of the deduced amino acid sequence of sp56 indicates that it contains multiple consensus repeats of 60 amino acids, termed Sushi domains, making sp56 a member of a protein superfamily that includes several proteins involved in protein–protein interactions. Even though sp56 is a lectin that binds oligosaccharides on ZP3, it does not have regions with significant homology to carbohydrate recognition domains (CRDs) of previously characterized lectins (7Bookbinder L.H Cheng A Bleil J.D Tissue- and species-specific expression of sp56, a mouse sperm fertilization protein.Science. 1995; 269: 86-89Crossref PubMed Scopus (137) Google Scholar). Although the data cited above support the idea that sp56 is a ZP3-binding protein, further experiments are required to learn more about its physiological role in the initial steps of sperm–egg binding. For example, it will be important to determine if sp56 is on the surface of live sperm, whether anti-sp56 monoclonal antibodies interfere with sperm–egg binding, and finally, since sp56 is a peripheral membrane protein, whether it associates with a transmembrane protein that links it to a signaling pathway. The third candidate ZP adhesion molecule is mouse p95, which became a candidate adhesion molecule because of its ability to bind radiolabeled ZP3 on ligand blots of total sperm proteins. As outlined below, mouse p95 is a tantalizing candidate because it is phosphorylated on tyrosine and because a cDNA for a putative human analog encodes a transmembrane tyrosine kinase. Thus, binding of ZP3 to mouse p95 could activate the protein tyrosine kinase activity of mouse p95 and initiate a signaling cascade leading to the acrosome reaction. While this is an appealing hypothesis, the molecular identity of mouse p95 and the relationship between mouse p95 and the putative human analog remain to be established. Evidence suggesting that mouse p95 participates in adhesion/signaling consists of the observations that its levels of tyrosine phosphorylation increase during capacitation (and again during the acrosome reaction) and that protein eluted from the 95 kDa portion of SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels of mouse sperm proteins has protein kinase activity that can be stimulated by ZP proteins in a manner sensitive to protein tyrosine kinase inhibitors (29Leyton L Saling P 95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding.Cell. 1989; 57: 1123-1130Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 30Leyton L Leguen P Bunch D Saling P.M Regulation of mouse gamete interaction by a sperm tyrosine kinase.Proc. Natl. Acad. Sci. USA. 1992; 89: 11692-11695Crossref PubMed Scopus (131) Google Scholar). Furthermore, a monoclonal antibody, LL95, raised against an electroeluted 95 kDa protein from mouse sperm membranes stains mouse sperm heads and inhibits sperm–egg binding (31Leyton L Tomes C Saling P LL95 monoclonal antibody mimics functional effects of ZP3 on mouse sperm evidence that the antigen recognized is not hexokinase.Mol. Reprod. Dev. 1995; 42: 347-358Crossref PubMed Scopus (18) Google Scholar). A critical issue in this story is the molecular identity of mouse p95, as the LL95 antigen has not yet been cloned and sequenced. Moreover, mouse sperm express multiple tyrosine-phosphorylated proteins that migrate at ∼95 kDa on SDS–PAGE and that appear to have different properties. One of these proteins, referred to as p95/hexokinase, is a testis-specific, tyrosine-phosphorylated hexokinase (26Kalab P Visconti P Leclerc P Kopf G.S p95, the major phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase with unique properties.J. Biol. Chem. 1994; 269: 3810-3817Abstract Full Text PDF PubMed Google Scholar), homologs of which have protein kinase activity and undergo autophosphorylation (23Herrero P Fernandez R Moreno F The hexokinase isoenzyme PII of Saccharomyces cerevisiae is a protein kinase.J. Gen. Microbiol. 1989; 135: 1209-1216PubMed Google Scholar). p95/hexokinase behaves as an integral membrane protein with a cell surface–exposed domain, and it is found on the head and flagella of sperm (58Visconti P.E Olds-Clarke P Moss S.B Kalab P Travis A.J Heras M Kopf G.S Properties and location of a tyrosine phosphorylated form of hexokinase in mouse sperm.Mol. Reprod. Dev. 1996; 43: 82-93Crossref PubMed Scopus (41) Google Scholar). Even though recent experiments with sperm from sterile mutant mice suggest a possible role for p95/hexokinase in fertilization, at present it is not considered a strong candidate as an adhesion/signaling molecule (41Olds-Clarke P Pilder S.H Visconti P.E Moss S.B Orth J.M Kopf G.S Sperm from mice carrying two t haplotypes do not possess a tyrosine phosphorylated form of hexokinase.Mol. Reprod. Dev. 1996; 43: 94-104Crossref PubMed Scopus (15) Google Scholar, 58Visconti P.E Olds-Clarke P Moss S.B Kalab P Travis A.J Heras M Kopf G.S Properties and location of a tyrosine phosphorylated form of hexokinase in mouse sperm.Mol. Reprod. Dev. 1996; 43: 82-93Crossref PubMed Scopus (41) Google Scholar). It should be noted that the relative migration on SDS–PAGE gels of the major 95 kDa proteins of mouse sperm depends on the presence or absence of reducing agents and varies from laboratory to laboratory. Hence, although monoclonal antibody LL95 and an anti-hexokinase antibody do not appear to recognize the same proteins (31Leyton L Tomes C Saling P LL95 monoclonal antibody mimics functional effects of ZP3 on mouse sperm evidence that the antigen recognized is not hexokinase.Mol. Reprod. Dev. 1995; 42: 347-358Crossref PubMed Scopus (18) Google Scholar), the structures and functions of the originally described mouse p95 (29Leyton L Saling P 95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding.Cell. 1989; 57: 1123-1130Abstract Full Text PDF PubMed Scopus (265) Google Scholar), the LL95 antigen (31Leyton L Tomes C Saling P LL95 monoclonal antibody mimics functional effects of ZP3 on mouse sperm evidence that the antigen recognized is not hexokinase.Mol. Reprod. Dev. 1995; 42: 347-358Crossref PubMed Scopus (18) Google Scholar), and p95/hexokinase (26Kalab P Visconti P Leclerc P Kopf G.S p95, the major phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase with unique properties.J. Biol. Chem. 1994; 269: 3810-3817Abstract Full Text PDF PubMed Google Scholar) remain to be clarified. Resolutions to these issues are needed before roles for these 95 kDa proteins can be ascribed with confidence. Moreover, if mouse p95 does play a role in initial sperm–egg interactions, it should be possible to demonstrate binding between ZP3 and p95 on live mouse sperm or on p95-transfected cells. In studies with human sperm, an antibody against a peptide derived from a newly cloned cDNA, hu9, immunoprecipitates a 95 kDa tyrosine-phosphorylated protein. The immunoprecipitated protein has protein kinase activity that can be stimulated by recombinant human ZP3 in immune complex assays (9Burks D.J Carballada R Moore H.D.M Saling P.M Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization.Science. 1995; 269: 83-86Crossref PubMed Scopus (159) Google Scholar). Synthetic peptides corresponding to regions of the extracellular domain of HU9 (referred to as zona–receptor kinase) inhibit sperm–egg binding. In the mouse, transcripts that hybridize with hu9 were detected exclusively in the luminal compartment of the seminiferous tubules. The hu9 cDNA encodes a transmembrane protein tyrosine kinase with a catalytic domain similar to that of the proto-oncogene, v-ryk. Although HU9 has properties consistent with a role in ZP3-mediated signal transduction, it remains to be shown that human ZP3 binds to HU9 on intact human sperm (or transfected somatic cells) or that HU9 is localized to the head region of the plasma membrane of live human sperm. In addition, although it is highly likely, it remains to be shown formally that HU9 has tyrosine kinase activity, as the antibody used for this purpose was directed against a peptide that is also present in c-mer, a highly related human transmembrane tyrosine kinase whose mRNA is expressed in testis (21Graham D.K Dawson T.L Mullaney D.L Snodgrass H.R Earp H.S Cloning and mRNA expression analysis of a novel human protooncogene, c-mer.Cell Growth Diff. 1994; 5: 647-657PubMed Google Scholar). Finally, although HU9 has properties that are reminiscent of those of mouse p95, further studies are needed to assess whether mouse p95 and HU9 are homologs. Studies of sperm in other species have yielded yet other candidate ZP adhesion molecules including rabbit sperm autoantigen and spermadhesins, proacrosin/acrosin, and zonadhesin in pig (62Wassarman P.M Towards a molecular mechanism for gamete adhesion and fusion during mammalian fertilization.Curr. Opin. Cell Biol. 1995; 7: 658-664Crossref PubMed Scopus (69) Google Scholar). Zonadhesin is particularly noteworthy as it shows high affinity species-specific binding and contains carbohydrate-binding domains (22Hardy D.M Garbers D.L A sperm membrane protein that binds in a species-specific manner to the egg extracellular matrix is homologous to van Willebrand factor.J. Biol. Chem. 1995; 270: 26025-26028Crossref PubMed Scopus (136) Google Scholar). Once all of these potential ZP adhesion molecules are tested by time, it would not be surprising if many of them remain as important players in fertilization. Sperm–egg or sperm–ZP(3) adhesion in vitro is an operational definition that will vary from laboratory to laboratory, and both low affinity–high capacity as well as high affinity–low capacity binding interactions almost surely will be important. The various candidates may represent redundant functions to ensure that this most important of events for a species occurs without fail. Alternatively, the proteins each may play a different role in adhesion and signaling, as has been shown for several adhesion molecules on immune cells (51Springer T.A Traffic signals for lymphocyte recirculation and leukocyte emigration the multistep paradigm.Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6277) Google Scholar) or as is the case for several proteins that play distinct roles in binding and responding to soluble ligands (48Schlessinger J Lax I Lemmon M Regulation of growth factor activation by proteoglycans What is the role of low affinity receptors?.Cell. 1995; 83: 357-360Abstract Full Text PDF PubMed Scopus (444) Google Scholar). That unique ZP adhesion proteins have been identified in sperm of other mammalian species may indicate that sperm–egg adhesion molecules have not been conserved in evolution; rather, it may be that variations in these gamete adhesion molecules drive speciation (20Goodenough U.W Armbrust E.V Campbell A.M Ferris P.J Molecular genetics of sexuality in chlamydomonas.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 21-44Crossref Scopus (43) Google Scholar, 52Swanson W.J Vacquier V.D Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa.Proc. Natl. Acad. Sci. USA. 1995; 92: 4957-4961Crossref PubMed Scopus (117) Google Scholar). While the initial interactions at the surface of the ZP essentially go unnoticed by the egg, the acrosome reaction that they initiate is critical for transforming the sperm into a fusogenic cell (68Yanagimachi, R. (1994). Mammalian fertilization. In The Physiology of Reproduction, E. Knobil and J.D. Neill, eds. (New York: Raven Press), pp. 189–317.Google Scholar). In addition to release of hydrolytic enzymes presumably required for sperm to penetrate the ZP, the acrosome reaction brings about remodeling of the sperm surface, with a portion of the original plasma membrane being replaced by the newly exposed inner acrosomal membrane. Although many of the details of this process remain unclear, membrane proteins undergo changes in location and activity required both for continued adhesion to the ZP as well as for fusion with the egg plasma membrane (38Myles D.G Molecular mechanisms of sperm–egg membrane binding and fusion in mammals.Dev. Biol. 1993; 158: 35-45Crossref PubMed Scopus (136) Google Scholar, 68Yanagimachi, R. (1994). Mammalian fertilization. In The Physiology of Reproduction, E. Knobil and J.D. Neill, eds. (New York: Raven Press), pp. 189–317.Google Scholar). Our understanding of the signal transduction mechanisms that couple sperm binding to the ZP with acrosomal exocytosis is at an early stage. One of the central features of the acrosome reaction is an influx of extracellular Ca2+. By analogy with other regulated exocytic events, this influx likely promotes the requisite fusion reaction between the acrosomal membrane and the overlying sperm plasma membrane (Figure 1B). It has been shown recently that interaction of sperm with ZP proteins initiates the sequential activation of a poorly selective cation channel and a voltage sensitive Ca2+ channel, thereby leading to large increases in intracellular Ca2+ (16Florman H.M Sequential focal and global elevations of sperm intracellular Ca2+ are initiated by the zona pellucida during acrosomal exocytosis.Dev. Biol. 1994; 165: 152-164Crossref PubMed Scopus (170) Google Scholar). While the mechanisms leading to this increase still are unclear, several elements of signaling pathways associated with Ca2+ flux in other systems have been identified in sperm. For example, protein kinase activity regulated by ZP interactions (29Leyton L Saling P 95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding.Cell. 1989; 57: 1123-1130Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 18Fraser L.R Na+ requirements for capacitation and acrosomal exocytosis in mammalian sperm.Int. Rev. Cytol. 1994; 149: 1-49Crossref PubMed Scopus (4) Google Scholar) could affect Na+/H+ exchange and thereby lead to changes in Ca2+ flux. Heterotrimeric G proteins of the class Gi1 and Gi2, which activate