Mammalian Fertilization: Molecular Aspects of Gamete Adhesion, Exocytosis, and Fusion

Abstract

Fertilization is defined as the process of union of two germ cells, egg and sperm, whereby the somatic chromosome number is restored and the development of a new individual exhibiting characteristics of the species is initiated. If fertilization fails to take place, both egg and sperm degenerate relatively rapidly in the female reproductive tract, since the two highly differentiated cells cannot survive long on their own. Among mammals, the process of union of germ cells includes several ordered steps (42Gwatkin R.B.L Fertilization Mechanisms in Man and Mammals. Plenum Press, New York1977Crossref Google Scholar, 81Wassarman P.M The biology and chemistry of fertilization.Science. 1987; 235: 553-560Crossref PubMed Scopus (295) Google Scholar, 95Yanagimachi, R. (1994). Mammalian fertilization. In The Physiology of Reproduction, E. Knobil and J.D. Neill, eds. (Raven Press, New York), pp. 189–317.Google Scholar, 72Snell W.J White J.M The molecules of mammalian fertilization.Cell. 1996; 85: 629-637Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). It begins in the oviduct with binding of free-swimming sperm to the ovulated egg extracellular coat, the zona pellucida (ZP) (Figure 1 and Figure 2), and ends a short time later with fusion of egg and sperm plasma membranes to form a single “activated” cell, the zygote. Along the way, several recognizable events take place, including the sperm acrosome reaction (a form of cellular exocytosis), penetration of the egg ZP by sperm, and the egg cortical reaction and zona reaction. The latter results in alteration of the ZP such that free-swimming sperm are unable to bind to fertilized eggs. Each of these events in the fertilization pathway has been studied in some detail.Figure 2Schematic Diagram of Sperm–Egg Interaction in MammalsShow full captionShown are morphological features of an acrosome-intact mammalian sperm bound to the ZP of an unfertilized egg by plasma membrane overlying the anterior region of the sperm head.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Shown are morphological features of an acrosome-intact mammalian sperm bound to the ZP of an unfertilized egg by plasma membrane overlying the anterior region of the sperm head. This review focuses on molecules currently thought to be involved in three steps of the mammalian fertilization process: (1) binding of sperm to eggs; (2) induction of the acrosome reaction by sperm; and (3) fusion of sperm and eggs. Much of the research discussed was carried out in mice. In recent years, considerable progress has been made toward delineating the molecular basis of each of these steps, especially in mice and other rodents. However, it is anticipated that some of the ideas presented may need to be modified as a result of ongoing research on a variety of mammals, including humans. The final steps of mammalian oogenesis and spermatogenesis prepare eggs and sperm, respectively, for fertilization. During ovulation, fully grown oocytes from antral (Graafian) follicles undergo “meiotic maturation,” a process that transforms fully grown oocytes into unfertilized eggs prepared to interact with sperm (86Wassarman, P.M., and Albertini, D.F. (1994). The mammalian ovum. In The Physiology of Reproduction, E. Knobil and J.D. Neill, eds. (New York: Raven Press), pp. 79–122.Google Scholar). Similarly, following deposition into and migration up the female reproductive tract, sperm undergo “capacitation,” a process that enables sperm to bind to eggs and to undergo the acrosome reaction (24Darszon A Liévano A Beltran C Ion channels key elements in gamete signaling.Curr. Topics Dev. Biol. 1996; 34: 117-167Crossref PubMed Google Scholar, 76Visconti P.E Kopf G.S Regulation of protein phosphorylation during sperm capacitation.Biol. Reprod. 1998; 59: 1-6Crossref PubMed Scopus (389) Google Scholar). Capacitation probably involves removal of inhibitory factors from sperm accompanied by membrane protein and lipid rearrangements and/or modifications. Apparently, some alterations are mediated, at least in part, by cAMP-dependent protein tyrosine phosphorylation, as well as by changes in pH and Ca2+ concentrations. Meiotic maturation of oocytes and capacitation of sperm propel gametes down a path that leads either to formation of a viable zygote or to degeneration of the cells. Typically, very few ovulated eggs are found in oviducts of females (e.g., humans, ∼1; mice, ∼10). Similarly, relatively few sperm are found at the site of fertilization (∼100–150) as compared to the number of sperm deposited into the female reproductive tract (∼107). A very low percentage of ejaculated sperm ever make their way to the position of unfertilized eggs in the oviduct (∼0.002%). Whether binding of sperm to eggs occurs due to a chance encounter of gametes in the oviduct or is promoted by a chemical gradient stimulus (“sperm chemotaxis”), as found with many nonmammalian species, remains to be resolved. In this context, it should be noted that there is good in vitro evidence for human sperm chemotaxis mediated by an egg follicular factor (28Eisenbach M Tur-Kaspa I Do human eggs attract spermatozoa?.BioEssays. 1998; in pressGoogle Scholar). It is well known that hybrids of certain mammalian species are viable; for example, mules derived from a cross between male donkeys and female horses. Does this mean that mammalian fertilization does not exhibit any species specificity? On the contrary, evidence from in vitro fertilization experiments strongly suggests that there are barriers to interspecies fertilization and that the egg ZP serves as a major barrier (95Yanagimachi, R. (1994). Mammalian fertilization. In The Physiology of Reproduction, E. Knobil and J.D. Neill, eds. (Raven Press, New York), pp. 189–317.Google Scholar). The ZP can interfere with interspecies fertilization by failing to permit the initial binding of sperm to eggs, induction of the acrosome reaction, or penetration of bound sperm through the egg extracellular coat. Although the restrictions on binding are not absolute (e.g., mouse sperm bind to hamster eggs and hamster sperm bind to mouse eggs), they provide for a relatively high degree of species-specific fertilization in vitro (e.g., guinea pig and human sperm do not bind to mouse eggs). Notably, removal of the ZP from unfertilized eggs, thereby exposing egg plasma membrane directly to sperm, virtually eliminates the barrier to fertilization between species in vitro. A reductionist’s view of the binding of sperm to eggs in mammals would have a molecule located on the sperm head (“egg-binding protein”) recognizing and binding to a complementary molecule located on the egg ZP (“sperm receptor”) in a species-specific manner. Such a situation is analogous to many other cellular adhesion events. Among these are binding of bacteria, animal viruses, and other pathogens to their cellular hosts, binding of pollen to the plant stigma, sexual agglutination in yeast, and binding of sperm to unfertilized eggs (e.g., sea urchin, abalone, and Xenopus) during fertilization. In each case, complementary molecules that support highly specific cellular adhesion are found on the surfaces of participating cells. In certain cases, the cellular adhesion is thought to be carbohydrate mediated. The plasma membrane of all mammalian eggs is completely surrounded by a ZP. Nearly 20 years ago it was demonstrated that the mouse egg ZP (∼6.2 μm thick containing ∼3.5 ng of protein) is composed of only three glycoproteins, called mZP1 (∼200 kDa; dimer), mZP2 (∼120 kDa), and mZP3 (∼83 kDa) (10Bleil J.D Wassarman P.M Structure and function of the zona pellucida identification and characterization of the proteins of the mouse oocyte’s zona pellucida.Dev. Biol. 1980; 76 (a): 185-202Crossref PubMed Scopus (451) Google Scholar, 82Wassarman P.M Zona pellucida glycoproteins.Annu. Rev. Biochem. 1988; 57: 415-442Crossref PubMed Scopus (467) Google Scholar). In mice, the three glycoproteins are synthesized exclusively by growing oocytes. Two of the glycoproteins, mZP2 and mZP3, interact with each other via noncovalent bonds to form long filaments bearing a 14–15 nm structural repeat that are interconnected by mZP1 (Figure 3). Each of these glycoproteins consists of a unique polypeptide that is heterogeneously glycosylated with both complex-type asparagine- (N-) linked and serine/threonine- (O-) linked oligosaccharides. Targeted disruption of the mZP3 gene by homologous recombination in embryonic stem (ES) cells has no effect on the phenotype of male mice, but it results in infertility in homozygous null females (55Liu 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. 1996; 93: 5431-5436Crossref PubMed Scopus (188) Google Scholar, 63Rankin T Familiari M Lee E Ginsburg A Dwyer N Blanchette-Mackie J Drago J Westphal H Dean J Mice homozygous for an insertional mutation in the ZP3 gene lack a zona pellucida and are infertile.Development. 1996; 122: 2903-2910PubMed Google Scholar). Ovaries from the homozygous null females (mZP3−/−) contain growing oocytes that completely lack a ZP, while oocytes from heterozygous null females (mZP3+/−) have a ZP that is about one-half the thickness (∼2.7 μm) of the wild type (90Wassarman P.M Qi H Litscher E.S Mutant female mice carrying a single mZP3 allele produce eggs with a thin zona pellucida, but reproduce normally.Proc. Roy. Soc. Lond. B. 1997; 264: 323-328Crossref PubMed Scopus (43) Google Scholar). These results are consistent with the proposed structural role of mZP3, as well as current models for ZP structure (88Wassarman P.M Mortillo S Structure of the mouse egg extracellular coat, the zona pellucida.Intl. Rev. Cytol. 1991; 130: 85-109Crossref PubMed Scopus (120) Google Scholar, 89Wassarman P.M Liu C Litscher E.S Constructing the mammalian egg zona pellucida some new pieces of an old puzzle.J. Cell Sci. 1996; 109: 2001-2004PubMed Google Scholar). Today, it is clear that the ZP of eggs from a wide variety of mammals, including humans, is composed of a small number of glycoproteins that are closely related (polypeptides ∼40%–90% similar) to mZP1–mZP3 (85Wassarman, P.M. (1999). Egg zona pellucida glycoproteins. In Guidebook to the Extracellular Matrix and Adhesion Proteins, T. Kreis and R. Vale, eds. (Oxford: Oxford University Press), in press.Google Scholar). For example, the positions of the 13 cysteine residues, as well as all of the recognizable domains of mZP3 polypeptide (Figure 4), are conserved in human ZP3 polypeptide. Even the vitelline envelope surrounding eggs from fish, birds, and amphibians contains glycoproteins whose polypeptides resemble mZP1–mZP3. Thus, there is a significant evolutionary link between glycoproteins of the vitelline envelope of nonmammalian eggs and glycoproteins of the ZP of mammalian eggs. Apparently, these glycoproteins play essential structural roles in assembling the extracellular coats during oogenesis. It will be of great interest in the future to compare the high-resolution structures of vitelline envelope and ZP glycoproteins. Only acrosome-intact sperm bind to the ovulated mouse egg ZP (33Florman H.M Storey B.T Mouse gamete interactions the zona pellucida is the site of the acrosome reaction leading to fertilization in vitro.Dev. Biol. 1982; 91: 121-130Crossref PubMed Scopus (221) Google Scholar, 12Bleil J.D Wassarman P.M Sperm-egg interactions in the mouse sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein.Dev. Biol. 1983; 95: 317-324Crossref PubMed Scopus (446) Google Scholar). Experimental evidence strongly supports the conclusion that, during binding of sperm to eggs, mZP3 serves as a receptor for sperm (Figure 3). For example, of the three glycoproteins that constitute the ZP, only purified mZP3 binds exclusively to heads of acrosome-intact sperm (i.e., to plasma membrane; ∼104 molecules of mZP3 per sperm head) and thereby prevents sperm from binding to ovulated eggs in vitro (11Bleil J.D Wassarman P.M Mammalian sperm-egg interaction identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm.Cell. 1980; 20 (b): 873-882Abstract Full Text PDF PubMed Scopus (502) Google Scholar, 13Bleil J.D Wassarman P.M Autoradiographic visualization of the mouse egg’s sperm receptor bound to sperm.J. Cell Biol. 1986; 102: 1363-1371Crossref PubMed Scopus (202) Google Scholar, 83Wassarman P.M Profile of a mammalian sperm receptor.Development. 1990; 108: 1-17Crossref PubMed Google Scholar, 58Mortillo S Wassarman P.M Differential binding of gold-labeled zona pellucida glycoproteins mZP2 and mZP3 to mouse sperm membrane compartments.Development. 1991; 113: 141-151PubMed Google Scholar, 87Wassarman P.M Litscher E.S Sperm-egg recognition mechanisms in mammals.Curr. Top. Dev. Biol. 1995; 30: 1-19Crossref PubMed Scopus (78) Google Scholar). Even at nanomolar concentrations, purified, unfertilized egg mZP3 is a very effective inhibitor of sperm binding in this competition assay. On the other hand, at similar concentrations, mZP3 from fertilized eggs or early embryos has no effect on binding of sperm to eggs in vitro. This is consistent with the failure of free-swimming sperm to bind to the ZP of fertilized eggs and preimplantation embryos. It can be concluded from these and other observations that, as a consequence of the zona reaction, mZP3 is altered such that free-swimming sperm can no longer recognize and bind to the glycoprotein (i.e., mZP3 is inactivated as a sperm receptor). What is it that acrosome-intact sperm recognize and bind to on mZP3? The ability of mZP3 to act as a sperm receptor in vitro is not significantly affected by exposure of the glycoprotein to high temperatures, detergents, denaturants, or reducing agents, or by limited proteolysis of the glycoprotein. Even after extensive proteolytic digestion of mZP3, the small glycopeptides produced retain activity as a sperm receptor, although higher than normal concentrations (∼50-fold) are required (35Florman H.M Bechtol K.D Wassarman P.M Enzymatic dissection of the functions of the mouse egg’s receptor for sperm.Dev. Biol. 1984; 106: 243-255Crossref PubMed Scopus (167) Google Scholar, 34Florman H.M Wassarman P.M O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity.Cell. 1985; 41: 313-324Abstract Full Text PDF PubMed Scopus (467) Google Scholar). These and other observations suggest that mZP3 polypeptide does not play a direct role in sperm receptor function. On the other hand, there is considerable data to suggest that mZP3 oligosaccharides do play a direct role in sperm receptor function (Figure 3). For example, chemical or enzymatic removal of all mZP3 oligosaccharides (N- and O-linked) results in complete inactivation of the glycoprotein as a sperm receptor. Furthermore, O-linked oligosaccharides recovered from mZP3 by mild alkaline hydrolysis under reducing conditions (34Florman H.M Wassarman P.M O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity.Cell. 1985; 41: 313-324Abstract Full Text PDF PubMed Scopus (467) Google Scholar, 14Bleil J.D Wassarman P.M Galactose at the non-reducing terminus of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein ZP3 is essential for the glycoprotein’s sperm receptor activity.Proc. Natl. Acad. Sci. USA. 1988; 85: 6778-6782Crossref PubMed Scopus (293) Google Scholar, 57Miller D.J Macek M.B Shur B.D Complementarity between sperm surface β1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding.Nature. 1992; 357: 589-593Crossref PubMed Scopus (399) Google Scholar) and certain O-linked related oligosaccharides synthesized in the laboratory (54Litscher E.S Juntunen K Seppo A Penttilä L Niemelä R Renkonen O Wassarman P.M Oligosaccharide constructs with defined structures that inhibit binding of mouse sperm to unfertilized eggs in vitro.Biochemistry. 1995; 34: 4662-4669Crossref PubMed Scopus (110) Google Scholar, 47Johnston D.S Wright W.W Shaper J.H Hokke C.H Van den Eijnden D.H Joziasse D.H Murine sperm-zona binding, a fucosyl residue is required for a high affinity sperm-binding ligand.J. Biol. Chem. 1998; 273: 1888-1895Crossref PubMed Scopus (113) Google Scholar) inhibit binding of sperm to eggs in vitro at micromolar concentrations. Collectively, these and other observations suggest that species-specific binding of sperm to eggs in mammals is a carbohydrate-mediated event. On the other hand, the identity of the sugars on mZP3 recognized by sperm remains unresolved, especially in view of results with homozygous null mice (73Thall A.D Maly P Lowe J.B Oocyte Galα1,3Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse.J. Biol. 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Biol. 1992; 154: 309-317Crossref PubMed Scopus (66) Google Scholar, 53Litscher E.S Wassarman P.M Characterization of a mouse ZP3-derived glycopeptide, gp55, that exhibits sperm receptor and acrosome reaction-inducing activity in vitro.Biochemistry. 1996; 35: 3980-3985Crossref PubMed Scopus (65) Google Scholar), exon swapping (50Kinloch R.M Sakai Y Wassarman P.M Mapping the mouse ZP3 combining site for sperm by exon swapping and site-directed mutagenesis.Proc. Natl. Acad. Sci. USA. 1995; 92: 263-267Crossref PubMed Scopus (93) Google Scholar), and site-directed mutagenesis (50Kinloch R.M Sakai Y Wassarman P.M Mapping the mouse ZP3 combining site for sperm by exon swapping and site-directed mutagenesis.Proc. Natl. Acad. Sci. USA. 1995; 92: 263-267Crossref PubMed Scopus (93) Google Scholar, 20Chen J Litscher E.S Wassarman P.M Inactivation of the mouse sperm receptor, mZP3, by site-directed mutagenesis of individual serine residues located at the combining-site for sperm.Proc. Natl. Acad. Sci. USA. 1998; 95: 6193-6197Crossref PubMed Scopus (120) Google Scholar) to identify the location of essential O-linked oligosaccharides on mZP3 polypeptide. Results of such studies suggest that these oligosaccharides are located on just two of five serine residues, serine-332 and serine−334, in a region of polypeptide near the carboxyl terminus encoded by exon-7 of the mZP3 gene (Figure 4). Interestingly, of the five serine residues, only these two are conserved from mouse to human ZP3. In this context, the numerous amino acid changes neighboring serine-332 and serine−334 that have occured during evolution may impose changes in the structure of O-linked oligosaccharides added to ZP3 and, thereby, affect species specificity of sperm–egg interaction (87Wassarman P.M Litscher E.S Sperm-egg recognition mechanisms in mammals.Curr. Top. Dev. Biol. 1995; 30: 1-19Crossref PubMed Scopus (78) Google Scholar). There is an extensive literature that deals with the search for egg-binding proteins (EBPs) on sperm that complement sperm receptors on the egg ZP (84Wassarman P.M Towards molecular mechanisms for gamete adhesion and fusion during mammalian fertilization.Curr. Opin. Cell Biol. 1995; 7: 658-664Crossref PubMed Scopus (69) Google Scholar, 72Snell W.J White J.M The molecules of mammalian fertilization.Cell. 1996; 85: 629-637Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). During the past 20 years, as many as two dozen different sperm proteins/glycoproteins have been implicated in the binding of sperm to eggs. A variety of mammalian species, from mice to humans, and experimental methodologies (e.g., Western blotting, affinity chromatography, chemical cross-linking, and solid-phase assays) have been used to identify these sperm components, which include a number of enzymes and lectin-like proteins. In several instances the components have been cloned and sequenced (e.g., β-galactosyltransferase, 71Shur, B.D. (1999). Cell surface galactosyltransferase. In Guidebook to the Extracellular Matrix and Adhesion Proteins, T. Kreis and R. Vale, eds. (Oxford: Oxford University Press), in press.Google Scholar; sperm protein-56, 16Bookbinder 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; zonadhesin, 39Gao Z Garbers D.L Species diversity in the structure of zonadhesin, a sperm-specific membrane protein containing multiple cell adhesion molecule-like domains.J. Biol. Chem. 1998; 273: 3415-3421Crossref PubMed Scopus (88) Google Scholar; and sperm protein-17, 64Richardson R.T Yamasaki N O’Rand M Sequence of a rabbit sperm zona pellucida binding protein and localization during the acrosome reaction.Dev. Biol. 1994; 165: 688-701Crossref PubMed Scopus (100) Google Scholar), in one case a high-resolution three-dimensional structure has been determined (spermadhesin, 68Romero A Romao M.J Varela P.F Kolln I Dias J.M Carvalho A.L Sanz L Töpfer-Petersen E Calvete J.J The crystal structures of two spermadhesins reveal the CUB domain fold.Nature Struct. Biol. 1997; 4: 783-788Crossref PubMed Scopus (126) Google Scholar), and, in at least one case, targeted mutagenesis of the gene has been carried out and homozygous null mice produced and characterized (β-galactosyltransferase, 56Lu Q Shur B.D Sperm from β1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly.Development. 1997; 124: 4121-4131Crossref PubMed Google Scholar). A partial list of candidate EBPs, together with a brief description of each protein, is presented in Table 1.Table 1Proposed Candidate EBPsCandidatesCommentsReferencesβ-galactosyltransferaseBinds to GlcNAc residues on mZP3 specifically57Miller D.J Macek M.B Shur B.D Complementarity between sperm surface β1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding.Nature. 1992; 357: 589-593Crossref PubMed Scopus (399) Google Scholar, 56Lu Q Shur B.D Sperm from β1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly.Development. 1997; 124: 4121-4131Crossref PubMed Google ScholarSperm protein-56Binds to mZP3 oligosaccharides; contains sushi and unique domains21Cheng A Le T Palacios M Bookbinder L.H Wassarman P.M 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, 16Bookbinder 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 ScholarZonadhesinBinds to the egg zona pellucida; contains multiple types of domains43Hardy D.M Garbers D.L A sperm membrane protein that binds in a species-specific manner to the egg extracellular matrix is homologous to von Willebrand factor.J. Biol. Chem. 1995; 270: 26025-26028Crossref PubMed Scopus (136) Google Scholar, 39Gao Z Garbers D.L Species diversity in the structure of zonadhesin, a sperm-specific membrane protein containing multiple cell adhesion molecule-like domains.J. Biol. Chem. 1998; 273: 3415-3421Crossref PubMed Scopus (88) Google ScholarSpermadhesinsExhibit carbohydrate-binding activity; possess a CUB domain26Dostalova Z Calvete J.J Sanz L Töpfer-Petersen E Boar spermadhesin AWN-1 oligosaccharide and zona pellucida binding characteristics.Eur. J. Biochem. 1995; 230: 329-336Crossref PubMed Scopus (100) Google Scholar, 68Romero A Romao M.J Varela P.F Kolln I Dias J.M Carvalho A.L Sanz L Töpfer-Petersen E Calvete J.J The crystal structures of two spermadhesins reveal the CUB domain fold.Nature Struct. Biol. 1997; 4: 783-788Crossref PubMed Scopus (126) Google ScholarZona receptor kinaseBinds to mZP3; identical to c-mer tyrosine kinase18Burks D.J Carballada R Moore H.D.M Saling P.M Interaction of tyrosine kinase from human sperm with the zona pellucida at fertilization.Science. 1995; 269: 83-86Crossref PubMed Scopus (159) Google Scholar, 17Bork P Sperm-egg binding protein or proto-oncogene?.Science. 1996; 271: 1431-1432Crossref PubMed Scopus (41) Google Scholar, 75Tsai J.-Y Silver L.M Sperm-egg binding protein or proto-oncogene.Science. 1996; 271: 1432-1434Crossref PubMed Scopus (23) Google ScholarMannose-binding proteinα-D-mannosidase that binds to mannose residues on ZP23Cornwall G.A Tulsiani D.R.P Orgebin-Crist M.-C Inhibition of the mouse sperm surface α-D-mannosidase inhibits sperm-egg binding in vitro.Biol. Reprod. 1991; 44: 913-921Crossref PubMed Scopus (133) Google ScholarGalactose-binding proteinRelated to asialoglycoprotein receptor1Abdullah M Kierzenbaum A.L Identification of rat testis galactosyl receptor using antibodies to liver asialoglycoprotein receptor purification and localization on the surface of spermatogenic cells and sperm.J. Cell Biol. 1989; 108: 367-375Crossref PubMed Scopus (62) Google ScholarSperm protein-17Sperm-specific autoantigen that binds to ZP64Richardson R.T Yamasaki N O’Rand M Sequence of a rabbit sperm zona pellucida binding protein and localization during the acrosome reaction.Dev. Biol. 1994; 165: 688-701Crossref PubMed Scopus (100) Google ScholarFertilization antigensBind to human ZP3; murine and human FA-1 and NZ-197Zhu X Naz R.K Fertilization antigen-1 cDNA cloning, testis-specific expression, and immunocontraceptive effects.Proc. Natl. Acad. Sci. USA. 1997; 94: 4704-4709Crossref PubMed Scopus (69) Google ScholarPhospholipase A2Inhibitors of and antibodies against PLA2 inhibit fertilization38Fry M Ghosh S East J Franson R Role of human sperm phospholipase A2 in fertilization effects of a novel inhibitor of phospholipase A2 activity on membrane perturbations and oocyte penetration.Biol. Reprod. 1992; 47: 751-759Crossref PubMed Scopus (32) Google Scholar, 65Riffo M Parraga M Study of the acrosome reaction and the fertilizing ability of hamster epididymal cauda spermatozoa treated with antibodies against phospholipase A2 and/or lysophosphatydylcholine.J. Exp. Zool. 1996; 275: 459-468Crossref PubMed Scopus (27) Google ScholarSperm agglutination antigen-1Human sperm surface antigen25Diekman A.B Westbrook-Case V.A Naaby-Hansen S Klotz K.L Flickinger C.J Herr J.C Biochemical characterization of sperm agglutination antigen-1, a human sperm surface antigen involved in gamete interaction.Biol. Reprod. 1997; 57: 1136-1144Crossref PubMed Scopus (50) Google Scholar Open table in a new tab Although a few candidate EBPs have been favored over others, even these remain controversial. For example, recently it was found that male mice which are homozygous null for β-galactosyltransferase (gt−/−; long and short forms of the protein) are fertile (56Lu Q Shur B.D Sperm from β1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly.Development. 1997; 124: 4121-4131Crossref PubMed Google Scholar). Although responses to mZP3 (e.g., induction of the acrosome reaction) are impaired in vitro, sperm from gt−/− mice bind to and fertilize eggs in vivo. These findings may at least minimize the role of β-galactosyltransferase in fertilization. Similarly, the realization that human zona receptor kinase (ZRK; Hu9), a protein tyrosine kinase, is identical with the proto-oncogene c-mer (17Bork P Sperm-egg binding protein or proto-oncogene?.Science. 1996; 271: 1431-1432Crossref PubMed Scopus (41) Google Scholar, 75Tsai J.-Y Silver L.M Sperm-egg binding protein or proto-oncogene.Science. 1996; 271: 1432-1434Crossref PubMed Scopus (23) Google Scholar) raises doubts about ZRK’s proposed role in binding of sperm to eggs (18Burks D.J Carballada R Moore H.D.M Saling P.M Interaction of tyrosine kinase from human sperm with the zona pellucida at fertilization.Science. 1995; 269: 83-86Crossref PubMed Scopus (159) Google Scholar, 70Saling P Carballada R Burks D Moore H Sperm-egg binding protein or proto-oncogene.Science. 1996; 271: 1434-1435Crossref PubMed Google Scholar). Finally, the recent finding that AM67, an acrosomal matrix protein, and sp56 are orthologs (37Foster J.A Friday B.B Maulit M.T Blobel C Winfrey V.P Olson G.E Kim K.-S Gerton G.L AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins.J. Biol. Chem. 1997; 272: 12714-12722Crossref PubMed Scopus (118) Google Scholar) casts some doubt on a role for sp56 in binding of acrosome-intact sperm to the ZP (16Bookbinder 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). These and other issues have contributed to the confusion that characterizes this area of research. What accounts for the large number of sperm components identified as EBPs? The following four possibilities may be contributing fa