Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
E European Society of Human Reproduction and Embryology Human Reproduction Update 1999, Vol. 5, No.4 pp. 314–329 Carbohydrate-based interactions on the route of a spermatozoon to fertilization Edda Töpfer-Petersen1 Institute of Reproductive Medicine, Veterinary School of Hanover, Germany Male and female intercommunication along the route which the spermatozoon takes to fertilization utilizes the information potential of carbohydrates. A hierarchy of carbohydrate-based binding events exists ranging from spermatozoa–oviduct interaction to primary and secondary binding between spermatozoon and oocyte. Before in-vivo fertilization can occur, spermatozoa are stored in the caudal part of the isthmus, in tight contact with the epithelium cells lining the oviduct. The sperm reservoir seems to be created by surface-associated sperm lectins recognizing epithelial glycoconjugates. With the changing conditions in the oviduct at the time of ovulation, spermatozoa may shed those sperm lectins, creating new surfaces which allow spermatozoa to be released from the epithelium, complete capacitation and interact with the oocyte in the appropriate manner. The first contact between both gametes occurs at the spermatozoa–zona pellucida interface. The ‘primary’ binding initiates the acrosomal exocytosis of the spermatozoa, followed by the ‘secondary’ binding of the acrosome-reacted spermatozoon that in consequence leads to sperm penetration through the zona pellucida. Primary and secondary binding events are directed by the cooperative interactions of multiple carbohydrate-recognition systems that may act in a hierarchical and redundant manner. The current perspective will focus on the role of carbohydrate-binding sperm proteins in the sequence of binding events during fertilization in the pig. Keywords: gamete recognition/oviduct/spermatozoa/zona pellucida/zona pellucida-binding proteins TABLE OF CONTENTS Introduction Formation of the oviductal sperm reservoir is a carbohydrate-mediated event Carbohydrates are the signals for gamete recognition Zona pellucida-binding proteins Uncapacitated and capacitated porcine spermatozoa bind to the zona pellucida in vitro Is carbohydrate-mediated gamete recognition really species-specific? Conclusions Acknowledgements References 314 315 317 321 324 325 326 326 327 Introduction Fertilization is a fundamental event which involves a highly coordinated sequence of cellular interactions between the male and female gamete, that is, between the sperm cell and the egg, in order to form a diploid zygote and, ultimately, the new individual. In mammals, fertilization occurs in the female 1Address reproductive tract. At ejaculation, millions of spermatozoa are deposited in the female reproductive tract, though only a few thousand enter the oviduct, a few reach the ampulla at the time of fertilization, and only one spermatozoon fertilizes the egg. To guarantee the meeting of the two highly specialized gametes at the right time, and in the right place, the oviduct and the egg itself coordinate sperm functions. On reaching the oviduct, spermatozoa are held back in the reservoir of the lower isthmus due to binding of the spermatozoa to the epithelium (reviewed by Hunter, 1988, 1996; Smith, 1998; Suarez, 1998) (Figure 1). Sperm interactions with the oviductal epithelium appear to increase the viability of the spermatozoa during storage, and suppress sperm motility (Smith, 1998; Suarez, 1998). Before fertilization can occur, however, spermatozoa must enter a functionally activated or capacitated state and develop a hyperactivated motility which enables them to respond to the egg in the appropriate manner (Bedford, 1983). The capacitation process appears to be coordinated temporally by the oviductal epithelium in a still unknown fashion. Close to the time when the egg is ovulated into the ampulla, spermatozoa start or continue the capacitation process and are released from for correspondence: Institute of Reproductive Medicine, Veterinary School, Hanover, Bünteweg 15, D-30559 Hanover, Germany. Tel: 0511 9538520; Fax: 0511 9538504; e-mail: [email protected] Spermatozoan–egg interactions in fertilization the oviductal epithelium, whereby the newly developed hyperactivated motility may help the detachment of spermatozoa and facilitate their swimming to the site of fertilization (Hunter, 1996; Suarez, 1996, 1998; Smith, 1998 and references therein). On approaching the oocyte, the spermatozoon must first be recognized by the oocyte. This interaction occurs when a spermatozoon first makes contact with the zona pellucida (ZP), the extracellular coat enveloping the oocyte. The ZP not only mediates the recognition between both gametes, but also regulates sperm functions, enabling the spermatozoon to complete fertilization. Capacitation is a prerequisite for the subsequent activation of the sperm transmembrane signalling system(s) by structures of the ZP, leading to the exocytosis of the sperm acrosome, referred to as the acrosome reaction. Thereby, the enzymatic equipment of the acrosome is activated, and is made available to aid sperm passage through the ZP, finally allowing fusion with the egg vitelline membrane. After fusion and oocyte activation have been completed by the spermatozoon, the sperm nucleus decondenses and delivers the male genome into the egg cytoplasm, thus marking the start of the programme for embryonic development. As one consequence of oocyte activation, the ZP is altered by components released from the oocyte cortical granules, contributing to the establishment of the egg-induced block to polyspermy (reviewed by Yanagimachi, 1994; Storey, 1995). Whereas the capacitation process is modulated by the oviduct, the ensuing physiologically significant acrosome reaction is coordinated by the ZP. It has long been accepted that recognition and initial binding between spermatozoa and egg involves the binding of multiple carbohydrate receptors of the sperm cell to the complementary oligosaccharide chains attached to the ZP proteins. Recently, sperm binding to the oviduct has also been shown to involve carbohydrate–protein interactions (Suarez, 1998). Thus, two important regulatory steps on the journey of the spermatozoon to union with the egg may be initiated by carbohydrates (Figure 1). In recent years, current knowledge of the mechanisms of spermatozoa–oocyte interaction has been excellently reviewed, covering different aspects of the role of carbohydrates in fertilization (for example Wassarman and Litscher, 1995; Chapman and Barratt, 1996; Clark et al., 1996; Snell and White, 1996; Benoff, 1997; Sinowatz et al., 1997; Tulsiani et al., 1997; McLesky et al., 1998). The current perspective will therefore focus on the role of ZP-binding proteins in fertilization under in-vitro conditions in the pig, and the assumed fate during in-vivo transit to the site of fertilization. Formation of the oviductal sperm reservoir is a carbohydrate-mediated event In mammals, millions of spermatozoa are stored in the cauda epididymis until, at ejaculation, they are deposited in the female reproductive tract. Most of the spermatozoa are lost during their 315 Figure 1. Schematic representation of the carbohydrate-based interactions on the route of a spermatozoon to fertilization. After reaching the oviduct, spermatozoa are stored in the oviductal reservoir created by carbohydrate-based interactions of spermatozoa and the oviductal epithelium. After capacitation, spermatozoa are released and swim to the site of fertilization. On approaching the oocyte, the initial binding and recognition is mediated by exposed carbohydrate of the oocyte zona pellucida and complementary carbohydrate/zona pellucida-binding proteins of the sperm surface. journey through the female reproductive tract, and only a small number reach the oviduct. The lower region of the isthmus is the site where the essential steps of the capacitation process are initiated, i.e. the sperm plasma membrane changes which are prerequisite for the onset of the acrosome reaction and motility hyperactivation. It has been shown across mammalian species that this region of the oviduct functions as a reservoir in which the spermatozoa are stored with tight contact to the ciliated oviductal epithelial cells lining the tract (Hunter, 1981, 1988, 1996; Smith, 1998; Suarez, 1998) (Figure 2A). In contrast to the observations in many mammalian species, there has been no conclusive evidence for a distinct oviductal sperm reservoir in humans. Human spermatozoa do not establish a tight binding to the homologous oviductal epithelium in vitro (Yeung et al., 1994; Murray and Smith, 1997). Nonetheless, the viability of human spermatozoa has been shown to be maintained by co-culture with oviductal epithelium cells (Yao et al., 1999). To date, no useful hypothetical model outlining the events of sperm transport in humans has been described. Role of the oviductal sperm reservoir The oviductal reservoir may serve a number of functions (Suarez, 1998). First, it may contribute to the prevention of polyspermic fertilization by controlling sperm transport to the ampulla. Second, during storage the sperm fertilizing capacity appears to be maintained to span the time between oestrus and fertilization. Third, the capacitation process is modulated to synchronize sperm function with the time of ovulation. Since the capacitated state reduces the life span of spermatozoa, the need to maintain sperm viability and control capacitation are mutually associated events. The caudal isthmus appears to be specialized for these purposes. Very recently, it has been shown in the pig (Hunter et al., 1998) that the caudal region of the 316 E.Töpfer-Peterson spermatozoa. Capacitation is controlled by ions, and in particular Ca2+ plays a critical role at this point. In mouse and other species, capacitation and the subsequent acrosome reaction in vitro are accompanied by a biphasic pattern of internal Ca2+ elevations, in which the first small peak is related to capacitation and the large influx of Ca2+ triggers acrosomal exocytosis (reviewed by Fraser, 1995). Under Ca2+-deficient conditions, spermatozoa fail to complete capacitation, indicating that Ca2+ has an important directive role in the progress of capacitation (Fraser, 1995). Sperm survival and capacitation in the oviduct may therefore be regulated by the control of Ca2+ uptake by spermatozoa. Equine spermatozoa attached to homologous oviductal epithelial cells show about 2- to 3-fold lower internal Ca2+ concentrations than freeswimming spermatozoa (Dobrinski et al., 1997). Alterations of the ionic environment at the time of ovulation (Fraser, 1995) may promote the transition to the capacitated state; however, the transmission appears to be dominated by active intervention of the oviductal epithelium. Binding of spermatozoa to the apical plasma membrane of the oviductal cells directly influences the functional state of the spermatozoa by maintaining low intracellular Ca2+, and in consequence slows down the rate of capacitation (Murray and Smith, 1997; Smith and Nothnick, 1997). Inhibition of spermatozoa–oviduct binding by antibodies raised against the oviductal apical plasma membranes reduces the controlling effect of the oviductal cells (Dobrinski et al., 1997). Previously, it was suggested (Bedford et al., 1983) that capacitation is a mechanism acquired by the spermatozoa to prevent certain functions from developing too quickly before fertilization can occur. Oviductal carbohydrates and sperm lectins may be involved in spermatozoa–oviduct binding Figure 2. Scanning electron micrographs. (A) The porcine utero-tubal junction and caudal isthmus of the oviduct of the pig, at 10 h after insemination. (B) Capacitated spermatozoa bound to the zona pellucida of ovarian oocyte of the pig in vitro. isthmus is more effective in neutralizing seminal plasma components and stimulating or regulating the capacitation process than other regions of the Fallopian tube. Although there are many lines of experimental evidence for specific effects of the oviductal reservoir on spermatozoa, little is known about the underlying mechanisms. Binding of the spermatozoa to, and their release from, the oviductal epithelium appear to be crucial regulatory steps related to the capacitation state (Smith, 1998). In a number of species including hamster (Smith and Yanagimachi, 1990), mouse (DeMott et al., 1995), cattle (Lefebvre and Suarez, 1996) and horse (Dobrinski et al., 1997), capacitated spermatozoa do not bind, or bind with a less significant frequency, to the oviduct than do uncapacitated The first indication that carbohydrate moieties are involved in spermatozoa–oviduct attachment comes from sugar inhibition experiments. Fetuin carrying sialylated N- and O-linked glycan chains effectively inhibits spermatozoa–oviduct binding in hamster (DeMott et al., 1995), whereas asialofetuin carrying galactose on the non-reducing terminus of the glycan chains, fucoidan and ovalbumin have been shown to have less, or no, effect. Monomeric sialic acid was also able to interfere with the binding, pointing to the role of sialic acid as the key signal for spermatozoa–oviduct recognition in hamster. In contrast, asialofetuin and galactose are the most effective inhibiting components in horse (Lefebvre et al., 1995) and fucose, in a α1,4-linkage to the penultimate carbohydrate residue, appears to be the determining sugar in cattle (Lefebvre et al., 1997; Suarez et al., 1998). In preliminary studies in pigs using explants isolated from the isthmic region of the oviduct, asialofetuin (in the micromolar range) has been shown to inhibit sperm binding, indicating that oligosaccharides with terminally located galactose may be critical for forming the Spermatozoan–egg interactions in fertilization sperm reservoir (R.Gelhaar, D.Waberski and E.Töpfer-Petersen, unpublished result). The sperm surface (Sinowatz et al., 1989) and the oviductal epithelium (Raychoudhury et al., 1993) possess a variety of externally orientated oligosaccharides linked to proteins and lipids that may serve as recognition signals for the binding events that take place in the isthmic region of the oviduct. In the context of the search for the receptors of the ZP carbohydrate ligands, a large number of carbohydrate-binding sites at the sperm surface (Sinowatz et al., 1989) and proteins with affinity for the ZP have been identified in a wide range of species (reviewed by Töpfer-Petersen et al., 1995; Benoff, 1997; Sinowatz et al., 1997; McLesky et al., 1998). Recently, carbohydrate-binding sites with affinity for terminal galactose have been identified in the tubal epithelium in rabbits, and these allude to the presence of lectin-like molecules or generally carbohydrate-binding proteins in the mammalian oviduct (Biermann et al., 1997). Although both the sperm surface and the oviductal epithelium possess the tools for the carbohydrate-mediated binding event, e.g. carbohydrate-ligands and carbohydrate-binding proteins, most data suggest that the recognition of exposed carbohydrate structures of the oviductal epithelial cells by lectin-like molecules associated with the sperm surface is the dominating mechanism creating the oviductal sperm reservoir. However, the contribution of other systems cannot be ruled out as yet. Candidates as oviduct-binding molecules are extrinsic secretory proteins which associate with the sperm plasma membrane during epididymal transit and/or ejaculation. In the hamster, fetuin-binding sites are lost from the acrosomal region of the sperm head during capacitation. This could be attributed to the loss of fetuin-binding proteins of 50, 32 and 27.5 kDa (DeMott et al., 1995). Coincidentally, spermatozoa lose their ability to bind to the oviductal epithelium. A galactose-binding protein has been isolated from equine sperm plasma membranes (Dobrinski et al., 1997), and fucose-binding sites have been localized at the head of motile bovine spermatozoa. Furthermore, the spermatozoa–oviduct binding and the binding of fucose-tagged probes to bovine spermatozoa require the presence of Ca2+, pointing to the role of a calciumdependent sperm lectin with a specificity for fucose in cattle (Suarez et al., 1998). In the pig (Figure 2A), candidate proteins are the spermadhesins which may mediate spermatozoa– oviduct binding. The spermadhesins form a novel protein family of 12–14 kDa with a carbohydrate specificity ranging from galactose in different complex oligosaccharide sequences to mannose-6-phosphate, or show no carbohydrate binding as aSFP, the bovine spermadhesin (discussed below). Fate of sperm lectins during sperm transport in pig oviduct Spermadhesins are secreted by the male genital tract of pig (and also of cattle and horse), comprising about 80% of total porcine seminal plasma proteins (reviewed by Töpfer-Petersen et al., 317 1998). During epididymal transit and at ejaculation, spermadhesins form, together with other proteins such as serine proteinase inhibitors and pB1 (a member of the bovine BSA1–3, and equine HSP1–2 family; Calvete et al., 1997b), a thick multi-molecular layer, particularly around the sensitive acrosomal region of the sperm head, thus probably preventing premature capacitation and the acrosome reaction (Töpfer-Petersen et al., 1998). At about 1–2 h after mating, sufficient spermatozoa are stored in the oviductal reservoir (Figure 2A) to ensure subsequent fertilization (Figure 2A), thereby leaving most of the seminal plasma components behind (Hunter, 1996 and references therein). Seminal plasma appears not to pass through the barrier of the utero-tubal junction, except for those proteins coating the sperm surface. This barrier function for seminal plasma was demonstrated by two-dimensional SDS–PAGE of oviductal fluid collected from the isthmus of inseminated sows (Einspanier et al., 1996; Calvete et al., 1997a). After deposition into the female genital tract, the spermatozoa entering the oviduct have an exposed surface loaded with a protein coat which may now interact with the oligosaccharides of the oviductal epithelium. Preliminary studies demonstrate that seminal plasma and the isolated spermadhesin fraction are able significantly to inhibit the binding of the spermatozoa to isthmic explants of the porcine oviduct in a concentration-dependent manner (D.Waberski, R.Gelhaar and E.Töpfer-Petersen, unpublished results). The ability of seminal plasma and its major components to interfere with oviduct–spermatozoa binding may refer to the contribution of spermadhesins in establishing the sperm reservoir in pig by recognizing galactose in complex oligosaccharides of the oviductal epithelium. During in-vitro capacitation, most of the protein coat is removed from the sperm surface. Commencing 1–2 h before ovulation, spermatozoa are released progressively from the isthmic region and proceed towards the site of fertilization in tightly controlled numbers (Hunter, 1996 and references therein). With the changing conditions in the oviduct at the time of ovulation, spermatozoa may shed their protein coat sequentially, thus creating new surface structures which could allow spermatozoa to be released from the epithelium, complete capacitation and interact with the ZP in the appropriate manner, leading to fertilization. Carbohydrates are the signals for gamete recognition The mammalian ZP surrounding the oocyte is a unique, highly organized three-dimensional matrix that is between 2 and 25 µm thick, and which protects the egg and the preimplantation embryo from physical damages; in addition, the ZP modulates sperm function. The penetration of the ZP is a crucial step in mammalian fertilization. Consequently, an inability of spermatozoa to pass this extracellular layer inevitably leads to infertility. Recognition and initial binding 318 E.Töpfer-Peterson between spermatozoa and the ZP are prerequisites for subsequent penetration (Figure 2B). The basic mechanism appears to be conserved throughout evolution from marine invertebrates to eutherian mammals, and is based on carbohydrate–protein interactions between the spermatozoa and the oocyte envelope. In most species, certain exposed oligosaccharide chains of the oocyte envelope and complementary carbohydrate-binding proteins on the spermatozoa–oocyte interface mediate, at least in part, the initial binding and the recognition between the spermatozoon and the egg cell (Sinowatz et al., 1997). Zona pellucida proteins The ZP matrix is composed of only three glycoproteins, which build a typical complex fibrillogranular structure by nonco-valent interactions. The ZP proteins are encoded by three different genes termed according to their decreasing molecular size, ZPA, ZPB and ZPC (Harris et al., 1994). The amino acid sequences of ZP glycoproteins in mammals and corresponding egg envelope proteins in widely divergent species (fish and amphibians) have been found to be highly conserved during evolution (Harris et al., 1994; Epifano et al., 1995; Hedrick, 1996). Post-translational modifications and processing of the protein backbone are species-specific events and result in heterogeneity of the attached oligosaccharides and in differing polypeptide chain lengths of the assembling egg envelope proteins. It appears that the ZP gene products display a conserved modular structure. A newly defined ZP module, a large domain spanning 260 amino acids, has been identified near the C-terminally located putative transmembrane domain; both of them are shared by all ZP proteins (Bork and Sander, 1992). In addition, a trefoil domain has been proposed in ZPB located next to the ZP-module (Bork, 1993). The strictly conserved cysteine residues, implying a similar organization of disulphide bonds, the same pattern of hydrophobicity, polarity and turn-forming structures within the ZP module and the trefoil domain are consistent with a highly conserved threedimensional structure across species. Whereas in mouse the synthesis of the three ZP proteins is restricted exclusively to the oocyte (Wassarman and Litscher, 1995; reviewed by Castle and Dean, 1996), in other species such as pig (Sinowatz et al., 1995; Kölle et al., 1996), cattle (Kölle et al., 1998; Totzauer et al., 1998), rabbit (Prasad et al., 1998), cynomolgus monkey (Martinez et al., 1996) and man (Grootenhuis et al., 1996), follicle cells participate in assembling the ZP matrix in a development-dependent manner. In pig and cattle, both ZPB and ZPC are predominantly expressed in primary follicles by the oocyte. During follicular development the follicle cells contribute to an increasing extent to ZP protein synthesis, and in the pig the granulosa cells and the corona radiata of tertiary and preovulatory follicles take over ZP protein synthesis almost completely (Sinowatz et al., 1995; Kölle et al., 1996, 1998). Fertilization appears to Figure 3. Scanning electron micrograph of the spherical structures which are formed occasionally by the zona pellucida glycoproteins of adult sows. After solubilization and storage overnight at 4°C, the zona pellucida glycoproteins are able to reorganize to a higher structure. Scale bar = 20 µm. terminate ZP gene expression in all species. The different biosynthetic patterns in mouse and in other species such as pig and cow, may have consequences for the organization of the three-dimensional ZP matrix in these species. In mouse, the relatively thin ZP coat is formed by long filaments of periodically arranged heterodimeric units of mZP2 (ZPA) and mZP3 (ZPC), randomly cross-linked by mZP1 (ZBA)-dimers (Wassarman and Mortillo, 1991). However, the mouse model may not hold for larger animals, and certainly not for the thick ZP of pigs and cattle (Totzauer et al., 1998; Kölle et al., 1998). Very recently it has been reported that porcine pZPB and pZPC in about equimolar ratios (1:1 up to 1:2) reconstitute to high-mass heteromultimeric complexes, whereas the highly purified proteins are predominantly monomeric (pZPB) or as pZPC form smaller aggregates with molecular masses of about 300 and 160 kDa (Yurewicz et al., 1998). Coincidentally with this report are observations that after heat-solubilization and storage overnight at 4°C, occasionally the native ZP glycoproteins collected from adult sows, but not from prepubertal sows (<100 kg), reorganize to spherical structures with a smooth surface (Figure 3) (E.Töpfer-Petersen and F.Sinowatz, unpublished results). The failure to develop the typical network-like architecture of the intact ZP under these in-vitro conditions may suggest that self-aggregation of the ZP glycoproteins is under the control of the synthesizing cells, e.g. the oocyte and the follicle cells. Spermatozoan–egg interactions in fertilization Carbohydrate structures of the zona pellucida Although carbohydrates play a crucial role in gamete recognition, our knowledge of the oligosaccharide structures of mammalian ZP glycoproteins is limited. One approach to characterize the oligosaccharide chains of the ZP is to employ lectins as tools. Comparative cytochemical studies demonstrate the species-specific variation in the lectin-binding pattern, which increases with the evolutionary distance of species (Skutelsky et al., 1994). Some carbohydrate structures are found in all species examined, such as mannose and N-acetylglucosamine, which are usually parts of the core region of N-linked oligosaccharides, whereas binding sites for α-galactose-recognizing lectins appear to be typical for the mouse and rat (Maymon et al., 1994). GalNAcβ1,4Gal sequences have been localized ultrastructurally in the innermost region of the intact murine ZP, indicating the regionalization of oligosaccharide structures within the three-dimensional structure of the zona matrix (Aviles et al., 1999). In humans, the occurrence of N-linked glycans of the bi-antennary complex type and/or the high mannose (concanavalin A, Con A) as well as of the tri-antennary core-fucosylated complex type (lentil lectin, LCA) and typical O-linked oligosaccharide chains have been shown by lectin-binding. Remarkably, most of the O-linked Galβ1,3GalNAc sequences appear to be blocked by α2,3-linked sialic acid (Maymon et al., 1994; Ozgur et al., 1998). Recently, the oligosaccharide sequences of the pig ZP proteins (Noguchi and Nakano, 1992; Noguchi et al., 1992; Hokke et al., 1994), of part of the mouse mZP2 and mZP3 (Noguchi and Nakano, 1993; Nagdas et al., 1994) and of the unfractionated cattle ZP glycoproteins have been reported (Katsuma et al., 1996). The ZP glycoproteins carry a complex pattern of N- and O-glycosidically linked oligosaccharides. ZPA is the largest ZP protein in the pig, with six potential N-glycosylation sites, whereas ZPB and ZPC possess three and five potential N-glycosylation sites respectively. Carbohydrate analysis suggested that the ZPB and ZPC proteins contain three and six additional O-glycosylation sites respectively (Yurewicz et al., 1991). The N-linked glycans in the pig, cattle and mouse possess basically the same structures. They belong to the fucosylated complex-type core structure elongated by nonbranched N-acetyllactosamine chains which in the case of the acidic glycans have sialic acid at the non-reducing end, and/or sulphate in the C-6 position of the N-acetylglucosamine residues of the lactosamine repeats. In the pig, additionally, sulphated residues have been localized at the C-6 position of N-acetylglucosamine residues in non-repeated antennae, and at the C-3 position of reducing terminal N-acetylglucosamine residues (Mori et al., 1998). However, species differ in the ratio of di-, tri- and tetra-antennary chains, and in the degree of sulphation and sialylation. Mouse and bovine ZP glycoproteins contain mainly acidic tri-and tetra-antennary chains. In both 319 species the sulphate contents are lower than in pig ZP glycoproteins, and the acidic properties are mainly due to the content of sialic acid. The most significant difference between the ZP matrices of these species is the structure of the neutral N-glycans. The major neutral N-type oligosaccharide in bovine ZP glycoproteins is a high-mannose-type oligosaccharide, whereas the major neutral N-glycans in the pig belong to the di-antennary fucosylated complex-type glycans containing N-acetyllactosamine chains (Figure 4). In both species, the neutral oligosaccharides constitute about 25% of the total carbohydrate portion, whereas in mice the content of neutral N-glycans is <5%. Terminal N-acetylgalactosamine and α-galactose residues, which are absent in porcine N-glycans, are minor components in the mouse and cow. The sulphated polylactosamine backbone of the O-linked oligosaccharides of porcine ZP glycoproteins exhibits the same structure as that of the N-glycans extending from the Galβ1,3GalNAc disaccharide core. Sialic acid can be linked with the nonreducing termini of the oligosaccharide chain and/or with the proximal N-acetylgalactosamine residue of the core disaccharide. Neutral structures terminate the non-reducing end of the carbohydrate chain with β-galactose and β-N-acetylglucosamine residues, and as minor components also with α-galactose and β-N-acetylgalactosamine residues. The amount of sulphated lactosamine repeats and the degree of sialylation in both N- and O-glycans contribute to the enormous heterogeneity of the ZP glycoproteins in all species. Studies on the attachment sites of individual oligosaccharides, their distribution within the three-dimensional structure of the intact ZP network, and their physiological relevance are still in their infancy, though first attempts have been made (Kudo et al., 1998). Sugar-mapping of the glycopeptides of pZPB has revealed that all three potential N-glycosylation sites, Asn203, Asn220 and Asn333 of the mature pZPB (cDNA-deduced residues 137–446) carry neutral bi-antennary N-glycans, whereas only Asn220 is additionally glycosylated with neutral tri- and tetra-antennary chains. At least one disulphide bond between the neighbouring cysteine residues Cys224 and Cys243 has been localized in the N-terminal half of pZPB (Kudo et al. 1998). Nonetheless, current knowledge of the ZP oligosaccharide structures in the pig makes this species an ideal model for studying the underlying molecular mechanisms of spermatozoa–oocyte recognition. Carbohydrate structures determining gamete recognition In most species, the nature and glycosylation sites of the individual oligosaccharides and the cognate carbohydratebinding protein that are involved in spermatozoa–zona binding and recognition are essentially unknown. In the mouse and hamster, the recognition signals and the ability to induce the spermatozoa acrosome reaction have been mapped to ZP3, the gene product of ZPC, whereas in the pig and rabbit 320 E.Töpfer-Peterson Figure 4. Schematic representation of the biologically active N-linked oligosaccharide of the zona pellucida glycoproteins in pig and cow. (A) The major neutral oligosaccharide of pig zona pellucida is a bi-antennary complex type N-glycan (Noguchi et al., 1992). (B) Only tri- and tetra-antennary complex type N-glycans of ZPB are able to inhibit spermatozoa–oocyte binding in the pig (Kudo et al., 1998). (C) The major neutral N-glycan of high-mannose-type exhibit prominent inhibitory activity of spermatozoa–egg binding and in-vitro fertilization (Nakano and Yonezawa, 1998). Gal = galactose; Man = mannose; Fuc = fucose; GlcNAc = N-acetylglucosamine. sperm-binding activity is associated with the glycoproteins coded by the ZPB genes (Yurewicz et al., 1991; Hedrick, 1996; Prasad et al., 1998). Interestingly in Xenopus laevis, the sperm receptor (gp69/64) is the homologue of mZP2, the ZPA gene product (Tian et al., 1999). By analogy with the mouse system, sperm receptor activity has been also postulated for human ZP3 (ZPC). While recombinant hZP3 possesses the ability to induce the acrosome reaction (Brewis et al., 1996), no direct evidence exists for the contribution of human ZP3 in spermatozoa–zona binding. Transgenic mice expressing human ZP3 restore fertility. However, as observed in wild-type mice, human spermatozoa do not bind to the chimeric ZP (Rankin et al., 1998). For the mouse, there is general consent that certain O-linked oligosaccharides clustered at the C-terminal sperm-combining site of mZP3 are critical to achieve high-affinity binding. On the contrary, the identity of the sugar moieties which determine the carbohydrate specificity of the spermatozoa–zona interaction, i.e. whether terminal α/β-linked galactose, β1,4-linked N-acetylglucosamine or fucose residues, is open to debate. The degree of sialylation and sulphation, however, appears to have no effect on bioactivity (Litscher et al., 1995; Litscher and Wassarman, 1996; Thaler and Cardullo, 1996; Liu et al., 1997; Johnston et al., 1998; Shur, 1998). A more differentiated model for sperm recognition in mouse has been presented (Johnston et al., 1998). These authors postulate a high-affinity binding site on spermatozoa recognizing α3-fucosylated oligosaccharides which also could be occupied with less affinity by α3-galactosyl-capped carbohydrates, and a second low-affinity binding site for terminal β-galactose-containing structures. In the case of the pig, the presentation of the bioactive oligosaccharide chains in heteromultimeric pZPB/ZPC complexes appears to be necessary to create high-affinity binding sites for spermatozoa (Yurewicz et al., 1998). Similar to the mouse system, there is considerable controversy regarding the structural entity of the carbohydrate ligand mediating porcine spermatozoa–zona binding. It has been shown (Yurewicz et al., 1991) that O-linked glycans attached to pZPB confer the bulk of sperm-binding activity in the intact ZP. In contrast, others (Yonezawa et al., 1995a,b; Nakano et al., 1996) have reported that neutral tri- and tetra-antennary N-glycans (Figure 4) linked to Asn220 located at the N-terminal region of endo-β-galactosidase-treated pZPB, but not the bi-antennary N-glycans and O-glycans of pZPB, play the critical role in mediating gamete-binding and recognition. As in the mouse, enzymatic removal of polylactosamine extensions of the Nand O-glycans has been shown not to diminish the biological activity of pZPB and the ZPB/ZPC complexes (Nakano et al., 1996; Yurewicz et al., 1998). Recently, data have become available regarding the bioactive carbohydrate structure in the bovine system. The bovine ZP also consists of three glycoproteins, homologous to pZPA, pZPB and pZPC as shown by Western blotting with anti-porcine ZPB and anti-porcine ZPC, peptide mapping, and N-terminal sequencing (Topper et al., 1997). The major neutral N-glycans of high-mannose-type having five mannose residues and the isolated bZPB exhibit prominent inhibitory activity of spermatozoa–egg binding and in-vitro fertilization (Figure 4). The sperm-binding capacity of fertilized eggs is diminished, indicating that the structure and/or the conformation of the sperm-binding site of the ZP changes during fertilization (Nakano et al., 1996; Nakano and Yonezawa, 1998). Studies in the human system are restricted by the limited availability of biological material, e.g. zona pellucidae, impeding detailed structural carbohydrate analysis. A series of monosaccharides (fucose, mannose, galactose and N-acetylglucosamine residues) has been found to inhibit sperm binding (Miranda et al., 1997). Conflict between the results of different researchers (Oehninger et al., 1990) has been linked (Miranda, 1998) to the uncontrolled capacitated states of the spermatozoa used in experiments. Complex glycoconjugates bearing selectin-like ligands, e.g. Sialyl-Lewisx(a) and fucosylated and sialylated unusual GalNAcβ1,4GlcNAc (lacdiNAc)-antennae of N-glycans, as in glycodelin A, are potent inhibitors, leading to the hypothesis that in humans a selectin-like mechanism may be a determinant for gamete recognition (reviewed by Oehninger et al., 1998). Chemical modifications affecting particularly the terminal sialic acid Spermatozoan–egg interactions in fertilization residues interfere partly with sperm binding (30–40% inhibition), whereas enzymatic release of sialic acid moieties (documented by the lectin from Maackia amurensis (MAA) recognizing α2,3-linked sialic acid) and the release of the bulky lactosamine repeats by endo-β-galactosidase significantly increase the binding (3- and 7-fold respectively), probably due to the demasking of potential binding sites (Ozgur et al., 1998). Furthermore, recognition and binding of clustered mannose or N-acetylglucosamine residues of the corresponding neoglycoproteins appears to induce the acrosome reaction in human spermatozoa, apparently in the proper manner (Brandelli et al., 1996; Jacob et al., 1998). As postulated for the mouse, so also in human, more than one binding siterecognizing carbohydrate with low (sialic acid, selectin-like?) and high affinities (mannose, N-acetylglucosamine?) at the sperm surface may act in concert or in a certain sequence with the multiple oligosaccharides of the ZP. All these data demonstrate that the acquisition of sperm receptor activity may be a consequence of at least partly species-specific, and perhaps tissue-specific glycosylation and oligosaccharide processing of the distinct ZP glycoproteins, and that the biologically active carbohydrate structures are not necessarily linked with the homologous zona proteins. Furthermore, recognition and binding between spermatozoa and egg appears to be the result of the cooperation of a multimeric receptor system (Shur, 1998). Zona pellucida-binding proteins Among the bulk of ZP-binding proteins (ZBP) that have been described to date, only a few are characterized biochemically and structurally. Sperm proteins known to have specific carbohydrate-binding sites include mouse β1,4 galactosyltransferase and sp56, rabbit sp17, guinea pig PH-20, and a human mannose-binding protein (MBP) (Benoff, 1997; Sinowatz et al., 1997; McLesky et al., 1998; Shur, 1998). Rabbit sp17 and the human MBP share some common features with the expanding group of Ca2+-dependent (C-type) lectins (Drickamer, 1993) containing some of the invariant amino acids of the C-type carbohydrate-recognition domain (CRD). Sp17 has been proposed to belong to the subfamily of lectins with specificity to galactose (Richardson et al., 1994) and the cDNA of human sperm MBP has been described to encode for a protein with seven putative mannose-like binding CRD (for details, see Benoff, 1997). The carbohydrate specificity and the binding domains of spermadhesins and proacrosin have been studied most intensively by determining the three-dimensional structure and protein modelling (see below), whereas other ZBP such as the zona receptor kinase (ZRK/hu9) and two porcine ZBP, P47 and zonadhesin, have been characterized structurally although the carbohydrate-binding has not yet been determined (Hardy and Garbers, 1995; Ensslin et al., 1998; for review see McLesky et al., 1998). ZBP are localized in different compartments of the sperm head. 321 Spermadhesins (Sinowatz et al., 1997; Töpfer-Petersen et al., 1998) and the newly described P47 (Ensslin et al., 1998) are associated peripherally to the sperm surface, whereas mouse galactosyltransferase (Gong et al., 1995), ZRK (McLesky et al., 1998) and porcine zonadhesin (Hardy and Garbers, 1995) are a membrane-spanning proteins. ZBP could also be integrated into the membrane and exposed to the surface at the time when capacitation and/or the acrosome reaction are proceeding (rabbit sp17, Richardson et al., 1994; human MBP, Benoff et al., 1993) or could be part of the acrosomal matrix, as demonstrated for porcine acrosin (Töpfer-Petersen et al., 1990), guinea pig AM67 and its mouse orthologue sp56 (Foster et al., 1997). Carbohydrate-binding proteins appear to be made available sequentially during the progress of the fertilization process and may act in oviductal binding, as primary and secondary receptors, or may be of still unknown function. The inadequate attribution of those carbohydrate/ZBP to their distinct roles in the sequence of binding events in vivo characterizes the problems in that research field and demonstrates the limitation of our in-vitro test systems. Zona pellucida-binding proteins in the pig Spermadhesins The spermadhesin family (Figure 5) has its greatest diversity of members in the pig. AQN-1, AQN-3, AWN (named after the first three N-terminal amino acids), PSP-I and PSP-II, its glycosylated (AWN and AQN-3) and N-terminally acetylated isoforms (AWN) are major secretion products of the seminal vesicle and comprise the bulk of the proteins in seminal fluid. By using reverse transcriptase–polymerase chain reaction (RT–PCR) and histochemical methods, they are also found to be expressed along the epididymal tract, although AWN is the only spermadhesin that binds tightly to epididymal spermatozoa. Spermadhesins are multifunctional proteins exhibiting a spectrum of ligand-binding affinities, e.g. carbohydrates/ZP glycoproteins, sulphated glycosaminoglycans, serine proteinase inhibitors, and o-phosphorylethanolamine. Although spermadhesins share more than 60% amino acid sequence identity, they are not functionally equivalent, each exhibiting a different set of affinities, modulated by the actual state of the protein, e.g. glycosylation and aggregation. Only non-aggregated monomeric AWN and AQN-3 show specific binding capacity toward phospholipids, glycosylation of AWN and AQN-3 abolishes the carbohydrate affinity, and the heparin-binding affinity is dependent on the formation of a PSPI/PSPII heterodimer (Töpfer-Petersen and Calvete, 1996; Sinowatz et al., 1997; Töpfer-Petersen et al., 1998). The most thoroughly studied feature of the spermadhesins is their carbohydrate-binding capacity. They bind to the ZP glycoproteins in a cation-independent manner with a Kd in the low micromolar range. AQN-1, AQN-3 and AWN display similar binding affinities for glycoproteins 322 E.Töpfer-Peterson Figure 5. Schematic representation of the major carbohydrate/zona pellucida binding proteins of porcine spermatozoa. Spermadhesins belong to the CUB-family; inset: the three-dimensional structure consists of five parallel and antiparallel β-strands (Romero et al., 1997). P47 is a mosaic protein with two tandemly organized N-terminal EGF-domains and two large C-terminal domains displaying similarity to the C1 and C2 region of blood-coagulating factors V and VIII (Ensslin et al., 1998). Zonadhesin contains five D-domains homologous to the von Willebrand factor, a mucin-like domain located next to the N-terminal region and a C-terminal transmembrane domain (Hardy and Garbers, 1995). Proacrosin is serine proteinase zymogen consisting of a light and a heavy chain. N-terminal processing by a single proteolytic clip between Arg23 and Val24 and C-terminal processing of the proline-rich region activates the mature and stable enzyme (Töpfer-Petersen et al., 1990). Figure 6. Schematic representation of the binding of uncapacitated and capacitated spermatozoa to the zona pellucida in the pig. Uncapacitated spermatozoa bind to oligosaccharides of the zona pellucida via the spermadhesins coating the sperm surface. After capacitation, most of the spermadhesins are released, exposing new zona pellucida binding proteins such as the processed zonadhesin and other receptor proteins which in concert are now able to interact with the zona pellucida, leading to the acrosome reaction. carrying Galβ1,3GalNAc and Galβ1,4 GlcNAc sequences in O- and N-linked oligosaccharides. Thereby, AWN, AQN-1 and AQN-3 differ slightly in their ability to recognize Galβ1,4 GlcNAc sequences in bi- (AQN-1) or tri/tetra-antennary Spermatozoan–egg interactions in fertilization (AQN-3, AWN) N-glycans. N-acetylneuraminic acid (α2,3/6) linked with the penultimate galactose residue may increase (AWN) or decrease (AQN-1) affinity. AWN recognizes Galβ1,3 GalNAc sequences about 50 times better than the N-acetyllactosamine unit in N-glycans (Dostalova et al., 1995; Calvete et al., 1996). In contrast, PSPII shows affinity to mannose-6-phosphate. By protein modelling on the basis of the crystal structure of the PSPI/PSPII heterodimer (see below), Arg43 of the PSPII molecule has been suggested to play a pivotal role in mannose-6-phosphate binding (Solis et al., 1998). Furthermore, glycosylation at Asn50 of AWN and AQN-3 interferes with their carbohydrate-binding ability, suggesting that the CRD is located around the conserved asparagine residue in position 50. The amino acid sequences do not show any discernible similarity to known CRD. However, they do belong to a family of 16 functionally diverse proteins, all of them sharing the CUB-domain within a modular structure (Bork and Beckmann, 1993). Spermadhesins, spanning 110–133 amino acids, form a subgroup with a single CUB-domain architecture. The crystal structure of the non-covalent heterodimeric PSPI/PSPII complex has been solved (Romero et al., 1997). The overall structure of the subunits consists of a β-sandwich comprising two sheets, each containing five parallel and antiparallel β-strands (Figure 6, inset). The conformation of the CUB-domain appears to be highly conserved in other spermadhesin molecules: also the bovine spermadhesin, aSFP, shows the same overall structure (Romero et al., 1997). The PSPI/PSPII complex is the first ZBP to be described in its three-dimensional structure which additionally reveals the architecture of the CUB-domain. The similar, though discrete, carbohydrate specificity as well as their fine modulation dependent on the actual state of the proteins, e.g. glycosylation and aggregation, as well as their distribution during different functional stages in vitro, imply that the spermadhesins are involved in the early events of fertilization. It has been shown that in vitro AWN is present on the sperm cell bound to the ZP, and that the bound sperm population was able to undergo the acrosome reaction (Figure 6) (Töpfer-Petersen and Calvete, 1996). However, whether spermatozoa that reach the site of fertilization will actually carry measurable amounts of AWN and AQN-3 molecules bound to the surface, and thus serve as primary receptors in vivo, is critical in assigning a biological role for spermadhesins in gamete interaction. P47, a novel ZBP P47 (Figure 5) is another tightly bound peripheral sperm membrane protein of 47 kDa that has been identified by affinity chromatography on immobilized ZP glycoproteins (Ensslin et al., 1998). P47 proteins disclose a mosaic structure organized by two tandemly arranged N-terminal epidermal growth factor (EGF)-like domains and two large C-terminal domains displaying similarity to the C1 and C2 regions of blood-coagulating factors V and VIII. The second EGF-like 323 domain displays an RGD tripeptide, a motif found in a number of integrin receptor ligands (Eble and Kühn, 1997). The C-terminal domains may function as anchors to the lipid bilayer of the sperm surface, as has been shown for the C2 domain of factor VIII encompassing a peptide with an α-helical structure that is supposed to bind to anionic phospholipids (Gilbert and Baleja, 1995). The amino acid sequence of this region appears to be highly conserved within the C-terminus of P47. P47 is first detected in testicular spermatozoa located at the apical rim of the acrosomal region. Its distribution varies only slightly during epididymal transit, though under in-vitro capacitation conditions it shows a granular appearance over the entire acrosome when tested by indirect immunfluorescence. Traces of P47 could still be detected at the acrosomal region of spermatozoa bound to the ZP and starting the acrosome reaction. P47 is able to bind biotinylated ZP glycoproteins, although it has not been tested whether it interacts with the carbohydrates or with the polypeptide backbone of the ZP proteins. P47 proteins belong to the MFGM-protein family, secretory proteins of the mammary gland with a still unknown function (Stubbs et al., 1990; Aoki et al., 1995; Anderson et al., 1997), and is homologous to rat O-acetyl ganglioside synthase (AGS; Ogura et al., 1996). P47 is not exclusive to the pig, but has been characterized by cDNA-sequencing in the testis of mouse, cattle, horse and human (Ensslin et al., 1998; M.Gentzel and E.Töpfer-Petersen, unpublished results), and also in the epididymis, mammary gland, uterus and several other bovine and porcine tissues, exhibiting an amino acid sequence identity of 60–100%. The role of P47-like protein in fertilization is highly speculative. The ZP affinity may implicate a role in primary binding, whereas the exposed RGD-sequence in the second EGF-domain may interact with integrin receptors on the sperm membrane or with an egg membrane integrin or serve as a ligand for EGF-receptors, and thus contribute to signalling events in fertilization. P47 is completely released during the in-vitro-induced acrosome reaction (T.Läkamp, M.Gentzel and E.Töpfer-Petersen, unpublished results), which may argue against a role in spermatozoa–oolemma interaction. Zonadhesin Porcine zonadhesin (Figure 5) has been shown to bind in a species-specific manner to the homologous ZP. The zonadhesin cDNA, cloned from pig testis, predicts a large protein with a modular structure containing five D-domains homologous to the von Willebrand factor, a mucin-like domain located next to the N-terminal region, and a C-terminal putative transmembrane domain (Hardy and Garbers, 1995). The expression of the zonadhesin gene is restricted to the testis, where it is translated primarily in haploid spermatids. The cDNA codes for a precursor protein which may be processed during epididymal transit and/or capacitation to generate the mature protein by removing the N-terminal and mucin-like 324 E.Töpfer-Peterson domains. After capacitation, the functional protein appears to contain two disulphide-bonded subunits, the N-terminal p45 and the p105 moieties. It is still unknown whether the zonadhesin molecule recognizes the ZP carbohydrates, or is directed against the polypeptide core of the ZP glycoproteins. The authors postulate that zonadhesin may recognize the sulphated oligosaccharide structure of the ZP via the heparin-binding sequences of the D-domains, thus contributing to species-specific recognition and subsequent signalling events. Recently, mouse tectorins—modular matrix proteins of the inner ear—have been found to display an arrangement of D-domains similar to that of zonadhesin, as well as a C-terminally located ZP module (Legand et al., 1997). It is interesting that in the mouse, the zonadhesin gene has been localized close to the Zp3 (ZPC) gene on chromosome 5 (Gao et al., 1997). Proacrosin Pro/acrosin (Figure 5) was one of the first sperm proteins to be biochemically and, in part, structurally characterized (Töpfer-Petersen et al., 1990). Acrosin is released as a consequence of the acrosome reaction and was considered to be essential for sperm penetration through the ZP matrix. It is the major ZBP of boar spermatozoa, interacting in a non-enzymatic manner with the ZP glycoproteins. In intact ejaculated spermatozoa, acrosin is stored in the acrosome as an inactive 53/55 kDa precursor molecule that is activated by a single proteolytic clip between Arg23 and Val24, accompanied by C-terminal processing to the stable 38 kDa protein. Activation and processing of acrosin occurs concomitantly with the acrosome reaction, and appears to be regulated on and by the ZP (Töpfer-Petersen and Cechova, 1990). About 5% of the latent proteolytic activity in boar spermatozoa is due to a 33 kDa zymogen presenting the same N-terminal amino acid sequence of 34 amino acid residues as in the proacrosin molecule (Töpfer-Petersen et al., 1990). Both the long and the short zymogen forms, as well as the active molecules show affinity toward the ZP. Limited autoproteolysis of pro/acrosin has led to the isolation of a 15 kDa N-terminal fragment still exhibiting about 66% of the ZP-binding activity (Töpfer-Petersen et al., 1990). Acrosin–ZP binding follows a polysulphate-recognition mechanism by which clustered basic amino acids on the pro/acrosin molecule interact with the sulphate groups within the lactosamine repeats of N- and O-glycans of the ZP. However, the binding is not restricted to ZP proteins but also occurs with other polysulphated glycans (Jones, 1991). Using recombinant techniques and site-directed mutagenesis (Jansen et al., 1995, 1998), binding has been mapped to the basic amino acids (particularly Arg250, Lys251, Arg253) within the 180 amino acids starting at Gly93, with essential contributions also being made by the basic amino acids His47, Arg50 and Arg51. Protein modelling on the basis of thrombin and chymotrypsin indicates that the essential basic amino acids appear to be located in the loops which are arranged near the active site. Studies on rabbit proacrosin led to similar results, demonstrating that the N-terminal region, containing three clustered Arg47, Arg50 and Arg51 mediates the sulphate binding (Richardson and O’Rand, 1996). After the acrosome reaction has been initiated, proacrosin may serve as a secondary receptor by temporarily anchoring acrosomereacted spermatozoa to the ZP. The polysulphate-binding domains appear to be involved not only in secondary binding, but also in the proacrosin–acrosin conversion (which is induced by ZP glycoproteins and other polysulphated glycans), as has been shown for porcine and human pro/acrosin (Töpfer-Petersen and Cechova, 1990; Moreno et al., 1998). Upon binding, the activation and processing of the enzyme is regulated by the ZP, resulting in the release of the enzyme and the spermatozoa. This fits into the proposed cyclic model of zona penetration (alternating cycles of binding, digestion and release reactions together with the forward motility is required to achieve penetration) which was first described in 1986 (O’Rand et al., 1986). The observation that proacrosin knock-out mice are fertile, though spermatozoa are slow in penetrating the ZP (Adham et al., 1997), may point to the important role of redundant systems involved in fertilization. Uncapacitated and capacitated porcine spermatozoa bind to the zona pellucida in vitro The oviductal epithelium and the ZP may share identical carbohydrate sequences, as could be shown by their lectin-binding pattern (Raychoudhury et al., 1993; Skutelsky et al., 1994). Under the conditions of different experimental approaches in vitro which have been widely used to identify ZBP (e.g. Western blotting, cross-linking studies, ZP-affinity chromatography and in-vitro inhibition assays), the sperm ZBP covering the surface of the uncapacitated spermatozoa may recognize carbohydrate structures of the ZP glycoproteins, although in the in-vivo situation they would rarely meet the ZP of the ovulated oocyte. In the pig, quantitative studies of spermatozoa–zona binding and penetration is seriously affected by the tendency of boar spermatozoa to bind to the ZP under non-capacitating conditions, and additionally by the considerable variation between semen samples (Petersen et al., 1984). Binding ability develops rapidly in either noncapacitating or capacitating conditions, while the penetration of the ZP appears to be a slow event and only occurs under conditions supporting capacitation (Harrison, 1997). Furthermore, it was reported (Harrison, 1997) that the proportion of zona-bound spermatozoa which are actually able to penetrate the ZP is much less than the proportion which has undergone capacitational changes in terms of CTC-staining, alterations of lectin-binding patterns (Harrison, 1997) and the recently observed increase of ZP binding sites (Harkema et al., 1998). By flow cytometry studies using solubilized fluorescence-labelled ZP glycoproteins it could be shown that, under non-capacitating conditions a low, but distinct number of Spermatozoan–egg interactions in fertilization ZP binding sites are localized at the plasma membrane overlying the acrosomal region which is involved in spermatozoa–zona binding. The zona-binding ability increases by incubation in media supporting capacitation with a maximum after 1–2 h in live, acrosome-intact cells. The complete omission of Ca2+ suppresses the increase of zona-binding ability, indicating that the development of ZP binding sites is related to capacitation (Harkema et al., 1998). Altogether, in the pig the number of zona-bound spermatozoa does not necessarily correlate with the possible ‘fertilizing’ subpopulation of spermatozoa that are able to respond immediately to the ZP. Moreover, boar spermatozoa appear to be able to bind to the ZP at different stages of capacitation, which may explain the temporal variation in developing penetration ability. Spermadhesins, predominantly forming the protein coat in ejaculated washed spermatozoa, appear to account for the binding of uncapacitated spermatozoa to intact ZP in vitro (Figure 6). As described above, their ligand-binding ability is influenced by the degree of glycosylation and aggregation. Moreover, serine proteinase inhibitors secreted by the seminal vesicle have been shown to bind specifically to members of the spermadhesin family, thereby partly masking the carbohydrate-binding domains (Sanz et al., 1992b; Calvete et al., 1993). The composition of the protein coat differs in individual ejaculates, and thus may influence zona-binding of the uncapacitated spermatozoa and also the progress of capacitation. Under capacitating conditions the spermadhesin aggregates and other bound substances, e.g. serine proteinases, may now be sequentially removed. This is accompanied by the observed increase of zona-binding ability that is due to the concomitant unmasking of other membrane-bound ZBP, e.g. AWN, AQN-3 and P47 and/or processing to the functional protein of integral zona-binding proteins such as zonadhesin (Figure 6). The sperm surface that actually responds to the ZP by activation of the signal transduction pathways and which leads to the acrosome reaction, may or may not carry membrane phospholipid-bound AWN and AQN-3. Regarding the behaviour of boar spermatozoa in vitro, the controversial finding concerning the biologically active oligosaccharide involved in spermatozoa–zona binding should be interpreted with care. Porcine ZP glycoproteins carry minor O-linked Neu5Acα2,3/Galβ1,3GalNAc tri- and disaccharides and sialylated bi-, tri- and tetra-antennary N-glycans with Galβ1,4GlcNAc sequences which may serve as ligands for the lectin-like spermadhesins. In fact, it could be shown that in vitro the spermadhesins recognize oligosaccharide structures on the three-dimensional network of intact zona pellucidae. AWN-coupled beads bind to the exposed oligosaccharide chains of ovarian oocytes (Figure 7) and AWN allowed to bind to intact ZP prevents both the beads and spermatozoa (Sanz et al., 1992a) from binding. The success of inhibition experiments with isolated oligosaccharides or glycopeptides of the ZP glycoproteins, e.g. O-glycans or N-glycans, may be dependent on the actual sperm subpopulation bound to 325 Figure 7. Scanning electron micrograph of the ovarian oocyte of the pig. Nucleosil C18 beads of 3 µm are loaded with the isolated AWN are able to interact with the zona pellucida. The binding could be inhibited by preincubation of the oocyte with AWN, and is inhibited competitively with the glycopeptides of fetuin (not shown). Bovine serum albumin (BSA)-loaded beads are not able to bind to the zona pellucida (not shown). the zonae pellucidae, and may be due to the inhibition of the binding of uncapacitated spermatozoa or of spermatozoa in various stages of capacitation. The undefined sperm population in terms of capacitational changes, which bind to zonae pellucidae in vitro, may be the reason for the apparently contradictory results that have been reported regarding the bioactivity of various carbohydrate structures. Is carbohydrate-mediated gamete recognition really species-specific? Species-specificity in mammals has been mostly linked to the carbohydrate-based recognition events at the spermatozoa–ZP interface. However, cross-binding has been observed between a variety of mammals. From mouse up to primates, spermatozoa are able to bind to human oocytes, though the reverse binding of human spermatozoa to oocytes of other species is restricted to near-related primates (Oehninger et al., 1993). It was reported recently (Wessa et al., 1999) that equine spermatozoa are able not only to bind and to penetrate the ZP of bovine ovarian eggs, but also to enter the egg’s cytoplasm. Capacitated porcine spermatozoa bind and have been found by electron microscopy to be mostly acrosome-reacted, though they do not pass bovine zonae pellucidae. The ZP exposes a variety of oligosaccharides containing basically similar structural elements in different species. Variable sperm lectins 326 E.Töpfer-Peterson may recognize those structures with or without consequence for sperm activation and fertilization. Thus, heterospecific incompatibility could occur at several steps in the fertilization cascade, e.g. spermatozoa–zona binding, induction of acrosome reaction and zona penetration, formation of the male pronucleus, and fusion of the male and female pronuclei to restore diploidy. The term ‘species-selectivity’ was created (Vacquier et al., 1995) to describe the situation in the sea urchin. Here, heterospecific gamete mixtures yielded the development of heterospecific zygotes varying from 0% to 100%. In the opinion of these researchers, ‘species-specificity’ means 100% species-exclusive and 0% heterospecific fertilization. These reflections may be also valid for gamete recognition in mammals. Conclusions Carbohydrates linked to proteins and lipids exercise a wide variety of functions and influences on the core molecule, e.g. protein folding, stabilization of conformation, targeting and sorting, as well as simple protection. One of the most fascinating features of complex sugar moieties, however, is their coding potential. The diversity of linkage sites and branching turn carbohydrates into ideal information-bearing molecules, having a vast informational potential within a short sequence, superior to that of other biological polymers such as DNA or proteins (Laine, 1997). The carbohydrate-encoded information must be deciphered by a protein with a complementary structure or CRD, e.g. glycosidases, glycosyltransferases, antibodies and lectins which transform the information into biological responses. In a wide variety of biological systems, cells and molecules use the information-storing potential of the sugar code for communication, e.g. recognition, adhesion and signalling events (Gabius, 1997). Thus, it is not surprising that along the route to fertilization, mammalian spermatozoa communicate with the female environment partly using the sugar codes. Carbohydrate/ZBP of the spermatozoa appear to be made available sequentially during the fertilization process. This suggests that there exists a hierarchy of binding events, ranging from spermatozoa–oviductal binding to primary and secondary binding between spermatozoa and oocytes. The binding of spermatozoa to the oviductal epithelium to create the sperm reservoir also appears to involve carbohydrates, although until now the role of the carbohydrates in transmitting oviductal signals to the spermatozoon is not clear. Moreover, the processes occurring in the oviduct are not species-specific events in as much as capacitation can occur in a heterologous oviduct, as well as under certain conditions in vitro. Nonetheless, the temporal and spatial regulation of sperm capacitation by the oviduct may be one way for male and female to communicate to ensure fertilization. However, the first contact between both gametes is the critical step in fertilization. Sperm binding to the ZP is directed by cooperative interactions of multiple complementary receptor systems which may act together in a hierarchical and redundant manner to guarantee survival of the species. The observations from studies with knock-out mice imply the existence of redundant systems rather than of single essential biological functions in fertilization. It has been demonstrated that the initial molecular interaction may involve low- and high-affinity components suggesting, therefore, a hierarchy of binding events at the spermatozoa–zona interface (Thaler and Cardullo, 1996). The interaction between the spermatozoa and the oocyte occurs at the surface of the three-dimensional ZP structure which is assembled by a defined array of polypeptide chains and arrangement of their oligosaccharides. In order to achieve high-affinity binding between spermatozoa and oocyte, a critical density of the different biologically active carbohydrates must be presented within the supramolecular architecture of the ZP matrix. It is a matter of speculation whether the combination of presented carbohydrates at the sperm-binding site of intact ZP may vary to some extent. The multivalent ligands which bind to the protein counterparts at the sperm surface, probably lead to the correct assembly of the proteins within the plane of the plasma membrane which is necessary to activate the signalling systems leading to acrosomal exocytosis. High affinity and multivalency may be the conditions for initiating the signal transduction pathway. This may explain why oligosaccharides or small glycopeptides are able to interfere with binding, but do not induce the acrosome reaction. Another question relates to whether the polypeptide backbone contributes to the induction of the acrosome reaction solely by clustering oligosaccharides or by direct protein–protein interaction. Recombinant ZP proteins undecorated with carbohydrates have been shown to activate acrosomal exocytosis in spermatozoa, thus emphasizing a role of the protein backbone in spermatozoa–zona interaction (Chapman et al., 1998). Although there is now a vast amount of information available on the events which occur when spermatozoa and oocyte meet, the linking pieces of the puzzle are still missing. Acknowledgements Our own studies were generously supported by the Deutsche Forschungsgemeinschaft. The author wishes to thank Dr Schwartz (Institute of Anatomy, University of Göttingen) for performing the electron microscopy. Drs Rump (Hannover), F.Sinowatz (Munich) and R.Ivell (Hamburg) are gratefully acknowledged for helpful discussions and critical reading of the manuscript, and Mrs E.Podajsky and Mrs Ch.Hettel for preparation of the photographs and figures. Spermatozoan–egg interactions in fertilization References Adham, I.M., Nayernia, K. and Engel, W. (1997) Spermatozoa lacking acrosin protein show delayed fertilization. Mol. Reprod. Dev., 43, 370–376. Anderson, M.H., Berglund, L., Rasmusson, J.T. and Petersen, T.E. (1997) Bovine PAS6/7 binds αvβ5 integrin and anionic phospholipids through two domains. Biochemistry, 36, 5441–5446. Aoki, N., Kishi, M., Taniguchi, Y. et al. (1995) Molecular cloning of glycoprotein antigen MGP53/57 recognized by monoclonal antibodies raised against bovine milk fat globule membrane. Biochim. Biophys. Acta, 1245, 385–391. Aviles, M., Castells, M.T., Martinez-Menarguez, J.A. et al. (1999) Cytochemical localization of GalNAc and GalNAcbeta1,4Galbeta1,4 disaccharide in mouse zona pellucida. Cell Tissue Res., 295, 269–277. Bedford, J.M. (1983) Significance of the need for sperm capacitation before fertilization in eutherian mammals. Biol. Reprod., 28, 108–120. Benoff, S. (1997) Carbohydrates and fertilization: an overview. Mol. Hum. Reprod., 3, 599–637. Benoff, S., Hurley, I., Cooper, G.W. et al. (1993) Fertilization potential is correlated with head-specific mannose-ligand receptor expression, acrosome status and membrane cholesterol content. Hum. Reprod., 8, 2155–2166. Biermann, L., Gabius, H.J. and Denker, H.W. (1997) Neoglycoprotein-binding sites (endogenous lectins) in the Fallopian tube, uterus and blastocyst of the rabbit during the preimplantation phase and implantation. Acta Anat., 160, 159–171. Bork, P. (1993) A trefoil domain in the major rabbit zona pellucida protein. Protein Sci., 2, 669–670. Bork, P. and Beckmann, G. (1993) The CUB domain. A widespread module in developmentally regulated proteins. J. Mol. Biol., 231, 539–545. Bork, P. and Sander, Ch. (1992) A large domain common to sperm receptors (Zp2 and Zp3) and TGF-β type III receptor. FEBS Lett., 300, 237–240. Brandelli, A., Miranda, P.V., Anon-Vazquez, M.G. et al. (1996) Voltage-dependent calcium channel and Gi regulatory protein mediate the human sperm acrosomal exocytosis induced by N-acetylglucosamine/mannosyl neoglycoproteins. J. Androl., 17, 522–529. Brewis, I.A., Clayton, R., Barratt C.L.R. et al. (1996) Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa. Mol. Hum. Reprod., 2, 583–589. Calvete, J.J., Sanz, L., Dostalova, Z. and Töpfer-Petersen, E. (1993) Characterization of AWN-1 glycosylated isoforms helps define the zona pellucida and serine proteinase inhibitor-binding region on boar spermadhesins. FEBS Lett., 334, 37–40. Calvete, J.J., Carrera, E., Sanz, L. and Töpfer-Petersen, E. (1996) Boar spermadhesins AQN-1 and AQN-3: oligosaccharide and zona pellucida characteristics. Biol. Chem., 377, 521–527. Calvete, J.J., Ensslin, M., Mburu, J. et al. (1997a) Monoclonal antibodies against boar sperm zona-pellucida-binding protein AWN-1. Characterization of a continuous antigenic determinant and immunolocalization of AWN-epitopes in inseminated sows. Biol. Reprod., 57, 753–742. Calvete, J.J., Raida, M., Gentzel, M. et al. (1997b) Isolation and characterization of heparin- and phosphorylcholine-binding proteins of boar and stallion seminal plasma. Primary structure of porcine pB1. FEBS Lett., 407, 201–206. Castle, P.E. and Dean, J. (1996) Molecular genetics of the zona pellucida: implications for immunocontraceptive strategies. J. Reprod. Fertil. Suppl., 50, 1–8. Chapman, N.R. and Barratt, C.L.R. (1996) The role of carbohydrate in sperm-ZP3 adhesion. Mol. Hum. Reprod. 2, 767–774. Chapman, N.R., Kessopoulou, E., Andrew, P.D. et al. (1998) The polypeptide backbone of recombinant human zona pellucida glycoprotein-3 initiates acrosomal exocytosis in human spermatozoa in vitro. Biochem. J., 330, 839–845. Clark, G.F., Oehninger, S. and Sepalla, M. (1996) Role of glycoconjugates in cellular communication in the human reproductive system. Mol. Hum. Reprod., 2, 513–517. DeMott, R.P. and Suarez, S.S. (1992) Hypoactivated sperm progress in the mouse oviduct. Biol. Reprod., 46, 779–785. 327 DeMott, R.P., Lefebvre, R. and Suarez, S.S. (1995) Carbohydrates mediate the adherence of hamster sperm to oviductal epithelium. Biol. Reprod., 52, 1395–1403. Dobrinski, I., Smith, T.T., Suarez, S.S. and Ball, BA. (1997) Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro. Biol. Reprod., 56, 861–869. Dostalova, Z., Calvete, J.J., Sanz, L. and Töpfer-Petersen, E. (1995) Spermadhesin AWN-1: oligosaccharide and zona pellucida binding characteristics. Eur. J. Biochem., 230, 329–336. Drickamer, K. (1993) Ca2+ dependent carbohydrate-recognition domain into C-type mannose-binding protein. Curr. Opin. Struct. Biol., 3, 393–400. Eble, J.A. and Kühn, K. (eds) (1997) Integrin–Ligand Interactions. Springer-Verlag, New York. Einspanier, R., Lauer, B., Wollenhaupt, K. and Schöneck, C. (1996) Influence of semen proteins on the route of fertilization. Reprod. Dom. Anim., 31, 107–112. Ensslin, M., Vogel, T., Calvete, J.J. et al. (1998) Molecular cloning and characterization of P47, a novel boar sperm associated zona pellucida-binding protein homologous to a family of mammalian secretory proteins. Biol. Reprod., 58, 1057–1064. Epifano, O., Liang, L.-F. and Dean, J. (1995) Mouse ZP1 encodes a zona pellucida protein homologous to egg envelope proteins in mammals and fish. J. Biol. Chem., 270, 27254–27258. Foster, J.A., Friday, B.B., Maulit, M.T. et al. (1997) AM67, a secretory component of guinea pig sperm acrosomal matrix is related to mouse sperm protein sp56 and the complement component 4-binding proteins. J. Biol. Chem., 272, 12714–12722. Fraser, L. (1995) Ionic control of sperm function. Reprod. Fertil. Dev., 7, 905–925. Gabius, H.-J. (1997) Animal lectins. Eur. J. Biochem., 243, 543–576. Gao, Z., Harumi, T. and Garbers, D.L. (1997) Chromosome localization of the mouse zonadhesin gene and the human zonadhesin gene (ZAN). Genomics, 41, 119–122. Gilbert, G.E. and Baleja, J.D. (1995) Membrane-binding peptide from the C2 domain of factor VIII forms an amphipathic structure as determined by NMR spectroscopy. Biochemistry, 35, 3022–3031. Gong, X., Dubois, D.H., Miller, D.J. and Shur, B.D. (1995) Activation of a G protein complex by aggregation of β-1,4 galactosyltransferase on the surface of sperm. Science, 269, 1718–1721. Grootenhuis, A.J., Philipsen, H.L.A., de Breet-Grijsbach, M.T. and Van Duin, M. (1996) Immunocytological localization of ZP3 in primordial follicles of rabbit, marmoset, rhesus monkey and human ovary using antibodies against human ZP3. J. Reprod. Fertil. Suppl., 50, 43–54. Hardy, D.M. and Garbers, D.L. (1995) 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., 44, 26025–26028. Harkema, W., Harrison, R.A.P., Miller, N.G.A. et al. (1998) Enhanced binding of zona pellucida proteins to the acrosomal region of intact boar spermatozoa in response to fertilizing conditions: a flow cytometric study. Biol. Reprod., 58, 421–430. Harris, J.D., Hibler, D.W., Fontenot, G.K. et al. (1994) Cloning and characterisation of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families. DNA Sequences, 4, 361–393. Harrison, R.A.P. (1997) Sperm plasma membrane characteristics and boar semen fertility. J. Reprod. Fertil. Suppl., 52, 195–211. Hedrick, J.L. (1996) Comparative studies and antigenic properties of zona pellucida glycoproteins. J. Reprod. Fertil. Suppl., 50, 9–17. Hokke, C.H., Damm, J.B., Penninkhof, B. et al. (1994) Structure of the O-linked carbohydrate chains of porcine zona pellucida glycoproteins. J. Biochem., 221, 491–512. Hunter, R.H.F. (1981) Sperm transport and reservoirs in the pig in relation to the time of ovulation. J. Reprod. Fertil., 63, 109–117. Hunter, R.H.F. (1988) Transport of gametes, selection of spermatozoa and gamete life span in the female reproductive tract. In Hunter, R.H.F. (ed.), The Fallopian Tubes: Their Role in Fertility and Infertility. Springer-Verlag, New York, pp. 53–86. Hunter, R.H.F. (1996) How, when and where do spermatozoa gain their fertilising ability in vivo? Reprod. Dom. Anim., 31, 51–54. 328 E.Töpfer-Peterson Hunter, R.H.F., Huang, W.T. and Holtz, W. (1998) Regional influences of the Fallopian tubes on the rate of boar sperm capacitation in surgically inseminated gilts. J. Reprod. Fertil., 114, 17–23. Jacob, A., Hurley, I., Mandel, F.S. et al. (1998) Human sperm non-nuclear progesterone receptor expression is a novel marker for fertilization outcome. Mol. Human. Reprod., 4, 533–542. Jansen, S., Quigley, M., Reik, W. and Jones, R. (1995) Analysis of polysulfate-binding domains in porcine proacrosin, a putative zona adhesion protein from mammalian spermatozoa. Int. J. Dev. Biol., 39, 501–510. Jansen, S., Jones, R., Jennecken, I. et al. (1998) Site-directed mutagenesis of boar proacrosin reveals residues involved in binding to zona pellucida glycoproteins. Mol. Reprod. Dev., 51, 184–192. Johnston, D.S., Wright, W.W., Shaper, J.H. et al. (1998) Murine sperm-zona binding, a fucosyl residue is required for a high affinity sperm-binding ligand. J. Biol. Chem., 273, 1888–1895. Jones, R. (1991) Interaction of zona pellucida glycoproteins, sulphated carbohydrates and synthetic polymers with proacrosin, the putative egg-binding protein from mammalian spermatozoa. Development, 111, 1155–1163. Katsuma, T., Noguchi, S., Yonezawa, N. et al. (1996) Structural characterization of the N-linked carbohydrate chains of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs. Eur. J. Biochem., 240, 448–453. Kölle, S., Sinowatz, F., Boie, G. et al. (1996) Localization of the mRNA encoding the zona protein ZP3α in the porcine ovary, oocyte and embryo by non-radioactive in situ hybridization. Histochem. J., 28, 441–447. Kölle S., Sinowatz F., Boie, G. and Palma, G. (1998) Differential expression of ZPC in the bovine ovary, oocyte and embryo. Mol. Reprod. Dev., 49, 435–443. Kudo, K., Yonezawa, N., Katsumata, T. et al. (1998) Localization of carbohydrate chains of pig sperm ligand in the glycoprotein ZPB of egg zona pellucida. Eur. J. Biochem., 252, 492–499. Laine, R. (1997) The information-storing potential of the sugar code. In Gabius, H.J. and Gabius, S. (eds), Glycosciences. Chapman & Hall, Weinheim, pp. 1–14. Lefebvre, R. and Suarez, S.S. (1996) Effect of capacitation on bull sperm binding to homologous oviductal epithelium. Biol. Reprod., 54, 575–582. Lefebvre, R., DeMott, R.P., Suarez, S.S. and Scamper, J.C. (1995) Specific inhibition of equine sperm binding to oviductal epithelium. Equine Reproduction VI. Biol. Reprod., Monograph 1, 689–696. Lefebvre, R., Lo, M.C. and Suarez, S.S. (1997) Bovine sperm binding to oviductal epithelium involves fucose recognition. Biol. Reprod., 56, 1198–1204. Legand, P.K., Rau, A., Keen, J.N. and Richardson, G.P. (1997) The mouse tectorins. J. Biol. Chem., 272, 8791–8801. Litscher, E.S. and Wassarman, P.M. (1996) Characterization of a mouse ZP3-derived glycopeptide, gp 55, that exhibits sperm receptor and acrosome reaction-inducing activity in vitro. Biochemistry, 35, 3980–3985. Litscher, E.S., Jutunen, K., Seppo, A. et al. (1995) Oligosaccharide constructs with defined structures that inhibit binding of mouse sperm to unfertilized eggs in vitro. Biochemistry, 34, 4662–4669. Liu, C., Litscher, E.S. and Wassarman, P.M. (1997) Zona pellucida glycoprotein mZP3 bioactivity is not dependent on the extent of glycosylation of its polypeptide or on sulfation and sialylation of its oligosaccharides. J. Cell Sci., 110, 745–752. Martinez, M.L., Fotenot, G.K. and Harris, J.D. (1996) The expression and localization of zona pellucida glycoproteins and mRNA in cynomolgus monkey (Macaca fasicularis). J. Reprod. Fertil. Suppl., 50, 35–41. Maymon, B.B., Maymon, R., Ben-Nun, I. et al. (1994) Distribution of carbohydrates in the zona pellucida of human oocytes. J. Reprod. Fertil., 102, 81–86. McLesky, S.B., Dowds, C., Carbadalla, R. et al. (1998) Molecules involved in mammalian sperm-egg interaction. Int. Rev. Cytol., 177, 57–113. Miranda, P.V. (1998) Human sperm-zona interaction: while trying to reproduce. Mol. Hum. Reprod., 4, 523–525. Miranda, P.V., Gonzalez-Echeverria, F., Marin-Briggiler, C.I. et al. (1997) Glycosidic residues involved in human sperm-zona pellucida binding in vitro. Mol. Hum. Reprod., 3, 399–404. Moreno, R.D., Sepuilveda, M.-S., de Ioannes, A. and Barros, C. (1998) The polysulphate binding domain of human proacrosin/acrosin is involved in both enzyme activation and spermatozoa-zona pellucida interaction. Zygote, 6, 75–83. Mori, E., Hedrick, J.L., Wardrip, N.J. et al. (1998) Occurrence of reducing terminal N-acetylglucosamine-3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoprotein. Glycoconj. J., 15, 447–456. Murray, S.C. and Smith, T.T. (1997) Sperm interaction with the fallopian tube apical plasma membrane enhances sperm motility and delays capacitation. Fertil. Steril., 68, 351–357. Nagdas, S.K., Araki, Y., Chayko, C.A. et al. (1994) O-linked trisaccharide and N-linked poly-N-acetyllactosamine glycans are present on mouse ZP2 and ZP3. Biol. Reprod., 51, 262–272. Nakano, M. and Yonezawa, N. (1998) Pig and bovine sperm receptors in the egg zona pellucida. Zygote, 6 (suppl. 1), 21–30. Nakano, M., Yonezawa, N., Hatanaka, Y. and Noguchi, S. (1996) Structure and function of the N-linked carbohydrate chains of pig zona pellucida glycoproteins. J. Reprod. Fertil. Suppl., 50, 25–34. Noguchi, S. and Nakano, M. (1992) Structure of the acidic N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur. J. Biochem., 209, 883–894. Noguchi, S. and Nakano, M. (1993) Structural characterization of the N-linked carbohydrate chains from mouse zona pellucida glycoproteins ZP2 and ZP3. Biochim. Biophys. Acta, 1158, 217–226. Noguchi, S., Hatanaka, Y., Tobita, T. and Nakano, M. (1992) Structural analysis of the N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur. J. Biochem., 204, 1089–1100. Oehninger, S., Coddington, C.C. and Hodgen, G.D. (1990) Antagonistic and agonistic properties of saccharide moieties in the hemizona assay. Fertil. Steril., 63, 377–386. Oehninger, S., Mahony, M.C., Swanson, J.R. and Hodgen, G.D. (1993) The specificity of human spermatozoa/zona pellucida interaction under hemizona assay conditions. Mol. Reprod. Dev., 35, 57–61. Oehninger, S., Patankar, M.S., Seppala, M. and Clark, G.F. (1998) Involvement of selectin-like carbohydrate binding specificity in human gamete interaction. Andrologia, 30, 269–274. Ogura, K., Nara, K., Watanabe, Y. et al. (1996) Cloning and expression of cDNA for O-acetylation of GD3 ganglioside. Biochem. Biophys. Res. Commun., 225, 932–938. O’Rand, M.G., Welch, J.E. and Fisher, S.J. (1986) Sperm membrane and zona pellucida interactions during fertilization. In Dhinsda, D.S. and Bahl, O. (eds), Molecular and Cellular Aspects of Reproduction. Plenum Press, New York, pp. 131–144. Ozgur, K., Patankar, M.S., Oehninger, S. et al. (1998) Direct evidence for the involvement of carbohydrate sequences in human sperm-zona pellucida binding. Mol. Hum. Reprod., 4, 318–324. Peterson, R.N., Russell, L.D. and Hunt, W.P. (1984) Evidence for specific binding of uncapacitated boar sperm to porcine zona pellucida in vitro. J. Exp. Zool., 231, 137–147. Prasad, S.V., Wilkins, B., Skinner, S.M. and Dunbar, B.S. (1998) Evaluating zona pellucida structures and function using antibodies to rabbit 55 kDa ZP protein expressed in baculovirus expression system. Mol. Reprod. Dev., 43, 519–529. Rankin, T.L., Tong, Z.B., Castle, P.E. et al. (1998) Human ZP3 restores fertility in zp3 null mice without affecting order-specific sperm binding. Development, 125, 2415–2424. Raychoudhury, S.S., Suarez, S.S. and Buhi, W.C. (1993) Distribution of lectin binding sites in the oviduct of cycling and hormone-treated pigs. J. Exp. Zool., 265, 659–668. Richardson, R.T. and O’Rand, M.G. (1996) Site-directed mutagenesis of rabbit proacrosin. J. Biol. Chem., 39, 24069–24074. Richardson, R.T., Yamasaki, N. and O’Rand, M.G. (1994) Sequence of a rabbit sperm zona pellucida binding protein and localization during acrosome reaction. Dev. Biol., 165, 688–701. Spermatozoan–egg interactions in fertilization Romero, A., Romao, M.J., Varela, P.F. et al. (1997) The crystal structure of two spermadhesins reveals the CUB domain fold. Nature Struct. Biol., 4, 783–787. Sanz, L., Calvete, J.J., Mann, K. et al. (1992a) The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa. FEBS Lett., 300, 213–218. Sanz, L., Calvete, J.J., Jonakova, V. and Töpfer-Petersen, E. (1992b) Boar spermadhesin AQN-1 and AWN are sperm-associated acrosin inhibitor acceptor proteins. FEBS Lett., 300, 63–66. Shur, B. (1998) Is sperm galactosyltransferase a signaling subunit of a multimeric gamete receptor? Biochem. Biophys. Res. Commun., 250, 537–543. Sinowatz, F., Voglmayr, J.K., Gabius, H.-J. and Friess, A.E. (1989) Cytochemical analysis of mammalian sperm membranes. In Progress in Histochemistry and Cytochemistry, vol. 19. Gustav Fischer Verlag, Stuttgart, New York. Sinowatz, F., Amselgruber, W., Töpfer-Petersen, E. et al. (1995) Immunocytological characterization of porcine zona pellucida proteins during follicular development. Anat. Embryol., 191, 41–46. Sinowatz, F., Töpfer-Petersen, E. and Calvete, J.J. (1997) Glycobiology of fertilization. In Gabius, H.J. and Gabius, S. (eds), Glycosciences. Chapman & Hall, Weinheim, pp. 595–610. Skutelsky, E., Ranen, E. and Shalgi, R. (1994) Variations in the distribution of sugar residues in the zona pellucida as possible species-specific determinants of mammalian oocytes. J. Reprod. Fertil., 100, 35–41. Smith, T.T. (1998) The modulation of sperm function by the oviductal epithelium. Biol. Reprod., 58, 1102–1104. Smith, T.T. and Nothnick, W.B. (1997) Role of direct contact between spermatozoa and oviductal epithelial cells in maintaining rabbit sperm viability. Biol. Reprod., 56, 83–89. Smith, T.T. and Yanagimachi, R. (1990) The viability of hamster spermatozoa stored in the isthmus of oviduct: the importance of sperm-epithelium contact for sperm survival. Biol. Reprod., 42, 450–457. Snell, W.J. and White, J.M. (1996) The molecules of mammalian fertilization. Cell, 85, 629–637. Solis, D., Romero, A., Jiminez, M. et al. (1998) Binding of mannose-6-phosphate and heparin by boar seminal plasma PSPII, a member of the spermadhesin protein family. FEBS Lett., 431, 273–278. Storey, B.T. (1995) Interactions between gametes leading to fertilization: the sperm’s eye view. Reprod. Fertil. Dev., 7, 927–942. Stubbs, J.D., Lekutis, C., Singer, K.L. et al. (1990) cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences. Proc. Natl Acad. Sci. USA, 87, 8417–8421. Suarez, S.S. (1996) Hyperactivated motility in sperm. J. Androl., 17, 331–335. Suarez, S.S. (1998) The oviductal sperm reservoir in mammals: mechanism of formation. Biol. Reprod., 58, 1105–1107. Suarez, S.S., Revah, I., Lo, M. and Kolle, S. (1998) Bull sperm binding to oviductal epithelium is mediated by a Ca2+-dependent lectin on sperm that recognize Lewis-a trisaccharide. Biol. Reprod., 59, 39–44. Thaler, C.D. and Cardullo, R.A. (1996) The initial molecular interaction between mouse sperm and the zona pellucida is a complex binding event. J. Biol. Chem., 271, 23289–23297. Tian, J., Gong, H. and Lennarz, W.J. (1999) Xenopus laevis sperm receptor gp69/64 glycoprotein is a homolog of the mammalian sperm receptor ZP2. Proc. Natl Acad. Sci. USA, 96, 829–834. 329 Töpfer-Petersen, E. and Calvete, J.J. (1996) Sperm-associated protein candidates for primary zona pellucida-binding molecules: structure-function correlation of boar spermadhesins. J. Reprod. Fertil. Suppl., 50, 55–61. Töpfer-Petersen, E. and Cechova, D. (1990) Zona pellucida induces conversion of proacrosin to acrosin. Int. J. Androl., 13, 190–196. Töpfer-Petersen, E., Cechova, D., Henschen, A. et al. (1990) Cell biology of acrosomal proteins. Andrologia, Suppl., 1, 110–121. Töpfer-Petersen, E., Calvete, J.J., Sanz, L. and Sinowatz, F. (1995) Carbohydrate- and heparin-binding proteins in mammalian fertilization. Andrologia, 27, 303–324. Töpfer-Petersen, E., Romero, A., Varela, P.F. et al. (1998) Spermadhesins: a new protein family. Facts, hypothesis and perspectives. Andrologia, 30, 217–223. Topper, E.K., Kruijt, L., Calvete, J.J. et al. (1997) Identification of bovine zona pellucida glycoproteins. Mol. Reprod. Dev., 46, 344–350. Totzauer, I., Kölle, S., Sinowatz, F. et al. (1998) Localization of the zona pellucida glycoproteins ZPB (αZP3) and ZPC (ZP3β) in the bovine ovary during pre-and postnatal development. Ann. Anat., 180, 37–43. Tulsiani, D.R.P., Yoshida-Komiya, H. and Araki, Y. (1997) Mammalian fertilization: a carbohydrate-mediated event. Biol. Reprod., 57, 487–494. Vacquier, V.D., Swanson, W.J. and Heilberg, M.E. (1995) What have we learned about sea urchin sperm binding? Dev. Growth Differ., 37, 1–10. Wassarman, P.M. and Mortillo, S. (1991) Structure of the mouse egg extracellular coat, the zona pellucida. Int. Rev. Cytol., 130, 85–110. Wassarman, P.M. and Litscher, E. (1995) Sperm-egg recognition mechanisms in mammals. Curr. Top. Dev. Biol., 30, 1–19. Wessa, E., Neumüller, C. and Sinowatz, F. (1999) Are sperm binding and sperm penetration through the zona pellucida really species-specific? Reprod. Dom. Anim. (in press). Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neill, J.D. (eds), The Physiology of Reproduction, 2nd edn. Raven Press, New York, pp. 189–317. Yao, Y.Q., Ho, P.C. and Yeung, W.S.B. (1999) Effects of human oviductal cell coculture on various functional parameters of human spermatozoa. Fertil. Steril., 71, 232–239. Yeung, W.S.B., Ng, V.K.H., Lau, E.Y.L. and Ho, P.C. (1994) Human oviductal cells and their conditioned medium maintain motility and hyperactivation of human sperm in vitro. Hum. Reprod., 9, 656–660. Yonezawa, N., Aoki, H., Hatanaka, Y. and Nakano, M. (1995a) Involvement of N-linked carbohydrate chains of pig zona pellucida in sperm-egg binding. Eur. J. Biochem., 233, 35–41. Yonezawa, N., Hatanaka, Y., Takeyama, H. and Nakano, M. (1995b) Binding of pig sperm receptor in the zona pellucida to the boar sperm acrosome. J. Reprod. Fertil., 103, 1–8. Yurewicz, E.C., Pack, B.A. and Sacco, A.G. (1991) Isolation, composition and biological activity of sugar chains of porcine zona pellucida 55K glycoproteins. Mol. Reprod. Dev., 30, 126–134. Yurewicz, E.C., Sacco, A.G., Gupta, S.K. et al. (1998) Heterooligomerisation-dependent binding of pig oocyte zona pellucida glycoproteins ZPB and ZPC to boar sperm membrane vesicles. J. Biol. Chem., 273, 7488–7494. Received on October 14, 1998; accepted on April 21, 1999