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Carbohydrates mediate sperm-ovum adhesion and triggering of the acrosome reaction

Daulat R.P. Tulsiani

Departments of Obstetrics & Gynecology and Cell Biology, Vanderbilt University School of Medicine, Room D-3243 MCN, Nashville, Tennessee 37232-2633, USA

Asian J Androl  2000 Jun; 2:  87-97


Keywords: sperm capacitation; sperm-ovum interaction; acrosome reaction; carbohydrates; fertilization
Abstract
The fertilization process is the net result of a complex sequence of events that collectively result in the fusion of the opposite gametes. The male gamete undergoes continuous morphological and biochemical modifications during sperm development in the testis (spermatogenesis), maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are able to recognize and bind to the bioactive glycan residue(s) on the ovum's extracellular coat, the zona pellucida (ZP). Sperm-zona binding in the mouse and several other species is believed to take place in two stages. First, capacitated (acrosome-intact) spermatozoa loosely and reversibly adhere to the zona-intact ovum. In the second stage tight irreversible binding occurs. Both types of bindings are attributed to the presence of glycan-binding proteins (receptors) on the sperm plasma membrane and their complementary bioactive glycan units (ligands) on the surface of the ZP. The carbohydrate-mediated adhesion event initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be prerequisite which allows the hyperactivated acrosome-reacted spermatozoa to penetrate the ZP and fertilize the ovum.  This review focuses on the role of carbohydrate residues in sperm-ovum interaction, and triggering of the acrosome reaction. I have attempted to discuss extensive progress that has been made to enhance our understanding of the well programmed multiple molecular events necessary for successful fertilization. This review will identify these events, and discuss the functional significance of carbohydrates in these events.

1 Introduction

For successful fertilization in several species, capacitated acrosome-intact spermatozoa recognize and bind to the ovum's extracellular coat, the zona pellucida (ZP) in a receptor-ligand-mediated manner[1-4]. Sperm-ovum interaction in the mouse and several other species, including man, is believed to take place in two stages.  First, the capacitated spermatozoa loosely and reversibly adhere to the ZP by means of plasma membrane overlying the acrosome, followed by a tight species-specific irreversible binding[1,2]. Both types of bindings are thought to be mediated by glycan-binding molecules (glycosyltransferases, glycosidases and/or lectin-like proteins) on sperm plasma membrane (receptors) and complementary terminal sugar residues (ligands) on glycan moiety(ies) of ZP[1-3]. In recent years considerable progress has been made in the identification and characterization of molecules on the sperm plasma membrane and complementary terminal sugar residues on ZP that are believed to be important for gamete interaction and triggering of sperm activation (i.e., induction of the acrosome reaction). The exocytosis of acrosomal contents during sperm activation is believed to be a prerequisite event which allows acrosome-reacted spermatozoa to penetrate the ZP and fertilize the ovum[5]. 

There has been a longstanding interest in the basic biology of the fertilization process. The success of in vitro fertilization in domestic animals and humans is a result of the knowledge gained in small laboratory animals.  Among mammals, the events leading to fusion of the opposite gametes are best illustrated in the mouse, although there is considerable information in other species, including man[1,2]. Successful fertilization in the mouse following mating involves several orchestrated steps.  These are: 1) sperm capacitation in the female genital tract, 2) binding of the capacitated spermatozoa to the ZP and induction of the acrosome reaction, 3) secondary binding of the acrosome-reacted sperm to ZP and its penetration, and 4) fusion of the spermatozoon with the ovum vitelline membrane (Figure 1). This review will focus on the recent advances made toward elucidating the molecular basis of the first two steps.

Figure 1. Progressive stages of sperm-ovum interaction in the mouse: 1, the acrosome-intact spermatozoon passes through cumulus cells and the innermost layer, the corona radiata; 2, sperm receptor(s) bind to glycan moiety(ies) of mZP3 and starts a signal transduction cascade resulting in acrosomal exocytosis; 3, the acrosome-reacted spermatozoon penetrate the ZP; and 4, the spermatozoon passes through the perivitelline space and fuses with the ovum plasma membrane.

1.1 Sperm capacitation

Mammalian spermatozoa released from the seminiferous tubules and those present within the proximal regions of the epididymis are non-motile and unable to bind to the ZP and undergo the acrosome reaction. They acquire progressive motility during transit through epididymis, a process termed epididymal maturation[1]. During this passage, the epididymis provides a specific intraluminal environment in which functionally immature sperm cells undergo morphological and biochemical alterations necessary to produce competent spermatozoa capable of undergoing capacitation and successful binding to the ZP[1]. During epididymal maturation, sperm plasma membrane, a vital component in the early events of fertilization undergoes extensive biochemical changes and remodeling[5-7]. These modifications are an essential part of the maturation process resulting in the production of a self-propelled, functionally-competent spermatozoon.

In addition to maturation in the epididymis, spermatozoa must undergo functional changes between the events of mating and fertilization[1,2]. During residence in the female reproductive tract, they undergo a series of biochemical and functional modifications collectively referred to as capacitation[1]. The net change during this process is a combined effect of multiple molecular modifications in the sperm plasma membrane proteins/glycoproteins and lipid components that are thought to modify the ion channels in the plasmalemma[8]. The preparatory alterations include removal of seminal plasma molecules adsorbed to the surface of ejaculated spermatozoa as well as modification and/or reorganization of sperm surface components[2]. The adsorbed seminal plasma factors are believed to prevent spermatozoa from undergoing premature acrosome reaction before completing their migratory journey through the female reproductive tract.  As a result of these alterations, capacitated spermatozoa become acrosomally responsive by yet unknown mechanisms. Experimental evidence from several studies strongly suggests that sperm loses cholesterol before becoming acrosomally responsive[9].

Although the significance of capacitation has been known for nearly 40 years[10,11], the molecular mechanisms responsible for the functional changes are not fully understood.  Most researchers agree that capacitation results from multiple molecular changes in sperm plasma membrane proteins/glycoproteins and lipid components that likely modify ion channels. The reported changes in cholesterol and phospholipid during  in vivo capacitation is consistent with this possibility[12].  As capacitation proceeds, a number of biochemical changes occur on spermatozoa. These modifications allow the transmembrane flux of ions that are believed to be important in initiating the events of capacitation, hyperactivation and acrosome reaction[2]. The former two events take place before sperm-ovum (zona) interaction, and the latter event, at least in the mouse and several other species including man, is initiated after sperm-ovum binding. It is, therefore, reasonable to assume that the three events are independent and involve region-specific alterations in sperm plasma membrane.

In most mammalian species studied, spermatozoa become hyperactive in the isthmus region of the oviduct, and the hyperactivated spermatozoa move to the ampullary environment, the site of in vivo fertilization[13-15]. A possible functional significance of hyperactivation could be that the hyperactivated beat pattern of a spermatozoon will definitely enhance its thrust (physical force) at the site of its binding to ZP and undergoing the acrosome reaction[16]. The hydrolytic action of glycohydrolases and proteases released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important in regulating the penetration of ZP and fusion with the ovum plasma membrane. 

All mammalian spermatozoa studied thus far undergo capacitation after residing in the female genital tract for a certain period of time. Similarly, spermatozoa recovered from the female genital tract after mating, or cauda epididymal spermatozoa following incubation with oviductal secretions, are able to fertilize an ovum in vitro[17,18]. The precise site of capacitation may be different in different species; however, several studies suggest that capacitation is most efficient when the spermatozoa pass through the uterus and oviduct. The oviductal secretions collected from the estrous females have been demonstrated to be most efficient in rendering the functional changes in spermatozoa in vitro[1,18]. 

Mammalian sperm can also be capacitated in vitro in a chemically defined medium supplemented with energy substrates, such as glucose and pyruvate, bovine serum albumin, and appropriate buffer[1,19]. It is noteworthy that albumin is the major protein in the female genital tract secretions and is an important component for in vitro and in vivo capacitation[19,20] protein is believed to facilitate capacitation by efflux of fatty acid/cholesterol/phospholipids from sperm plasma membrane. The precise mechanism as to how the loss of cholesterol/phospholipids regulates the functional changes is not yet known; however, it is generally believed that the loss of fatty acids/cholesterol increases fluidity and permeability of the sperm plasma membrane and initiate the events of capacitation and the acrosome reaction.

As capacitation proceeds, a number of intracellular and cell surface changes occur on spermatozoa. The known changes include a) increased adenylate cyclase and cAMP[21,22], b) increase in intracellular pH[23], c) calcium influx[24], d) loss of sperm surface components[25], e) modification/alteration of sperm plasma membrane[26], and f) tyrosine phosphorylation of a subset of sperm proteins[27,28]. Recent studies suggest that the tyrosine phosphorylation is dependent on increased cAMP, a result consistent with the suggestion of a relationship between cAMP-dependent protein kinase A and tyrosine kinases and/or phosphatases[27-29]. Whether any of the above changes are necessary for capacitation is not yet known. The molecular mechanism(s) as to how these changes affect the functional state of spermatozoa is currently under intensive investigation.

One important surface alteration that happens following capacitation is a change in the lectin-binding properties of spermatozoa[30-33], a result indicative that the terminally exposed carbohydrate moieties are altered during this process. A possible explanation for these changes in vivo could be the association of an oviductal glycoprotein secreted from oviductal epithelial cells, and reported to bind to the posterior region of the spermhead, midpiece, and flagellum[2,34]. A second possibility is that the existing sperm plasma membrane glycoproteins are modified in vivo by glycosyltransferases present in the uterus and oviduct secretions, and which display a temporal surge in the genital tract during the esterous cycle[35]. The plausible modification of the endogenous sperm surface glycoproteins by addition of sugar residues to the terminally exposed glycan moieties will be expected to alter the lectin-binding properties of capacitated spermatozoa. Alternatively, the glycohydrolases present within the sperm acrosome of noncapacitated spermatozoa could translocate (redistribute) on the sperm surface of capacitated sperm as has been reported for hyaluronidase[36]. These enzymes not only hydrolyze glycosyl residues from glycoproteins, they themselves are glycoproteins[6]. The appearance of intra-acrosomal glycohydrolases (glycoproteins) on the sperm surface following capacitation is expected to alter the lectin-binding properties of spermatozoa.

Finally, several studies have provided evidence suggesting a role for proteoglycans and glycosaminoglycans in altering the functional state of spermatozoa. Heparin, the most effective glycosaminoglycan, binds to spermatozoa from several species, including man[37]. The binding is said to be sensitive to pH, ionic strength, and temperature, and thus has characteristics of a typical receptor-ligand interaction.  However, the receptor for glycosaminoglycan has not been identified, and the molecular mechanisms responsible for these interactions are not fully understood. 

From the preceding discussion, it is obvious that capacitation is a net result of multifaceted changes in mature spermatozoa.  Although molecular details of these changes may vary among species, the final result is the development of hyperactivated motility, zona binding ability, and responsiveness of the bound spermatozoa to undergo the acrosome reaction.

2 Sperm-Zona (Egg) binding

The capacitated spermatozoa bind to the ZP in a highly precise manner. Extensive studies in the mouse seem to suggest that sperm binding is a two-step process. First, capacitated sperm loosely and reversibly adhere to ZP by means of plasma membrane overlying the acrosome, followed by a tight and irreversible binding[1]. Both types of bindings are attributed to the presence of glycan-binding proteins (receptors) on the sperm plasma membrane and their complementary glycan chains (ligands) on ZP glycoconjugates[38,39]. Thus sperm-ovum interaction is a carbohydrate-mediated receptor-ligand binding event. This type of binding is analogous to cellular adhesion events of bacteria, viruses, and many pathogens to their respective host cells.

2.1 Zona pellucida glycoconjugates

ZP is the extracellular glycocalyx which surrounds all mammalian ovums. The ZP in all species studied is a relatively simple structure composed of three families of glycoproteins, designated as ZP1, ZP2, and ZP3, and the pig has a fourth form as well (Table 1). The main functions of ZP in fertilization have been generally accepted as the mediation of the relative species specificity of sperm binding, induction of the sperm acrosome reaction, secondary binding events, blocking of polyspermy, and protection of growing embryo from fertilization to implantation[1]. It is now clear that the proteins which make up the zona pellucida are highly conserved among mammalian species, suggesting that their organization may also be conserved.  In the mouse, the three glycoproteins are synthesized and secreted by the growing oocytes. Two of the glycoproteins, mouse ZP2 (mZP2) and mZP3, interact noncovalently to form filaments of structural repeats that are interconnected by mZP1 forming a three-dimensional network of cross-linked filaments[40].  Such a structure may explain the elasticity of ZP and the relative ease of its penetration by the hyperactivated acrosome-reacted spermatozoa.

Table 1. Characteristics of mammalian ZP glycoproteins.

Species

Zona glyco-
conjugate

Molecular mass (kDa)

No. of N-glycosylation sites

No. of N-linked oligosaccharides

Molecular mass of O-linked oligosaccharide (kDa)

Bonnet Monkey1

ZP1

51, 40 (dimer)

---

---

---

ZP2

68

---

---

---

ZP3

50

---

---

---

Hamster2

ZP1

103(dimer)

---

---

---

ZP2

208

---

---

---

ZP3

56

4

---

---

Human2

ZP1

90-110

---

---

---

ZP2

64-78

6

---

---

ZP3

57-73

4

---

---

Mouse2

ZP1

185-200

6

+

---

ZP2

90-140

7

6

+

ZP3

83

6

3 or 4

2-3

Porcine2

ZP1

82-90

7

---

---

ZP2

61-65

---

---

---

ZP3

55(,)

5

---

---

ZP4

21-25

2

---

---

Rabbit2

ZP1

85

---

---

---

ZP2

75

7

---

---

ZP3

55

6

---

---

Rat3

ZP1

205

4

---

---

ZP2

119

8

---

0

ZP3

115

6

---

2

1Gupta SK, Govind CK, Senthil D, Srivastava M, Jethanandani P, Kaul R, Mittal A, Sivapurapu N, Sheela P. Molecular characterization of non-human primate zona pellucida glycoproteins.  In: Gupta SK, editor.  Reproductive Immunology. New Delhi: Narosa Publishing House; 1999.  p 33-44.  
2Tulsiani DRP, Yoshida-Komiya H, Araki Y. Mammalian fertilization: a carbohydrate mediated event.  Biol Reprod 1997; 57: 487-494.
3Akatsuka K, Yoshida-Komiya H, Tulsiani DRP, Orgebin-Crist MC, Hiroi M, Araki Y. Rat zona pellucida glycoproteins: Molecular cloning and characterization of the three major components. Mol Reprod Dev 1998; 51: 454-467.

In recent years, considerable progress has been made in understanding structure-function of various zona components. In particular, work on mZP has resulted in identification of primary (mZP3) and secondary (mZP2) binding sites for homologous spermatozoa[41]. This conclusion was based on the experimental evidence suggesting that only mZP3 is able to inhibit sperm-ovum binding in an in vitro assay in a dose-dependent manner. The observed inhibition is apparently caused by competition of the added mZP3 for the complementary receptor(s) on the plasma membrane overlying capacitated sperm head.  Moreover, when the radioiodinated mZP2 or mZP3 was incubated with capacitated (acrosome-intact) or acrosome-reacted spermatozoa, the former glycoprotein showed higher binding to the acrosome-reacted spermatozoa, whereas the latter glycoconjugate showed higher binding to capacitated sperm head[42]. Further support that the primary binding sites are present on mZP3 comes from the experimental evidence demonstrating that mZP3 from fertilized ovums is no longer able to inhibit sperm-ovum binding. This result is consistent with the assumption that the loss of primary binding sites on ZP of the fertilized ovums is due to modification of mZP3.

Several lines of evidence listed in previous review articles[2,40] strongly suggest that glycan units of mZP3 provide the primary ligand site(s) for the sperm receptors. Two of these evidences are highly specific and are repeated in this report. First, sperm binding activity of mZP3 is sensitive to trifluoromethane sulfonic acid treatment, an acid known to break glycosidic bonds between the monosaccharide residues of N-linked and O-linked oligosaccharides without altering the protein backbone[40]. Second, the ability of mZP3 to competitively inhibit sperm-ovum binding is unaffected by digestion with pronase. The proteolysis digests protein backbone of mZP3, but the resulting glycopeptides (N-linked and O-linked ranging in size from 1.5-6.0 kDa) are able to inhibit sperm-ovum binding in vitro in a dose-dependent manner[40]. It should be noted that the binding of capacitated spermatozoa starts a cascade of signaling events resulting in acrosomal exocytosis. The ability of mZP3 to serve as the primary ligand mainly depends on glycan chains; however, its ability to induce the acrosome reaction depends on glycan units as well as the protein backbone. Consistent with this possibility is the finding that the pronase generated ZP3 glycopeptides retain bioactivity but do not induce the acrosome reaction unless the bound glycopeptides (glycan units) are cross-linked on the sperm surface by anti-ZP3 IgG[43,44]. Studies published from another laboratory also suggest that binding of multiple glycan units of mZP3 to the sperm surface galactosyltransferase causes its aggregation and triggering of the acrosome reaction[45]. Our recent studies demonstrate that specific sugar residues (mannose, N-acetylglucosamine, and N-acetylgalactosamine) can induce the acrosome reaction, but only when covalently conjugated to a protein backbone[20,46]. The potential implication of multiple monosaccharide residues in initial sperm-ovum binding is consistent with a report demonstrating that initial molecular interaction between sperm and mZP3 is a complex binding process which reflects multiple sperm surface receptors with multivalent ZP3[47]. The implication of several sugar residues in initial sperm-ovum binding and induction of the acrosome reaction is also consistent with this possibility. The sugar residues suggested to have a role in initial binding of the opposite gametes are: -D-galactose[48], -D-galactose[49,50], -N-acetylglucosamine[3,51], mannose[52,53], and sialic acid[54]. Although a terminal fucosyl residue has not been implicated in initial binding of opposite gametes in the mouse, its presence appears to be obligatory for an oligosaccharide to bind to spermatozoa with high affinity[55]. Taken together, these studies suggest a possible interaction between terminal sugar residues on mZP3 or neoglycoproteins prior to the induction of the acrosome reaction. The ZP3 or protein conjugated sugar residues are thought to induce the acrosome reaction by cross-linking or aggregating its receptor on the sperm plasma membrane. 

Work from my group[52,53] and others[56,57] suggests that N-linked (asparagine-linked) glycans may be bioactive molecules that are recognized by sperm surface receptor(s). It is interesting to notice that N-linked glycans contribute nearly half of the molecular mass of mZP3 and over 40% of mZP2[58]. Thus a discussion on the complexities in the structure of N-linked glycans will contribute to a better understanding of their role in sperm-ovum interaction. The N-linked glycans may be either of high mannose, hybrid type or complex (bi-, tri-, tetraantennary) structures[5]. The three types of glycans contain the basic structure composed of a branched trimannose region to an N,N'-diacetylchitobiose which is attached to the amide nitrogen of an asparagine residue on the protein. In the high mannose oligosaccharide, the core structure is substituted by -linked mannosyl residues whereas in complex structures, the core structure is elongated by the presence of trisaccharide (sialic acid-galactose-N-acetylglucosamine).  The hybrid type glycan is a combination of high mannose and complex type where one antenna of the core structure contains only mannosyl residues and the other antenna contains one or two trisaccharide units on 1,3-branch[59].

In addition, many glycoproteins, including mZP2, mZP3[58], and porcine ZP3[60] contain N-linked poly-N-acetylglucosaminyl glycans. These glycans contain repeat units of disaccharide (3Gal1,4GlcNAc1) present on complex-type tri- and tetraantennary structures (Figure 2). The fact that these glycans were demonstrated by us to contribute 23 kDa and 16 kDa to the molecular mass of mZP2 and mZP3, respectively[58], suggests that the two zona components may contain a variety of structurally variable polylactosaminyl chains. Indeed, current evidence indicates that polylactosaminyl glycans present in many cell surface glycoconjugates[61-63] may contain four variable terminal sugars (Figure 2), suggesting that polylactosaminyl glycan chains on mZP2 and mZP3 may also be quite complex structures with many variables. From the above discussion, it is obvious that the number of N-linked glycan species identified in glycoproteins is very large and could run in the hundreds. However, although individual cells are able to synthesize many N-linked glycan chains, the process is highly specific and is controlled in such a way that the glycan chains at a particular glycosylation site have one or a small number of closely related structures[64].  Nonetheless, the fact that a large number of glycan structures are possible makes it difficult to identify and chemically characterize the bioactive glycan residue(s). The efforts are further hampered by the small amounts of mouse zona glycoproteins that can be purified and subjected to structure-function studies.

Figure 2. Structure of N-linked polylactosaminyl glycan chains present on a number of well-defined cell-surface glycoproteins (61-63) and perhaps mZP3. A, tetraantennary; B, triantennary; and C, triantennary with side chains. R represents one of the following structures: sialyl 2,6; Gal 1,3; GlcNAc 1,3; and Gal 1,4 GlcNAc. The sum of m+n+o+p may vary from 4-10 disaccharide units.

My laboratory has met reasonable success in the structural analysis of N-linked glycans present on the mZP2 and mZP3. In these studies, purified mZP2 and mZP3 were exhaustively treated with N-glycanase, and the released N-glycan chains were radiolabeled by reduction with3HNaBH4 as described[65]. The3Hlabeled glycan units when subjected to gel filtration on a column of Bio-Gel P-4, separated into several peaks indicating that both zona glycoproteins contain a variety of N-linked glycans. Interestingly, the radioactive peaks present in the mZP2 and mZP3 were quite different, a result indicating the qualitative and quantitative differences in the N-linked glycan chains. The peak fractions containing3Hlabeled glycans present in the mZP2 and mZP3 were pooled and fractionated by serial lectin column chromatography. The following immobilized lectins were used. Serotonin, specific for sialic acid; Ricinus communis (RCA-1), specific for N-acetylglucosamine; Griffonia simplicifolia (GS-1), specific for -galactose; Concanavalin A, specific for -mannose (high mannose/hybrid type glycans), and Griffonia simplicifolia (GS-11), specific for -galactose. The structure of the various3Hglycans eluted from the lectin columns was established by sizing column chromatography before or after digestion with exo-/endo-glycohydrolases. Data from these studies revealed that the mZP2 and mZP3 contain a variety of bi-,tri- and tetra-antennary complex-type, poly-N-acetyllactosaminyl complex-type, and high mannose/hybrid-type glycan chains[65]. However, whereas the mZP3 was found to contain polylactosaminylated glycan with terminal -galactosyl residue, no such structure was detected on the mZP2. These results provide additional new information on the complex nature of glycan chains present on the mZP2 and mZP3. The presence of sulphate, phosphate, and several monosaccharide residues on nonreducing terminus contributes to the extensive heterogeneity in the structure of glycan chains[66]. 

It is noteworthy that, whereas some studies suggest that O-linked glycan units are the bioactive molecules[47], several lines of evidence suggest that N-linked glycans also have a role in sperm-ovum interaction. First, treatment of zona-intact mouse ovums with almond glycopeptidase F (N-glycanase), an endoenzyme that hydrolyzes  -aspartyl-glucosamine of all classes of N-linked glycans, greatly reduced sperm-ovum binding[57]. This study implies that N-linked glycan chains are also important. Second, our own studies have provided evidence suggesting that N-linked high mannose/hybrid type glycans on the mouse[52] and rat[53] zona-intact ovums may be recognized by sperm surface mannosidase. The enzyme is a glycosidase. Its catalytic mechanism of action has been discussed in a previous review article[2]. The catalytic mechanism includes the formation of an enzyme: substrate (carbohydrate) intermediate before cleavage of the sugar residues. Since purified sperm surface mannosidase cleaves negligible amounts of3Hmannosyl residues from3Hmannose-containing glycoproteins after 4 h of incubation at 37[67], it is surmised that an intermediate of sperm (enzyme): zona (substrate) is formed that leads to the nexti step in fertilization before a significant amount of mannosyl residues is cleaved. The evidence for the presence of high mannose/hybrid-type oligosaccharide on mZP2 and mZP3[68] is consistent with this suggestion.

It should be noted that, like mZP3, the porcine ZP glycoprotein (pZP3) has been reported to contain sperm binding activity[60,69]. The 55 kDa molecule is also highly glycosylated, containing N-linked and O-linked oligosaccharides, and poly-N-acetyllactosaminyl glycans[60,69-72]. A recent report by Noguchi et al[69] presented structural analysis of N-linked glycan chains of pZP3 released and radiolabeled following hydrazinolysis. The3Hglycans were separated into neutral (28%) and acidic (72%) chains by anion-exchange HPLC. Competitive sperm-ovum binding assay in vitro in the absence or presence of glycan provided evidence suggesting that a mixture of neutral N-glycan units are important in sperm-ovum recognition and binding[69]. Collectively, the mouse, rat, and porcine data seem to suggest that both O-linked and N-linked glycan units of ZP3 glycocongugate are important for ligand activity.     

2.2 Sperm receptors for zona pellucida

Although the ZP is a relatively simple structure consisting of 3 or 4 glycoproteins, the sperm plasma membrane overlying the acrosome is more complex structure containing several dozen proteins/glycoproteins. For nearly two decades, investigators have applied multiple approaches to identify and isolate the complementary receptor molecules in several species. Their efforts have resulted in the recognition of several putative receptor molecules (for review, see 2, 4, and 73). 

Why are there so many putative receptors on the sperm plasma membrane? The following factors may contribute to the long list of proposed multiple receptor and ligand molecules. First, several receptor-ligand interactions may occur between spermatozoa and zona-intact ovum before a committed sperm-ovum binding. The multiple receptors may participate either individually or as multimeric receptor complexes. The experimental evidence, suggesting that the initial molecular mechanism between spermatozoa and ZP is a complex binding event that reflects interaction between multiple sperm proteins with multivalent ZP3[46], is consistent with the above possibility. Second, since sperm-ovum interaction is relatively species-specific, it is possible that different molecules are involved in different mammalian species. Alternatively, multiple sperm receptors may interact with complementary ligands in a well programmed sequence; the precise order of these interactions or the dominant receptor-ligand interaction may vary among species and may contribute to the species-specificity of fertilization. Regardless of the mechanism underlying sperm-ovum interaction, the research of numerous investigators strongly suggests the occurrence of multiple receptors on spermatozoa and multiple ligands on homologous ZP. 

2.3 The sperm acrosome and acrosome reaction     

The acrosome plays an important role at the site of sperm-zona (ovum) binding during the fertilization process. The organelle is a Golgi-derived secretory granule which is formed during an early stage of spermiogenesis[74]. It is a sac-like structure surrounded by inner and outer acrosomal membranes and filled with a host of enzymes such as acid glycohydrolases, proteases, esterases, acid phosphatases, aryl sulfatases, etc[5]. It is noteworthy that over 35 years ago, de Duve proposed that penetration of vestments surrounding the ovum may be mediated by the release of hydrolytic enzymes from the sperm acrosome[75]. Today, most researchers agree that the powerful hydrolytic enzymes released during the acrosomal exocytosis, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoa[16], are important factors that regulate the penetration of ZP and fusion of the gametes. 

The carbohydrate-mediated sperm-ovum binding initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents (i.e., induction of the acrosome reaction (AR).  This step is believed to be a prerequisite which enables the acrosome-reacted spermatozoa to penetrate the ZP and fertilize the ovum.  In most species, including man, multiple fusions between the sperm plasma membrane and outer acrosomal membrane (i.e., the AR) is triggered by components of the ZP[5]. In addition to ZP, a number of physiological and nonphysiological substances have been used to induce the AR in epididymal and ejaculated spermatozoa. These inducers have been described in two earlier reports[5,76] and will not be repeated here.

In the mouse, the binding of capacitated spermatozoa to the terminal sugar residue(s) on mZP3 starts a cascade of signaling events prior to the fusion of membranes and induction of the AR[5,74]. As stated above, the ability of mZP3 to be the natural agonist of the AR depends on the glycan moiety(ies) as well as the polypeptide portion of the molecule. The multiple glycan moieties are recognized by the sperm surface receptor(s) prior to its aggregation and triggering of the AR. Our studies with neoglycoproteins demonstrated the need for the sugar residues as well as the protein backbone[20]. Combined, the studies emphasize the role of sugar moiety(ies) in initial recognition by sperm surface receptors, and the role of the protein backbone in the cross-linking and aggregation of receptors prior to triggering the AR. The involvement of multiple sugar residues and  the protein backbone strongly supports the concept that the ZP3/neoglycoprotein-induced acrosome reaction is a net result of multivalent interactions.

What is the molecular mechanism of the calcium-dependent induction of the AR? There is a good evidence that ZP3 stimulation of spermatozoa activates G proteins, particularly G1-like proteins[77,78]. Inactivation of G1-like proteins with pertussis toxin blocks triggering of the AR by the solubilized mZP/mZP3. The bacterial toxin blocks ZP3-induced Ca2+ influx, leading to the suggestion that G1-like proteins regulate the Ca2+ influx and raise the levels of free calcium. The increase in intracellular Ca2+ is thought to be necessary prior to the AR.

Another important change that triggers the AR is a rise in internal sperm (pH)i. ZP3-stimulated spermatozoa display an elevated pH that is believed to be regulated by several ion channels on sperm plasma membrane and outer acrosomal membrane (for review, see 5). The potential mechanisms as to how these ion channels regulate intracellular free Ca2+ and elevate the sperm (pH)i have been described in the above review and will not be repeated here. Two recent reports have demonstrated that cholesterol efflux during sperm capacitation causes elevation in sperm (pH)i[79,80]. In addition, several reports have provided evidence suggesting that rise in sperm pH in response to the zona agonist may also activate adenylyl cyclase, protein kinases, tyrosine kinases, and phospholipases[4,5]. How the ZP3-induced changes in spermatozoa and the activation of various macromolecules regulate the signal transduction pathway prior to the acrosome reaction is not yet known.

3 Conclusion

This review supports a role for glycan-binding enzymes (glycosyltransferases and glycohydrolases) or lectin-like molecules on sperm plasma membrane and complementary bioactive glycan units on the surface of ZP in sperm-ovum binding. Although the sequence of events during fertilization varies among species, the mechanisms underlying sperm capacitation, sperm-ovum interaction, and induction of the acrosome reaction share many similarities. These events are best understood in the mouse, although there is some understanding in other species, including man. In the mouse, irreversible binding of capacitated spermatozoa to the glycan moiety(ies) of homologous ZP triggers signal transduction pathway resulting in induction of the AR. The evidence for the presence of a large diversity in the structure of glycans on ZP3 suggests that successful sperm-ovum interaction is a net result of several ligand (glycan)-receptor interactions. Consistent with this suggestion is the finding that initial molecular interaction between spermatozoa and ZP3 is a complex binding event that reflects multiple sperm proteins with multivalent ZP3. In this review, I have attempted to highlight current advances to explain mechanisms underlying capacitation, sperm-ovum interaction, and induction of the AR. I hope that some of these advances will allow new strategies to regulate these events and alter sperm function.

4 Acknowledgements

The author acknowledges the contributions of the present and past collaborators during various phases of the studies from my laboratory. I am grateful to Mrs. Loreita Little for editorial assistance and to Drs. Marjorie D. Skudlarekand Malika Bendahmane for critically reading this review.

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The work was supported in part by grants HD25869 and HD34041 from the National Institute of Child & Human Development.  
Correspondence to:  Dr Daulat R.P. Tulsiani, Departments of Obstetrics & Gynecology and Cell Biology, Vanderbilt University School of Medicine.  
Tel: +1-615-343 1993 Fax: +1-615-322 4358  

E-mail: daulat.tulsiani@mcmail.vanderbilt.edu
Received 2000-04-01     Accepted 2000-05-08