The Biological and Functional Significance of the Sperm Acrosome and Acrosomal Enzymes in Mammalian Fertilization

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

240, 151–164 (1998)

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REVIEW The Biological and Functional Significance of the Sperm Acrosome and Acrosomal Enzymes in Mammalian Fertilization1 Daulat R. P. Tulsiani,2 Aida Abou-Haila,3 Christoph R. Loeser,4 and Ben M. J. Pereira5 Center for Reproductive Biology Research and Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633

The mammalian spermatozoon undergoes continuous modifications during spermatogenesis, maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are capable of binding the zona-intact egg and undergoing the acrosome reaction. The fertilization process is a net result of multiple molecular events which enable ejaculated spermatozoa to recognize and bind to the egg’s extracellular coat, the zona pellucida (ZP). Sperm–egg interaction is a species-specific event which is initiated by the recognition and binding of complementary molecule(s) present on sperm plasma membrane (receptor) and the surface of the ZP (ligand). This is a carbohydrate-mediated event which initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be a prerequisite which enables the acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg. This review focuses on the formation and contents of the sperm acrosome as well as the mechanisms underlying the induction of the acrosome reaction. Special emphasis has been laid on the synthesis, processing, substrate specificity, and mechanism of action of the acid glycohydrolases present within the acrosome. 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 factors regulating the penetration of ZP. We have discussed the most recent studies which have attempted to explain signal transduction pathways leading to the acrosomal exocytosis. q 1998 Academic Press 1

Supported in part by Grants HD 25869 and HD 34041 from the National Institute of Child Health and Human Development. 2 To whom correspondence and reprint requests should be addressed: Fax: (615) 343-7797. 3 Present address: Laboratoire de Biologie Cellulaire, Universite Rene Descartes, Paris, France. 4 Recipient of a fellowship from the Deutsche Forschungsgemeinschaft, Germany. 5 Present address: Department of Biosciences and Biotechnology, University of Roorkee, India.

Key Words: mammalian fertilization; sperm acrosome; acrosome reaction; zona pellucida; sperm–egg interaction.

INTRODUCTION

Mammalian fertilization is the result of a complex set of molecular events which enable the capacitated spermatozoon to recognize and bind to the egg’s extracellular coat, the zona pellucida (ZP). In order to fertilize an egg, mammalian spermatozoa undergo a series of biochemical and functional changes during: (a) development in the testis (spermatogenesis); (b) maturation in the epididymis; and (c) capacitation in the female genital tract. Only capacitated spermatozoa are able to recognize and bind to the ZP [1, 2]. There is overwhelming evidence that sperm–ZP interaction is a carbohydrate-mediated receptor-ligand binding event [2, 3] which initiates a signal transduction pathway resulting in the exocytosis of acrosomal contents. The exocytotic event is believed to be a prerequisite that enables acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg. Since the fertilization process takes place in a complex microenvironment within the oviduct [1], it is important to list the in vivo events leading to successful fertilization. Ejaculated spermatozoa are unable to fertilize an egg. They undergo biochemical and functional changes during their residence in the female genital tract, including removal of seminal plasma proteins adsorbed to the sperm plasma membrane (PM), modification and/or reorganization of sperm PM molecules, and many internal changes [4]. The multifaceted modifications, collectively referred to as capacitation, vary from species to species, but the net result in all species examined is sperm hyperactivity and their ability to traverse the investments that surround the egg PM [1]. These investments include the matrix of the cumulus oophorus (cumulus cells) and the ZP (Fig. 1). Spermatozoa penetrate the ovulated egg surrounded

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FIG. 1. Progressive stages of sperm–egg interaction in the mouse: 1, the acrosome-intact spermatozoon passes through the matrix of the cumulus cells and the innermost layer, the corona radiata; 2, binding of sperm receptor(s) to glycan moiety(ies) of ZP3 initiates a signal transduction pathway resulting in acrosomal exocytosis; 3, the acrosome-reacted spermatozoon penetrate the ZP; 4, the spermatozoon passes through the perivitelline space and fuses with the egg PM.

by follicular cells and an intercellular matrix consisting of acidic mucopolysaccharides, hyaluronic acid, and proteins. The hyaluronic acid, a polymer composed of repeat disaccharide units containing glucuronic acid and N-acetylglucosamine in b1,3-linkage, is present in most of the extracellular ground substance of connective tissues, synovial fluid in joints, the vitrous humor of the eye, and in/or surrounding the cell coat. Each disaccharide unit is attached to the next by b1,4-linkage forming alternate b1,3- and b1,4-linkages (Fig. 2).

The polymer is covalently linked to a protein backbone and participates in maintaining the level of hydration within the cumulus cells. The carboxyl groups in hyaluronic acid are completely ionized, and the polymer has a net negative charge at physiological pH. This property enables hyaluronic acid to become soluble in aqueous medium forming an extremely viscous solution. It is generally accepted that hydrolytic enzymes, such as arylsulfatase, b-glucuronidase, N-acetylglucosaminidase, and hyaluronidase, and perhaps proteases aid in the process of dispersion of the cumulus mass by hydrolyzing hyaluronic acid and/or polypeptide substrates. The enzyme hyaluronidase catalyzes the hydrolysis of b1,4-linkages making the polymer solution less viscous (Fig. 2). Any reasonable hypothesis suggesting a role for the sperm enzymes in hydrolysis and dispersion of the cumulus mass must include possible mechanisms by which the hydrolytic enzymes could be present on the sperm PM so that they can react with their substrates on the cumulus oophorus. Interestingly, epididymal luminal fluid and seminal fluid are rich in most of the hydrolytic enzymes [5–7]. Thus, one likely possibility is that these enzymes tightly bind to the sperm surface during epididymal transit and ejaculation and remain bound during interaction of spermatozoa with the cumulus mass in the oviduct. A second possibility is that the hydrolytic enzymes present in the acrosome diffuse during capacitation and are exposed on the sperm surface during sperm–cumulus interaction. Alternatively, some of the capacitated spermatozoa may undergo spontaneous acrosome reaction releasing the acrosomal contents (hydrolytic enzymes, proteases, etc.). These powerful enzymes could disperse the cumulus cells allowing the acrosome-intact spermatozoa a clear passage through the cells. Following penetration of the cumulus mass, the acrosome-intact spermatozoon in many species binds to ZP in a highly precise manner [8–10]. The ZP in the

FIG. 2. Chemical structure of two repeating (disaccharide) units of hyaluronic acid. The enzyme, hyaluronidase, hydrolyzes b1, 4linkage(s) present between N-acetylglucosaminyl and D-glucuronide residues.

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mouse, rat, and many other species is composed of three glycoproteins, namely, ZP1, ZP2, and ZP3, and the pig seems to have a fourth form as well [for review see 2]. The three glycoproteins, at least in the mouse, are synthesized and secreted during oogenesis [11]. The secreted molecules interact noncovalently to form a three-dimensional network of cross-linked filaments forming the ZP. In mammals, ZP mediates several events, including: (i) relative species specificity during irreversible binding of the capacitated spermatozoa to the ZP; (ii) induction of the sperm acrosome reaction; (iii) block to polyspermy; and (iv) protection of the developing embryo from fertilization to implantation [1]. Several excellent reviews on mammalian fertilization have been published in recent years [2, 13–18]. However, most of the reviews have a much broader scope and do not focus on a particular event. The present review emphasizes the biochemical and functional significance of the mammalian sperm acrosome and acrosomal enzymes and will highlight three main aspects: First, the formation of the acrosome during sperm development in testis; second, the acrosomal contents and their modification during sperm maturation; and third, the mechanisms underlying the acrosome reaction. Our intention is also to discuss briefly the sperm capacitation and sperm–egg interaction which in most mammalian species is a prerequisite for the induction of the acrosome reaction. It is important to point out that many details of sperm capacitation, sperm–egg interaction, and sperm activation are still lacking. However, most researchers agree that binding of sperm receptor(s) to a glycan chain(s) present on the mouse ZP3 (mZP3) starts a signal transduction cascade leading to the acrosomal exocytosis [12, 18]. MALE GAMETE

Formation and Surface Modification of Spermatozoa during Spermatogenesis Male germ cells at all stages of development along with the somatic Sertoli cells are present in the seminiferous tubules of the adult mammalian testis [19]. The spermatozoon, a self-propelled cell composed of a head and tail varying in shape and size depending on the species, is the product of a proliferation and differentiation process termed spermatogenesis. This process is dependent on a specific environment provided by the somatic cells of the testis and requires both endocrine and auto/paracrine regulation as well as direct cell–cell interactions [20, 21]. Major morphological and cytological changes occur during the process of spermatogenesis leading to three main phases: First, a mitotic phase where spermatogonia replicate DNA and divide to give rise to primary spermatocytes. The second is a meiotic phase in which primary spermatocytes undergo genetic recombination

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and reduction divisions producing briefly a secondary spermatocyte and then the haploid round spermatid. The differentiation of the round spermatid to a hydrodynamically shaped spermatozoon with a head containing the haploid condensed nucleus and acrosome, the midpiece and the principal piece, occurs during the third phase termed spermiogenesis. Several cellular organelles (nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, centriole, etc.) simultaneously undergo structural and biochemical transformations during the differentiation of the spermatid which acquires a series of shape changes from round to elongate as it metamorphoses into a spermatozoon [22]. The nucleus elongates, shifts toward the cell surface, and takes on a compact and species-specific microanatomy. Chromatin condenses, which is accompanied by the replacement of testis-specific histones with spermatozoal proteins (protamines) rich in arginine and cysteine. The mitochondria migrate from the peripheral cytoplasm, elongates, and are positioned into a helical arrangement along the midpiece of the forming tail. In addition, new structures such as axoneme, cytoskeletal elements, and acrosome are formed from cytoplasmic organelles (e.g., centrioles, ER, and Golgi apparatus) and take position in the developing spermatozoa. The acrosome begins to form at the early phase of spermiogenesis (round spermatid). During this phase, a flagellum elongates from one pole of the cell defining an initial axis of polarity. The acrosome originates from Golgi-derived granules and becomes situated above the nucleus anterior to the flagellum. It is believed that membranous vesicles, whose products appear to be processed through the Golgi apparatus and delivered via coated vesicles, fuse with each other to yield a single acrosomic granule, which is closely associated with the nuclear envelope. As spermiogenesis continues, portions of the membrane extend laterally and flattens over the surface of the nucleus as a ‘‘cap’’ conforming to its shape while the acrosomal contents accumulate concomitant with nuclear condensation [23]. The acrosomal membrane close to the nuclear envelope is termed the inner acrosomal membrane whereas the membrane surrounding the acrosomal contents and underlying the sperm PM is termed the outer acrosomal membrane. The elongation of the spermatid begins in the late stages of spermiogenesis, when the sperm cell flattens as the chromatin condenses and the acrosome grows but becomes compact. The PM at the anterior pole of the cell becomes closely apposed to the acrosome. A thin rim of cytoplasm encounters the nucleus, whereas the bulk of cytoplasm is within the cytoplasmic lobe which detaches from the late spermatid to yield the residual body, leaving behind a small mass referred to as the cytoplasmic droplet. The sperm cell is released from its attachment from the Sertoli cell and the intercellular bridges connecting the spermatids into the lu-

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men of the seminiferous tubule [19, 22]. At this stage, the spermatozoon becomes an isolated cell which undergoes continuous changes during its transit through the male and female reproductive tracts until it fuses with the egg. Despite many advances in our understanding of various phases of spermatogenesis [19] and acrosome formation [22], little is known about the events that regulate the formation of sperm cells. Biochemical and immunological approaches have led to the recognition of several cell surface antigens on pachytene spermatocytes during prophase of meiosis [24]. Additional surface antigens are first detected on round spermatids following meiotic reduction division [25]. Very few surface proteins have been detected at the spermatogonial stage on premeiotic germ cells [24, 26]. These studies suggest that the stage-specific appearance of cell surface macromolecules on the diploid and haploid cells are important events in the development of male gamete. In addition to proteins that are synthesized by the developing germ cells themselves and incorporated into the sperm membrane, there is evidence that sperm surface proteins/glycoproteins undergo modifications during spermiogenesis. Three mechanisms have been described: (i) elimination or masking of the protein [24, 27]; (ii) modification of the existing glycoproteins [24, 25, 28]; and (iii) selective proteolysis [29, 30], a process known to convert enzymatically inactive precursor forms to an enzymatically active mature form [31, 32]. Posttesticular Modifications of Spermatozoa Testicular spermatozoa are nonmotile and unable to fertilize an egg. They acquire progressive forward motility and fertilizing capacity during passage through the epididymis, which provides specific intraluminal environment for the morphological and biochemical modifications necessary to produce a functionally mature spermatozoon [1]. Elucidation of maturation-associated changes on spermatozoa and sperm PM has been an important goal for reproductive biologists. The sperm PM, a vital component during the early events in fertilization, undergoes extensive modifications as spermatozoa transit the epididymis. Although many details of these modifications are poorly understood, it is generally believed that the process involves changes on sperm surface glycoproteins. The carbohydrate-related sperm surface modifications can be explained in a number of ways: (i) adsorption/association of the macromolecules from luminal fluid to the sperm PM; (ii) exposure of previously masked molecules due to reorganization of sperm PM [33]; and (iii) modification of preexisting glycoconjugates by the action of glycosidases [5, 34, 35], glycosyltransferases [7, 36], and proteases [31, 38–40]. Although the enzymes involved in glycoprotein biosynthesis (glycosyltransferases) are optimally active

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around neutral pH and are believed to be functional in the microenvironment of epididymal lumen [7, 40], the hydrolytic enzymes (glycosidases) have acidic pH optima and are expected to be functional only in the acidic environment within lysosomes/acrosomes. Several years ago we examined the kinetic properties of the luminal fluid b-galactosidase using a synthetic substrate (p-nitrophenyl b-galactopyranoside) and two glycoprotein substrates [35]. Interestingly, we demonstrated that while the enzyme optimally cleaves the synthetic substrate at acidic pH, it shows maximum activity toward glycoprotein substrates in the neutral environment. The pH-dependent substrate preference for the hydrolysis of synthetic and natural substrates may explain how the b-galactosidases (and perhaps other ‘‘acidic’’ glycosidases) could be functional in the neutral environment of the epididymal lumen [35]. It is important to point out that the maturation-associated modifications of spermatozoa are not limited to the effects of glycan modifying enzymes. Several sperm surface molecules are reported to be modified by proteolytic processing. For instance, rat sperm PM mannosidase is synthesized in the testis as an enzymatically inactive/less active precursor form of 135 kDa and is proteolytically processed to an enzymatically active mature form of 115 kDa during development in the testis and maturation in the epididymis [30]. Similar proteolytic processing has been reported for guinea pig sperm PM proteins (PH-20 and PH-30 [38]), mouse sperm antigen M42 [41], rat sperm glycoprotein CE9 [42], and rat sperm surface antigen 2B1 [37]. In addition to the alteration of surface proteins/glycoproteins, spermatozoa undergo internal modifications of several acrosomal enzymes. These include sperm b-galactosidase [5], a-L-fucosidase [43], acrosin [44], and acrogranin [45]. Thus, it is reasonable to suggest that the processing of sperm PM antigens and acrosomal glycohydrolases and protease(s) (intraacrosomal processing) is an important part of the maturation process. LYSOSOMAL AND ACROSOMAL ENZYMES

Over 30 years ago de Duve proposed that the penetration of investments surrounding an egg may be mediated by the release of acidic glycohydrolases from the sperm acrosome [46]. The hydrolytic enzymes were first reported to be localized in an electron dense organelle called the lysosome, a bag-like structure which normally functions in intracellular digestive and defensive mechanisms. The sperm acrosome resembles a lysosome in three ways. First, the acrosome, like the cell lysosome is derived from the Golgi apparatus. Second, both organelles stain a bright orange-red color with acridine orange indicating an acidic pH within the organelles. Finally, the two organelles contain several common enzymes such as acid glycohydrolases, esterases, and arylsulfatases. Despite these similarities, the

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acrosome has some distinctive features. The sperm acrosome is a sac-like structure surrounded by inner and outer acrosomal membranes containing the acrosomal contents. Immediately after sperm–egg (zona) interaction, at least in the mouse, the outer acrosomal membrane fuses with the overlying PM, releasing the acrosomal contents (acrosomal exocytosis). Because of its exocytotic property, the acrosome is considered more like a secretory granule. The acrosome is also different from the cellular lysosome in that it contains specific enzymes like acrosin [39] and acrogranin [45]. It is important to mention that, like the acrosome, the cytoplasmic or protoplasmic droplet is also derived from the Golgi apparatus during spermatogenesis. It contains several glycosidases common to the lysosome and acrosome [5, 47]; it is initially attached to the sperm midpiece, but is shed shortly after ejaculation. Glycohydrolases Mammalian spermatozoa contain a host of acid glycohydrolases with catalytic and immunological properties similar to the enzymes present within lysosomes. Thus any information about the biosynthesis and targeting of the enzymes present in lysosomes will provide insight into the acrosomal biogenesis. The lysosomal glycohydrolases are synthesized on polysomes bound to the endoplasmic reticulum (ER), and they enter and are modified in the ER and are further modified in the Golgi apparatus. These enzymes are glycoproteins with glycan portion contributing up to 10% of the molecular mass [48]. The glycan portion of acid glycosidases, as in other glycoproteins, is mainly N-linked (asparaginelinked oligosaccharide). The precursor form of the Nlinked oligosaccharide is preassembled in the ER as a branched structure containing two N-acetylglucosaminyl (GlcNAc), nine mannosyl (Man), and three glucosyl (Glc) residues while the oligosaccharide chain is attached to dolichol pyrophosphate in the ER membrane. Following the en bloc transfer of Glc3Man9GlcNAc2 , to the nascent polypeptide chain in the ER, three glucosyl residues are removed by glucosidases I and II [48]. The Man9GlcNAc2 derivatives are converted to Man5GlcNAc2 derivatives by a-1,2-specific mannosidases [50–52]. The Man5GlcNAc2 is N-acetylglucosaminylated to form GlcNAcMan5GlcNAc2 species [52]. Golgi mannosidase II then removes terminal a-1,3- and a-1,6-mannosyl residues to yield GlcNAcMan3GlcNAc derivatives [51, 53] which are converted by several glycosyltransferases to form the complex glycan (Fig. 3). It is important to keep in mind that a single glycohydrolase often contains multiple glycan chains of high mannose type, complex type (bi-, tri-, and tetraantennary), and perhaps a hybrid type. The latter glycan is biantennary where one antenna contains only mannosyl residues and the other antenna contains sialyl, galactosyl, and N-acetylglucosaminyl residues attached to a mannosyl residue [53].

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FIG. 3. The pathway for processing of N-linked glycoproteins. The processing involves: 1, removal of three glucosyl residues by glucosidases I and II (Glc I and Glc II); 2, removal of four a1, 2linked mannosyl residues by mannosidase I (Man I); 3, addition of Nacetylglucosaminyl residue by glucosaminyl transferase I (GlcNAc. Trans. I); 4, removal of a1,3- and a1,6-linked mannosyl residues by Golgi mannosidase II (Man II); 5, addition of sugars by glucosaminyltransferase II and glycosyltransferases. G, glucose; M, mannose; GlcNAc, N-acetylglucosamine; Gal, galactose; SA, sialic acid; Asn, aspargine residue of peptide backbone.

It has been known for some time that lysosomal glycohydrolases are phosphorylated on a mannosyl residue during the processing of N-linked glycans [48, 54]. As these enzymes pass through the Golgi apparatus, they are marked with GlcNAc 1-phosphate on the high mannose oligosaccharide which is hydrolysed by Golgi N-acetylglucosamine 1-phosphodiester a-N-acetylglucosaminidase, exposing mannose-6-phosphate (M6P) residue [55]. The newly synthesized glycohydrolases are transported to lysosomes by virtue of the exposed M6P-ligand (Fig. 4). The ligand is recognized by M6P receptors localized in the trans-Golgi cisternae which regulate the delivery of the hydrolytic enzymes. There are two classes of known M6P receptors: one a 215kDa cation independent (CI) class and the other a 46-

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FIG. 4. Phosphorylation of high mannose oligosaccharide present on lysosomal (and perhaps acrosomal) glycohydrolases. The oligosaccharide (I) is phosphorylated by formation of GlcNAc-phosphodiester (II). Subsequent removal of N-acetylglucosaminyl residue(s) to form either high mannose oligosaccharide with two M6Ps (III) or processed to form hybrid structure with one M6P G(III*). M, mannose; N, Nacetylglucosamine; P-GlcNAc, N-acetylglucosamine phosphodiester; Gal, galactose; SA, sialic acid; Asn, asparagine.

kDa cation-dependent (CD) type. The two receptors are trans-membrane N-glycosylated glycoproteins which recognize and bind to M6P ligands on the newly synthesized glycohydrolases and segregate them in transport vesicles. These vesicles bud-off from the Golgi-associated compartments (transport vesicles) and deliver their enzymes by fusing with prelysosomal (endosome) structures [56]. The affinity of the M6P receptors for the phosphorylated hydrolases is high between pH 6.0 and 7.0, but very low at pH values below 6.0. Thus, when transport vesicles fuse with acidic prelysosomal structures, the hydrolases dissociate from the receptor, leaving glycohydrolases in the acidic vesicles, while the M6P receptors recycle back to the Golgi region [57]. The signal required for the targeting of glycohydrolases to the lysosome is well documented but less clear for the acrosome and the cytoplasmic droplet. Although it is known that acrosomal enzymes are transported into this organelle during spermiogenesis, our understanding of the specific signal(s) needed for acrosomal targeting is limited. The mouse testicular germ cells and Sertoli cells have been demonstrated to contain CI

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and CD-M6P receptors [58] suggesting the potential for involvement of these receptors. Whether the germ cells and Sertoli cells use a common targeting pathway is not known at the present time. The acrosomal glycohydrolases (Table 1), like the lysosomal glycohydrolases, have a high substrate specificity and will not hydrolyze even a closely related glycosidic linkage(s). The enzymes catalyze hydrolytic cleavage of terminal sugar residues from the glycan portion of glycoproteins and glycolipids [59]. These enzymes are named on the basis of their sugar substrate. Thus, a glucosidase will hydrolyze only a glucosyl residue(s) and a mannosidase will cleave only a mannosyl residue(s). Under in vivo conditions, the hydrolytic enzymes function sequentially in such a way that the product of the first enzymatic cleavage becomes the substrate for the next enzyme (Fig. 5). The mechanism of action of glycohydrolases is believed to follow the model advanced for lysozyme. In this model, there are two carboxylic acid moieties in the active site: one ionized and other protonated [60]. The former moiety stabilizes the resulting oxocarbon-

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TABLE 1

TABLE 2

Glycohydrolase Activities Present in Mammalian Sperm Acrosome

Proteinase Activities in Mammalian Spermatozoa

Enzyme

Sugar linkagea

Hyaluronidase a-L-Fucosidase a-D-Galactosidase b-D-Galactosidase b-D-Glucuronidase b-N-Hexosaminidase a-D-Mannosidase b-D-Mannosidase Neuraminidase Aryl sulfatases A, B, and C

(GlcNAc-Gluc)n Fuc-GlcNAc Gal-Gal Gal-GlcNAc Gluc-GlcNAc GlcNAc-(Gal)GalNAc Man-Man Man-GlcNAc NANA-(Gal)GalNAc Sulfates

a GlcNAc, N-acetylglucosamine; Gluc, glucuronide; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Man, mannose; NANA, N-acetylneuraminic acid.

ium ion, either by ion pair interaction or by covalent bonding, whereas the latter moiety facilitates departure of the cleaving group. The common catalytic mechanism of all hydrolytic enzyme is the formation of an enzyme:substrate (sugar) intermediate before the cleavage of the glycosidic bond and the release of the sugar residue. Proteinases A number of proteinases have been identified on spermatozoa (Table 2). These include the extensively studied serine-specific proteinase (acrosin/proacrosin) to less studied cysteine proteinases (cathepsins) and metalloproteinase. The guinea pig sperm acrosome possess dipeptidylpeptidase II. The enzyme (apparent molecular mass of 130 kDa) is optimally active at an acidic pH of 4.5–5.5 [61]. In addition, a dipeptidyl carboxypeptidase [62] and calpain II [63] are reported to be present in the sperm acrosome. Whether these en-

Enzyme

Specificity

Localization

References

Acrosin Acrolysin Cathepsin D Cathepsin L Cathepsin S-like Metalloprotease

Serine Amino proteinase Carboxyl Cysteine Cysteine Not known

Acrosome Acrosome Acrosome Acrosome Spermatozoa Acrosome

[106] [107] [108] [109] [110, 111] [112]

zymes are necessary for normal functioning of spermatozoa is not yet known. Acrosin, a sperm-specific serine-like proteinase, is believed to be present in the sperm acrosome as well as on the surface of spermatozoa. The enzyme has been suggested to have a role in sperm-ZP binding and the penetration of the ZP by proteolytic cleavage of zona glycoconjugates [39]. Synthesized in an enzymatically inactive precursor form proacrosin, the enzyme is processed to an enzymatically active form during spermatogenesis [64] and undergoes intraacrosomal processing during epididymal maturation [44]. Although acrosin/proacrosin is the most widely studied acrosomal enzyme, the experimental evidence suggesting its role in the sperm penetration of the ZP and/or its involvement in secondary binding to ZP2 is circumstantial rather than definitive. Furthermore, a recent study demonstrated that sperm from mice carrying a targeted mutation of the acrosin gene can bind to the egg ZP and fertilize the egg [65]. This result is consistent with the suggestion of the investigators that acrosin, at least in mice, is not essential for fertilization. In addition to the glycohydrolases and proteinases, the sperm acrosome contains esterases, sulfatases, phosphatases, and phospholipases. These enzymes have been described in sufficient details in a recent book chapter [66] and will not be discussed here.

FIG. 5. Structure of an N-linked biantennary complex-type oligosaccharide attached to an asparagine residue. The glycohydrolases shown in the figure are all exo-hydrolases and cleave only at terminal sugar residue(s).

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ACROSOME REACTION

Ejaculated spermatozoa must undergo biochemical and functional changes as they traverse the female reproductive tract before undergoing the acrosomal reaction (AR). The preparatory changes are collectively termed as capacitation. Although the importance of sperm capacitation has been known for several decades [2], the molecular mechanisms underlying these changes are not fully understood. Most researchers agree that capacitation results from multiple molecular alterations in sperm PM proteins/glycoproteins and lipid components that are responsible for modifying ion channels in the plasmalemma of spermatozoa [1]. It is, therefore, reasonable to assume that capacitation is a net result of multiple biochemical changes on the sperm PM [2]. These modifications are important to increase fluidity and permeability of the sperm PM and to allow the transmembrane flux of ions that are necessary for initiating the events of capacitation, hyperactivation and the AR [68–70]. Inducers of the Acrosome Reaction It is generally accepted that a glycan moiety(ies) of ZP3 provides the primary ligand site(s) for the acrosome-intact spermatozoa. In addition to ZP, a number of physiological and nonphysiological substances have been used to induce the AR in epididymal and ejaculated spermatozoa [70]. The physiological inducers are the substances that spermatozoa will encounter during in vivo fertilization. Progesterone, a hormone produced during ovulation, has been suggested to induce the AR by interacting with the sperm PM in a receptor-mediated manner [73–75]. Prostaglandins, sterol sulfate, and glycosaminoglycans present in the follicular fluid and cumulus cell secretions have been suggested to induce the AR [74, 75]. However, the mechanisms underlying the interactions of these substances with spermatozoa are not clear and the putative receptors on the sperm PM have not been identified. There is a long list of nonphysiological substances known to induce the AR. The inducers include calcium ionophore, lectins, neoglycoproteins, etc. The artificial inducers have been described in a recent paper [76] and will not be described here. Molecular Mechanisms of the Acrosome Reaction The potential mechanisms of the AR have been discussed in greater detail in a recent review [77] and will be described briefly. As stated earlier, in most species, including man, fusion between the PM and outer acrosomal membrane is triggered by component(s) of the ZP [78]. The ability of mZP3 to serve as an AR inducer depends on the glycan moiety(ies) as well as the polypeptide portion of the molecule [79]. Consistent with this possibility is the finding that the mZP3 glycopep-

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tides bind to the sperm PM but do not induce the AR unless they are cross-linked on the sperm surface by ZP3 antibody [80]. This result is in agreement with the proposed aggregation of the sperm surface receptor(s) prior to the induction of the AR. Studies carried out in another laboratory also suggest that binding of multivalent ligands (glycans) of mZP3 to the sperm surface galactosyltransferase causes its aggregation and the triggering of the AR [81]. If aggregation of the sperm surface receptor(s) is the key requirement for the AR, then it should be possible to induce the AR by aggregation of the binding sites on spermatozoa in the absence of ZP glycoproteins. Recent evidence suggests that a heat and acid stable proteinase inhibitor of seminal vesicle origin binds to the acrosomal cap region of the capacitated spermatozoa. The in vitro binding of the protease inhibitor to the sperm surface did not induce the AR. The exocytotic event, however, can be initiated by immunoaggregation of the inhibitor with anti-inhibitor Fab-fragment [82]. Taken together, these results support the conclusion that the aggregation mechanism (with or without ZP) may be important for the initiation of the AR. In the mouse, the binding of spermatozoa to the terminal sugar residue(s) on mZP3 starts a cascade of signaling events. A possible mechanism has been discussed [1, 78]. According to this mechanism, interaction of sperm receptors with glycan binding sites on ZP3 elevates cytoplasmic levels of second messengers through appropriate transducers and effectors. The second messengers are either ions, such as Ca2/ [83], or small molecules like cAMP [18, 84, 85] and IP3 [87– 89] in the sperm cytoplasm that are believed to initiate a train of events triggering the exocytosis. The evidence for the presence of G-proteins on the sperm surface and the importance of ZP3 in inducing the AR, at least in the mouse, suggests that ligandreceptor- G-protein second messenger system plays an important role in triggering the AR [78, 89]. Interestingly, solubilized mZP or purified mZP3 has been reported to activate G-proteins identified on sperm membranes [90, 91]. Pertussis toxin, which inactivates the Gi-like proteins expressed in the developing acrosome [92], blocks activation of G-proteins and triggering of the AR by the solubilized mZP [93]. The Ca2/ influx that triggers the AR is believed to be mediated by ion channels that are regulated by G-proteins. The pertussis toxin blocks mZP3-induced Ca2/ influx and pH changes, leading to the suggestion that Gi-like proteins may regulate Ca2/ influx and internal pH [94]. Most investigators agree that an important change that triggers the AR is a rise in sperm cytoplasmic levels of Ca2/ and pH [83, 94]. In this context, the mechanisms regulating the ion conductance deserve some comments. Evidence accumulated over the years suggests the occurrence of several types of ion channels on the sperm acrosomal and plasma membranes (Table

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TABLE 3 Channel Proteins Proposed to Be Involved in Transport of Ions across Sperm Membranes Channel protein

Ions(s) transported Ca2/ Mg2/ Na//K/ Na//Ca2/ Na//H/ Cl0/HCO30 K/

Transport ATPases

Na//Ca2/ exchanger Na//H/ exchanger Cl0/HCO30 exchanger K/ channel Ca2/ channels Voltage dependent T Type L Type F Type IP3 gated channel

References [104, 105, 103] [114, 115] [116, 117] [118] [119] [120] [121]

Ca2/ Ca2/ Ca2/ Ca2/, Co2/, Mn2/, Ni2/ Ca2/

[102, 122] [123, 124] [125] [103] [86]

3). Regardless of the type of channel involved and the mode of its operation, it is important to emphasize that ion specificity and the direction of their movement across the sperm membranes are important contributing factors that elevate the pH and the level of intracellular Ca2/ ions preceeding the AR. The possible mechanisms which are likely to regulate the rise in intracellular pH and the elevation in the levels of free Ca2/ ions are shown in Figs. 6 and 7, respectively.

159

Several sperm surface molecules have been suggested to have a role in the induction of the AR. These include: (i) a 95-kDa sperm plasma membrane protein which is increasingly phosphorylated as a consequence of its aggregation by the mZP3 [95]; (ii) protein tyrosine kinases, a family of intrinsic signal transducing receptors which undergo autophosphorylation of tyrosine residues in response to ligand (mZP3) binding. Inhibitor of tyrosine kinase activities (tyrphostins) inhibit the AR [96]. The result is consistent with the suggestion that tyrosine phosphorylation is a necessary step for the exocytotic event; (iii) at least three different effector molecules, phospholipase C [97], phospholipase A2 [98], and adenylyl cyclase [75, 99, 100], are activated during the AR. The precise mechanism by which phospholipase C activates phospholipase A2 has been described in recent reviews [73, 77]. The order in which these macromolecules fit in the puzzle of a signaling mechanism for the AR is not yet known. An understanding of the cytoskeletal elements present between the inner layer of sperm PM and outer layer of the outer acrosomal membrane is important prior to a discussion as to how the rise in intracellular Ca2/ and elevated pH may trigger the AR. The cytoskeleton is rich in actin, a protein characteristic of muscle fibers. In capacitated spermatozoa, the protein occurs in filamentous form (F-actin) and has been suggested to provide a scaffolding to keep the phospholipase C

FIG. 6. Model illustrating a possible mechanism for the rise in sperm cytoplasmic pH preceding the acrosome reaction: A, unphosphorylated Na//H/ exchange channel protein allows H/ ions to pass through the PM of the acrosome-intact sperm in exchange for Na/ ions; B, phosphorylation of the channel protein either by 95-kDa protein or by an increase in Ca2/ or diacylglycerol (DAG) reverses the direction of ion movement elevating the intracellular pH.

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FIG. 7. Model illustrating possible mechanisms that could explain an increase in sperm cytoplasmic Ca2/ preceding the acrosome reaction. 1, import of extracellular Ca2/ by voltage-sensitive [102] and voltage-insensitive [103] calcium channel proteins; 2, retention of Ca2/ by the inhibition of Ca2/ transport ATPases present on sperm PM and outer acrosomal membrane [104, 105]; 3, IP3-induced opening of Ca2/ channel(s) present on outer acrosomal membrane and the entry of Ca2/ from acrosome (Ca2/ ) pool [86]; 4, release of Ca2/ perhaps bound to intracellular sperm proteins (?).

bound to the sperm PM and to provide a physical barrier for the fusion of the PM and the outer acrosomal membrane [101]. In response to the increased Ca2/ and pH, the F-actin depolymerizes to form soluble monomeric actin (G-actin) which disperses bringing the PM closer to the outer acrosomal membrane [101]. At the same time, phospholipase A2 cleaves fatty acids from the phospholipids to produce lysophospholipids which promote membrane fusion (Fig. 8). Inhibitors of phospholipase A2 are capable of blocking the AR in vitro, a finding suggesting that the enzyme plays a role in destabilizing the sperm plasma membranes causing them to fuse. A recent study has reconstructed these events in a cell free system using purified and labeled plasma membrane and outer acrosomal membranes from capacitated bovine spermatozoa [77]. By using appropriate inhibitors and monitoring the change in resonance energy transfer, the authors have demonstrated that phospholipase C activity and depolymerization of membrane bound F-actin are essential for the membrane fusion event and the triggering of the AR. CONCLUSIONS

This review discusses the importance of the sperm acrosome and its enzymatic contents in the penetration of the egg. Most investigators agree that complementary molecules initiate sperm–egg recognition and spe-

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cies-specific irreversible binding by a receptor–ligand mechanism. Although the sequence of events varies among species, the mechanisms underlying events of sperm capacitation, sperm–egg interaction, and the induction of the acrosome reaction share many similarities. These events are best understood in the mouse, although there is some information in other species, including the pig, guinea pig, hamster, and human. In the mouse, the acrosome reaction is triggered following adhesion of spermatozoa to ZP, a complex binding event which appears to reflect the interaction of multiple sperm surface antigens with multiple glycans (ligands) on ZP3. Thus, it seems logical to conclude that the acrosome reaction is a net result of multiple molecular changes in the sperm PM and outer acrosomal membrane proteins/glycoproteins and lipid components. These modifications allow the transmembrane flux of ions which is believed to be important in triggering the acrosome reaction. It is important to emphasize that recent advances in assisted (in vitro) fertilization procedures and intracytoplasmic sperm injection techniques appear to sideline the importance of capacitation, sperm–egg interaction, and the acrosome reaction. However, the significance of these events in the unassisted (in vivo) fertilization process should not be undermined. In this review, we have attempted to highlight current advances to explain potential mechanisms underlying the acrosome

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membrane during epididymal maturation. Biol. Reprod. 48, 417–428. 8. Saling, P. M., and Storey, B. T. (1979). Mouse gamete interactions during fertilization in vitro: Chlortetracycline as fluorescent probe for the mouse sperm acrosome reaction. J. Cell Biol. 83, 544–555. 9. Storey, B. T., Lee, M. A., Muller, C., Ward, C. R., and Wirtshafter, D. G. (1984). Binding of mouse spermatozoa to the Zona pellucida of mouse egg in cumulus: Evidence that the acrosome remain substantially intact. Biol. Reprod. 31, 1119– 1128. 10. Phillips, D. M., and Shalgi, R. (1982). Sperm penetration into rat ova fertilized in vivo. J. Exp. Zool. 221, 373–378. 11. Wassarman, P. M. (1988). Zona pellucida glycoproteins. Annu. Rev. Biochem. 57, 415–442.

FIG. 8. Diagram illustrating destabilization of the sperm PM preceding the acrosome reaction. The sperm PM, like other biological membranes, consists of lipids and proteins. Phospholipids, the most abundant lipids in sperm membrane, are amphipathic molecules containing a hydrophilic (polar) head group and two hydrophobic (nonpolar) tail groups made up of 14–24 carbon fatty acids. Since the cross-sectional area of the polar head equals the combined area of the two tail groups, the phospholipids spontaneously arrange themselves to form a stable lipid bilayer. The loss of a single fatty acid residue from the nonpolar tails results in the formation of lysophospholipid and destabilization of the membrane structure.

12. Wassarman, P. M. (1995). Mammalian fertilization: egg and sperm (glyco) protein that support gamete adhesion. Am. J. Reprod. Immunol. 33, 253–258. 13. Snell, W. J., and White, J. M. (1996). The molecules of mammalian fertilization. Cell 85, 629–637. 14. Shur, B. D. (1992). Glycosyltransferases as cell adhesion molecules. Curr. Opin. Cell Biol. 5, 854–863. 15. Myles, D. G. (1993). Molecular mechanism of sperm-egg membrane binding and fusion in mammals. Dev. Biol. 158, 35–45. 16. Chapman, N. R., and Barratt, C. L. R. (1996). The role of carbohydrate in sperm-ZP3 adhesion. Mol. Hum. Reprod. 2, 767– 774. 17. Benoff, S. (1997). Carbohydrates and fertilization: An overview. Mol. Hum. Reprod. 3, 599–637.

reaction. An understanding of the sequence of events preceding the acrosome reaction will allow new strategies to regulate these events. The authors are indebted to Drs. Benjamin Danzo, Marjorie D. Skudlarek, and Oscar Touster for critically reading the manuscript and to Dr. J. J. Lareyre for assistance with several computer graphics.

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Received October 10, 1997 Revised version received December 18, 1997

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