Developmental Biology 238, 332341 (2001) doi:10.1006/dbio.2001.0380, available online at http://www.idealibrary.

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A Mouse Acrosomal Cortical Matrix Protein, MC41, Has ZP2-Binding Activity and Forms a Complex with a 75-kDa Serine Protease
Ichiro Tanii, 1 Tadasuke Oh-oka, Kazuya Yoshinaga, and Kiyotaka Toshimori
Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical College, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan

Sperm with a large acrosome such as that of guinea pigs and hamsters have a subdomain structure in the anterior acrosome, but the mouse acrosome looks homogeneous and its matrix has not been precisely analyzed. The intra-acrosomal protein MC41 is localized in the cortical region of the mouse anterior acrosome, suggesting a subdomain structure in the mouse acrosome. Thus, the present study was undertaken to analyze the mouse acrosomal matrix using an anti-MC41 antibody. When mouse sperm were treated with 2% Triton X-100, Triton-insoluble matrix components remained in the acrosomal cortical region. Immunogold for MC41 labeled the Triton X-100 and high-salt-insoluble matrix components, demonstrating that MC41 is a subdomain-specic acrosomal matrix protein. We further examined interactions of MC41 with acrosomal proteases and zona proteins. A serine protease of 75 kDa was associated with MC41 under low-salt conditions, presumably forming a complex. Far Western blotting technique indicated that MC41 bound to both ZP2 and ZP2 f in the presence of high-salt-soluble sperm proteins. In acrosome-reacting sperm, MC41 was present on the hybrid vesicles formed by the fusion of the plasma and outer acrosomal membranes. Presumably, MC41 has a signicant role in secondary spermzona binding during the acrosomal reaction. 2001 Academic Press Key Words: acrosomal matrix; subdomain structure; serine protease; zona pellucida; secondary binding; acrosome reaction; spermzona interaction; fertilization; mouse.

INTRODUCTION
During the early process of fertilization, attachment of mammalian sperm to zona pellucida induces the acrosome reaction that permits sperm to penetrate the zona pellucida and accomplish successful fertilization (Yanagimachi, 1994). Recent studies have demonstrated that the mammalian sperm acrosome is not a plain sac containing a variety of hydrolases, but is organized into functional subdomains by nonenzymatic structural proteins. Early electron microscopic studies noted a subdomain structure in the anterior acrosome of guinea pigs and hamsters (Fawcett and Hollenberg, 1963; Yanagimachi and Noda, 1970). Cytochemical studies using phosphotungstic acid staining have visualized a carbohydrate-rich region in the anterior acrosomal cortex in several mammalian species, such as the hamster, guinea
To whom correspondence should be addressed. Fax: 85-1363. E-mail: itanii@post1.miyazaki-med.ac.jp.
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pig, bull, boar, and ram, showing biochemical differences between the acrosomal cortex and medulla (Courtens, 1978; Holt, 1979; Sinowatz and Wrobel, 1981). Detergentinsoluble acrosomal structural proteins have been identied in the hamster and bovine sperm and have been termed the acrosomal matrix (Olson et al., 1985; Olson and Winfrey, 1991) and, more recently, subdomain-specic acrosomal matrix proteins in the guinea pig (Westbrook-Case et al., 1994; Foster et al., 1997). Thus, acrosomal matrix proteins are likely to serve as a structural framework which functionally divides the acrosome into subdomains. Although the most widely accepted model for sperm egg interaction in mammals was proposed from studies using mice, mouse sperm does not show any dened acrosomal subdomain structure, and therefore the mouse acrosomal matrix has not been precisely elucidated. Hence, it is unclear whether the acrosomal subdomain structure is universal in mammalian sperm. Some acrosomal hydrolases have not only hydrolytic
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and water in accordance with the guidelines for animal welfare of our college. Mice were sacriced by cervical dislocation before use.

activity for the extracellular matrix surrounding the egg, such as hyaluronic acids and the zona pellucida, but also binding activity to the zona pellucida. PH-20 is a hyaluronidase present on sperm heads (Phelps et al., 1988; Gmachl et al., 1993; Lin et al., 1994). Since the anti-PH-20 antibody inhibits the binding of acrosome-reacted sperm but not acrosome-intact sperm to the zona pellucida, PH-20 is involved in acrosome-reacted spermzona binding (Myles et al., 1987; Hunnicutt et al., 1996). Acrosin is a major acrosomal serine protease showing a high afnity for zona protein (Topfer-Petersen and Henschen, 1987; Mori et al., 1993; Hardy and Garbers, 1994). A specic inhibitor of -N-acetylglucosaminidase inhibits sperm penetration through the zona pellucida and, similarly, ligand sugars of -D-mannosidase inhibit spermzona binding (Cornwall et al., 1991; Miller et al., 1993). Accordingly, acrosomal hydrolases have important roles in spermzona interactions. It is noteworthy that acrosomal hydrolases bind to the acrosomal matrix in the bovine, hamster, and guinea pig sperm (Hardy et al., 1991; NagDas et al., 1996a,b). The nding that an acrosomal matrix-binding protein, acrosin, is released slowly during the acrosome reaction, compared to the rapid release of dipeptidyl peptidase II, an acrosomal matrix nonassociated protein (Talbot and DiCarlantonio, 1985; Hardy et al., 1991), led to the hypothesis that the binding of acrosomal hydrolases to the acrosomal matrix regulates their release. In other words, by binding to the acrosomal matrix, the acrosomal hydrolases can have a long interaction with the zona pellucida. To understand the process of early sperm egg interaction in fertilization, therefore, it is important to consider acrosomal enzymematrix interactions, as well as acrosomal enzymezona interactions. We previously showed that an intra-acrosomal protein, MC41, is restricted to the cortical region of the mouse anterior acrosome (Tanii et al., 1995), suggesting that a subdomain structure is also present in the mouse acrosome. In the present study, we examined the mouse acrosomal matrix using an anti-MC41 antibody, revealing that MC41 is a component of a subdomain-specic acrosomal matrix. In the course of analysis of MC41-associated protease, we identied a serine protease of 75 kDa. The anti-MC41 antibody inhibits fertilization rate of zona-intact eggs but not zona-free eggs in vitro (Saxena et al., 1999), suggesting that MC41 is involved in spermzona interaction. Therefore, we further studied the binding of MC41 to zona proteins. The role of MC41 in spermzona interaction will be discussed.

Materials
We previously reported the production of a monoclonal antibody (IgG 1) against the intra-acrosomal antigen MC41 and characterized the properties of the antigen (Tanii et al., 1992a,b). The medium used in this study was a modied KrebsRinger bicarbonate solution, TYH medium, consisting of 119.37 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl 2, 1.19 mM KH 2PO 4, 1.19 mM MgSO 4, 25.07 mM NaHCO 3, 1 mM pyruvate, 5.56 mM glucose, and 0.4% bovine serum albumin (BSA), adjusted to pH at 7.2 (Toyoda et al., 1971).

Preparation of Sperm and Zona Proteins

Sperm were obtained by squeezing the cauda epididymis and vas deference of male mice. Sperm were suspended in TYH medium and washed by centrifugation at 250g for 10 min. They were then treated with a solution containing 2% Triton X-100, 20 mM TrisHCl (pH 7.5) under low- or high-salt conditions (1 M NaCl) on ice for 2 h and centrifuged at 400g for 10 min. Supernatants were kept as Triton extracts. Sperm pellets were washed in a 20 mM Tris buffer (pH 7.5) and treated with 0.1% SDS, 20 mM TrisHCl (pH 7.5) at RT for 10 min to extract acrosomal matrix proteins. After centrifugation at 400g for 10 min, and subsequently at 10,000g for 10 min, the SDS extracts were diluted 10 times with a 20 mM Tris buffer (pH 7.5) containing 1% Triton X-100 to replace SDS with Triton X-100 for protein renaturing. Triton and SDS extracts were used for zymography and Far Western blot analysis, and sperm pellets were used for electron microscopic study. Oocyte cumulus complexes were obtained from the oviducts of supraovulated female mice as described before (Saxena et al., 1999). Cumulus cells were dispersed by treatment with 1 mg/ml hyaluronidase (type IV, Sigma Chemical Co., St. Louis, MO) in BSA-free TYH medium for 35 min at 37C and removed from zona-intact eggs by transferring the eggs to a fresh BSA-free TYH medium three times. Two-cell embryos were obtained by incubation of cumulusintact eggs with capacitated sperm for 24 h. Zona proteins were solubilized by treating zona-intact eggs or two-cell embryos with a BSA-free acidic TYH medium (pH 3.0) at RT. When the zona pellucida disappeared under a microscope, zona-free eggs were removed by transferring them. The remaining solution containing the solubilized zona proteins was neutralized by adding 1 M TrisHCl (pH 8.0) and kept in a deep-freezer at 80C until use.

Immunoelectron and Conventional Electron Microscopy

Various sperm samples including intact sperm, detergent-treated sperm, and acrosome-reacting sperm, were prepared for immunolocalization of MC41 and conventional morphological study. For immunoelectron microscopy, sperm were xed in periodate lysineparaformaldehyde (PLP) xative. Fixed sperm were washed in PBS (136.8 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2HPO 4, 1.5 mM KH 2PO 4, pH 7.4), dehydrated, and embedded in Lowicryl K4M (Polysciences, Inc., Warrington, PA) at 35C. Ultra-thin sections were made on an LKB microtome and mounted on nickel grids. The sections were treated in a blocking solution containing 1% BSA in PBS and incubated with the anti-MC41 antibody and then with 5 nm gold-conjugated antimouse IgG (British BioCell International Ltd., Golden Gate, UK). Counterstaining was done with uranyl

MATERIALS AND METHODS

Animals
Female (9 weeks) and male (12 weeks) mice (ICR strain) were purchased from Japan SLC Inc. (Hamamatsu, Japan) and maintained in the Laboratory Animal Center for Experimental Research in Miyazaki Medical College. They were allowed free access to food

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acetate/methyl cellulose absorption staining (Roth et al., 1990). For conventional electron microscopy, sperm were xed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4C overnight and postxed with 1% OsO 4 in the buffer at 4C for 1 h. The samples were washed in the buffer, dehydrated, and embedded in Epon 812 resin. Ultra-thin sections were counterstained with uranyl acetate and lead citrate, then observed under a transmission electron microscope (model H7100, Hitachi, Tokyo, Japan) at a 75-kV accelerating voltage.

Tanii et al.

Immunoblotting and Zymographic Analysis of Proteases

To determine the molecular weight of MC41 in the SDS extracts, 10 vol of ice-cold acetone was added to the sperm extractions with 0.1% SDS, and precipitated proteins were collected by centrifugation. The pellets were dissolved in a buffer containing 1% SDS, 62.5 mM TrisHCl (pH 6.8), 1 mM PMSF, 20% glycerol, and bromophenol blue. The solubilized proteins were separated by SDSPAGE (Laemmli, 1970) under reducing or nonreducing conditions. Proteins were blotted on to polyvinylidiene uoride (PVDF) membranes. The blots were incubated with the anti-MC41 antibody, then with horseradish peroxidase-conjugated antimouse IgG M (ICN Pharmaceuticals Inc.-Cappel Product, Costa Mega, CA), and stained with 4-chloro-1-naphthol. MC41-associated proteins were isolated by immunoprecipitation as follows. Sperm protein extracts (1 ml) were mixed with 20 g of anti-MC41 antibody, then incubated at RT for 10 min. A protein A-insoluble cell suspension (10 l, Sigma) was then added to the mixture and incubated for 5 min. The precipitated immunocomplexes were washed three times with 25 mM Tris buffer (pH 7.5) containing 0.5 M NaCl, 5 mM EDTA, and 1% Triton X-100, and then once with 10 mM TrisHCl (pH 7.5) and 5 mM EDTA by centrifugation. The pellets were dissolved in a buffer containing 2% SDS, 62.5 mM TrisHCl (pH 6.8), 7% glycerol and bromophenol blue. To check the presence of MC41 in the immunoprecipitates, proteins isolated by immunoprecipitation were applied to 10% SDS gel under nonreducing conditions, and then immunoblotting was performed. To detect proteins exhibiting protease activity, zymography was performed as described previously (Siegel and Polakoski, 1985). Briey, the solubilized proteins were separated by SDSPAGE using a 0.1% gelatin-containing gel under nonreducing conditions. Proteins separated on the 10% polyacrylamide gels were renatured by removing SDS and then incubated in a reaction solution containing 50 mM Tris buffer (pH 7.5) in the presence or absence of 10 mM CaCl 2 at 37C for 18 h. The gels were stained with Coomassie brilliant blue R-250. To inhibit serine protease activity, p-aminobenzamidine (Sigma) was added to the reaction solution.

0.05% Tween 20 at RT for 30 min. The blots were washed once in TBSTween and then incubated with sperm protein extracts at 4C for 2 h. The blots were successively incubated with the anti-MC41 antibody, a biotin-conjugated anti-mouse IgG (EY Laboratories, Inc., San Mateo, CA), and horseradish peroxidase-conjugated streptavidin (Amersham) at RT for 1 h each. MC41 bound onto membranes was visualized with an ECL Western blotting detection kit (Amersham). In the control experiment, immunodepletion of MC41 from the sperm protein extracts was done by immunoprecipitation with the anti-MC41 antibody, and we used a remaining supernatant, in which no MC41 was detected by immunoblotting.

Induction of the Acrosome Reaction

Cauda sperm were suspended in a TYH medium at a concentration of 6 10 5 cells/ml and incubated in a CO 2 incubator at 37C with an atmosphere of 5% CO 2 in air for 90 min to achieve capacitation. To induce the acrosome reaction, solubilized zona proteins from eggs were added to sperm suspension at the concentration of 1 zona/ l. After a 60-min incubation, the reaction was stopped by adding a PLP xative. Under these conditions, 58% of sperm underwent the acrosome reaction as estimated by indirect immunouorescence using the anti-MC41 antibody. Fixed sperm were embedded in Lowicryl K4M resin for immunoelectron microscopy as described above.

RESULTS
Properties of Subdomain Structure in the Mouse Sperm Anterior Acrosome
Immunoelectron microscopy demonstrated that MC41 was localized at the cortical region of the anterior acrosome in the mouse (Fig. 1A) as described previously (Tanii et al., 1995). To dene the biochemical properties of the subdomain structure, cauda sperm were treated with 2% Triton X-100 under low- or high-salt (1 M NaCl) conditions for 2 h on ice, and the remaining structures were observed under an electron microscope. Electron-dense materials remained at the acrosomal cortex after the Triton X-100 treatment under both low- and high-salt conditions. The Tritoninsoluble anterior acrosomal components, referred to as the acrosomal cortical matrix, were labeled with an immunogold probe for MC41 (Fig. 1B). The Triton X-100 treatment caused no apparent change in the density of immunogold labeling. Salt condition did not affect the labeling either. The solubility of the acrosomal cortical matrix was examined. Indirect immunouorescence using anti-MC41 antibody was done to determine whether the acrosomal cortical matrix was dissolved. Immunoreactivity disappeared after the treatments with 0.1% SDS and 10 mM NaOH (data not shown). Sonication was also effective for solubilization, but 6 M urea treatment was ineffective (data not shown). Electron microscopy demonstrated that the acrosomal cortical matrix components were thoroughly dissolved by the SDS treatment (Figs. 2A and 2B). Immunoblotting demonstrated that MC41 was extracted with 0.1% SDS in conjugation with the acrosomal cortical matrix (Fig. 2C). Multiple immunoreactive bands were found at mo-

Binding Assay of Acrosomal Proteins to Zona Proteins by Far Western Blotting

Zona proteins isolated from eggs or two-cell embryos were applied to a gradient acrylamide gel (515%), separated by SDS PAGE, and transferred onto nitrocellulose membranes (Hybond ECL, Amersham International plc, Buckinghamshire, UK). The blotted proteins were renatured by successively changing a buffer solution containing guanidineHCl at a concentration of 6 M, 3 M, 1.5 M in 10 mM Hepes (pH 7.9), 50 mM NaCl, and 1 mM EDTA. Nonspecic binding was blocked with 5% nonfat dry milk in TBSTween consisting of 10 mM TrisHCl (pH 7.5), 154 mM NaCl,

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centration of 5 mM (Fig. 3A, e). The band was found in both the absence and the presence of CaCl 2 in the reaction solution (data not shown), indicating that the protease activity was not due to metalloprotease. In all extracts, immunoreactive bands for the anti-MC41 antibody were found by immunoblotting, showing that the immunoprecipitation was working correctly in the SDS extracts as well as in the Triton extracts (Fig. 3B). The immunoreactive bands did not correspond to the band with hydrolyzing activity in zymography, indicating that MC41 by itself had no protease activity.

Binding Property of MC41 to Zona Proteins

Mouse zona pellucida consists of three glycoproteins of 200, 120, and 80 kDa, referred to as ZP1, ZP2, and ZP3, respectively, as described previously (Bleil and Wassarman, 1980). The binding activity of MC41 to zona proteins was examined by using the Far Western blotting technique. Zona proteins isolated from eggs or two-cell embryos were separated by SDSPAGE under nonreducing conditions, and three polypeptides corresponding to ZP1, ZP2, and ZP3 were distinguishable (Fig. 4a). Then zona proteins were blotted to nitrocellulose membranes and incubated with sperm proteins containing MC41, which were prepared with 0.1% SDS following pretreatment of sperm with Triton X-100 under low- or high-salt conditions, as described in the above section. When the Triton pretreatment was carried out under low-salt conditions, MC41 was detectable on a band corresponding to the position of ZP2 (Fig. 4d). By contrast, no band was detected on the membrane when the Triton pretreatment was conducted under high-salt conditions (Fig. 4, f). Immunodepletion of MC41 from the sperm protein extract abolished the immunoreactive band (Fig. 4g). It is well known that ZP2 is subject to partial proteolysis as a result of fertilization, and the processed form of ZP2 is called ZP2 f (Bleil et al., 1981). It was therefore of interest to determine whether MC41 retains the ability to bind to ZP2 f. We prepared ZP2 f from two-cell embryos and checked the conversion from ZP2 to ZP2 f by SDSPAGE under reducing conditions. Zona proteins from eggs were separated into two bands of 120 and 80 90 kDa (Fig. 4, b), whereas, in zona proteins from two-cell embryos, the 120kDa band completely disappeared, and lower band intensity was markedly increased (Fig. 4c). This indicated that ZP2 was converted to ZP2 f. In SDSPAGE under nonreducing conditions, both ZP2 and ZP2 f migrated to the same position at 120 kDa. Far Western blotting showed that the immunoreactivity for MC41 was distinctly detectable on the band of 120 kDa corresponding to ZP2 f (Fig. 4e).

FIG. 1. Immunogold labeling for MC41 on intact and Triton X-100-treated sperm. (A) Intact sperm. Gold particles for MC41 specically bind to anterior acrosomal cortical materials. (B) Sperm treated with 2% Triton X-100. The majority of acrosomal cortical matrix materials are resistant to Triton X-100 treatment, and gold particles specically bind to the Triton X-100-insolubule acrosomal cortical matrix. Scale bars, 0.2 m.

lecular weights of not less than 200 kDa under both nonreducing and reducing conditions. Higher bands detected under nonreducing conditions disappeared after treatment with a reducing agent.

Serine Protease Associated with MC41

To detect protease activities in MC41-associated proteins, zymography was done after immunoprecipitation of the protein extracts prepared under the following four different protocols. Sperm proteins were extracted with (1) 2% Triton X-100 under low-salt conditions, (2) 2% Triton X-100 under high-salt conditions with 1 M NaCl, (3) 0.1% SDS following pretreatment with 2% Triton X-100 under low-salt conditions, (4) 0.1% SDS following pretreatment with 2% Triton X-100 under high-salt conditions with 1 M NaCl. Gelatin-hydrolyzing activity was detected at a molecular weight of 75 kDa in the SDS extract of sperm pretreated with Triton X-100 under low-salt conditions (protocol 3) (Fig. 3A, c). In contrast, no band with hydrolyzing activity was detected in the SDS extract when Triton X-100 pretreatment was carried out under high-salt conditions with 1 M NaCl (protocol 4) (Fig. 3A, d). No MC41associated protease was contained in the Triton extracts (protocols 1 and 2) (Fig. 3A, a and b). The proteolytic band of 75 kDa was markedly reduced by addition of p-aminobenzamidine, a serine protease inhibitor, at a con-

Behavior of MC41 during the Acrosome Reaction

As the above results indicated, MC41 interacted with the zona pellucida. However, MC41 is not a plasma membrane surface protein. Hence, it was interesting to examine when

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FIG. 2. Solubilization of the mouse acrosomal cortical matrix. (A, B) Electron micrographs. (A) Sperm were treated with 2% Triton X-100. Acrosomal cortical matrices (arrows) remain on the sperm head. (B) Sperm were treated with 2% Triton X-100 and subsequently with 0.1% SDS. Note that Triton-insoluble acrosomal cortical matrices were completely solubilized by SDS treatment. Scale bars, 2 m. (C) Immunoblot analysis with the anti-MC41 antibody for total sperm proteins in the SDS extract. (a) Nonreducing conditions. (b) Reducing conditions. Multiple immunoreactive bands are found at molecular weights of not less than 200 kDa. The numbers on the left indicate molecular weight markers ( 10 3).

and how MC41 is exposed outside during the acrosome reaction. Immunolocalization of MC41 was examined in zona-induced acrosome-reacting sperm. In the early stage of the acrosome reaction, the sperm plasma membrane and outer acrosomal membrane were fused, forming hybrid vesicles. Then the acrosomal contents were dispersed. Hybrid vesicles appeared connected with each other by acrosomal matrix components and xed to some region of the sperm head. Immunogold labeling for MC41 was found on the hybrid vesicles (Fig. 5A). In the later stage of the acrosome reaction, most hybrid vesicles were detached from the sperm head. MC41 was still found on the hybrid vesicles remaining around the sperm head, but no gold labeling was found on the inner acrosomal membrane (Fig. 5B).

DISCUSSION
Nature of the Subdomain Structure in the Mouse Anterior Acrosome
Domain-specic acrosomal matrix proteins have not been identied in the mouse. The present study demonstrated the subdomain structure in the mouse anterior acrosome and identied MC41 as a domain-specic matrix protein in the mouse. Recently, sp56 has been reported as a mouse acrosome matrix protein (Kim et al., 2001). However, unlike MC41, this protein is localized on both the cortex and the medulla of the acrosome (Foster et al., 1997).

Guinea pig and hamster sperm exhibit distinct subdomain structures with different electron densities in the anterior acrosome (Fawcett and Hollenberg, 1963; Yanagimachi and Noda, 1970). Three subdomain structures can be observed in guinea pig sperm, termed M1, M2, and M3 from dorsal to ventral, and two domain-specic matrix proteins have been isolated: AM50 (50 kDa) in the M3 region, and AM67 (67 kDa) in the M1 region (Westbrook-Case et al., 1994; Foster et al., 1997). AM67 has been identied as a guinea pig orthologue of mouse sp56 (Foster et al., 1997). We recently reported the localization of MC41 in guinea pig sperm to the outer acrosomal membrane matrix-associated materials at the dorsal margin of the apical segment (Yoshinaga et al., 2001). This denes another acrosomal subdomain in the guinea pig in addition to M1, M2, and M3. Compared with the localization of sp56 in the mouse and AM67 in the guinea pig, MC41 is localized at a more restricted region. The molecular weight of guinea pig MC41 in cauda epididymal sperm is 165 kDa (Yoshinaga et al., 2001). Based on localization and molecular weight, MC41 is a different matrix protein from AM50 and AM67. In hamster sperm, two matrix proteins, AM22 (22 kDa) and AM29 (29 kDa), have been isolated and are localized in the cortical region of the anterior acrosome (Olson et al., 1988). In addition, in bovine sperm, a stable acrosomal matrix assembly is formed by antigenically related multiple polypeptides of 38 19 kDa (Olson et al., 1997). The polypeptides are homologous to an acrosome-specic protein, SP-10 (Freemerman et al., 1993; Olson et al., 1997), which has been

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copy demonstrated that MC41 was still present on the Triton X-100-resistant acrosomal matrix (Fig. 1). A previous study showed that a MC41-reactive molecule of 200 kDa was soluble in a weak detergent, such as Triton X-100 and octylglucopyranoside, by immunoblotting (Tanii et al., 1992a, 1995), and therefore it is possible that some modication or denaturation causes the release of a relative amount of a 200-kDa molecule from the detergentinsoluble acrosomal matrix assembly.

A Serine Protease of 75 kDa Associated with MC41

Serine proteases have important roles in fertilization. Specic inhibitors for serine proteases block several steps in sperm egg interactions: sperm binding to the zona pellucida (Saling, 1981; Bleil et al., 1988), zona penetration (Yamagata et al., 1998), and the sperm egg fusion process (Takano et al., 1993). Acrosin is the most abundant serine protease in the acrosome and has the zona-binding activity (Topfer-Petersen and Henschen, 1987; Hardy and Garbers, 1994). It is therefore assumed to play a major role in sperm penetration into the zona pellucida (Urch et al., 1985; Jones and Brown, 1987; Liu and Baker, 1993). However, sperm from acrosin-decient mice can penetrate the zona pellucida and fertilize eggs, although showing delayed fertilization (Baba et al., 1994; Adham et al., 1997). This means that

FIG. 3. Analysis of proteolytic activities in MC41-associated proteins. (A) Zymography was done after immunoprecipitation of the sperm protein extracts prepared under four different protocols (a d). Sperm proteins were extracted with (a) 2% Triton X-100 under low-salt conditions, (b) 2% Triton X-100 under high-salt conditions with 1 M NaCl, (c) 0.1% SDS following pretreatment with 2% Triton X-100 under low-salt conditions, (d) 0.1% SDS following pretreatment with 2% Triton X-100 under high-salt conditions with 1 M NaCl. Proteins isolated by immunoprecipitation using the anti-MC41 antibody were separated by nonreducing SDSPAGE containing gelatin. Gels were stained with Coomassie brilliant blue. Note the 75 kDa proteolytic band (arrow) in lane c. (e) Proteolytic activity was inhibited by p-aminobenzamidine (5 mM). Sample is the same as in lane c. (B) Immunoblotting of MC41-associated proteins isolated by immunoprecipitation. Samples (a d) were the same as the samples in corresponding lanes in (A). Immunoreactive bands were detected in all lanes. The numbers on the left indicate molecular weight markers ( 10 3).

identied in several mammalian species, including humans. Thus, the subcompartment structure constructed by a framework of acrosomal matrix proteins is presumably a common architecture in mammals. The mouse cortical acrosomal matrix was resistant to nonionic detergent such as Triton X-100 and high salt, but was unstable with strong ionic detergents such as SDS and mechanical disruption by sonication. The biochemical stability of the mouse acrosomal matrix is similar to those of the bovine, hamster, and guinea pig acrosomal matrices as reported previously (Olson et al., 1985, 1988, 1997; Noland et al., 1994; NagDas et al., 1996b). Multiple MC41immunoreactive bands of not less than 200 kDa were detected in the SDS extract. These immunoreactive polypeptides may form a stable acrosomal matrix assembly by a disulde bond, since the upper immunoreactive bands detected under the nonreducing condition disappeared under reducing conditions (Fig. 2). Immunoelectron micros-

FIG. 4. Binding of MC41 to the zona proteins by Far Western blotting. Same quantity of zona proteins isolated from eggs (a, b, d, f, g) and two-cell embryos (c, e) was applied on SDS gel (515% acrylamide) under nonreducing (a, d g) or reducing (b, c) conditions. (a c) Silver staining of gel. (d g) Far Western blotting. Sperm protein extracts prepared with 0.1% SDS following pretreatment of Triton X-100 under low-salt conditions (d, e, g), or high-salt conditions (f) were overlaid on nitrocellulose membranes, to which zona proteins were blotted. Bound MC41 was visualized by immunostaining with the anti-MC41 antibody. Note that the position of the immunoreactive bands in lane d and e correspond to that of ZP2. (g) Immunodepletion of MC41 from sperm protein extract was done as a control experiment. No immunoreactive band was detected on the membrane.

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FIG. 5. Immunolocalization of MC41 on zona-induced acrosome-reacting sperm. (A) Early stage of the acrosome reaction. The outer acrosomal and plasma membranes were fused, forming hybrid vesicles, and the acrosomal matrix is mostly dispersed. Gold particles label the hybrid vesicles. (B) Later stage of the acrosome reaction. Gold particles label some hybrid vesicles remaining around the sperm head. No labeling was seen on the inner acrosomal membrane. AA, anterior acrosome; ES, equatorial segment. Scale bars, 0.2 m.

acrosin is not the only molecule involved in spermzona binding and zona penetration. It seems reasonable to suppose that more than two serine proteases play roles in sperm egg interactions. The genes coding two novel serine proteases of calculated molecular masses of 40,765 and 40,252 Da have been cloned using a cDNA library of acrosin-decient mouse testes (Kohno et al., 1998). This study identied a 75-kDa protease in the mouse sperm extract. This was a serine protease because the protease activity was independent of calcium and was inhibited by a serine protease-specic inhibitor, p-aminobenzamidine. This serine protease has not yet been reported, based on its molecular weight. It has been reported that some acrosomal hydrolases are associated with the acrosomal matrix in several species. Proacrosin and N-acetylglucosaminidase are bound to hamster acrosomal matrix proteins (NagDas et al., 1996a). Proacrosin is also associated with the guinea pig and bovine acrosomal matrix (Hardy et al., 1991; Noland et al., 1994; NagDas et al., 1996b). This study demonstrated that the 75-kDa serine protease was bound to a mouse acrosomal matrix protein, MC41, presumably forming a complex. The 75-kDa protease was easily dissociated from MC41 under high-salt conditions. Similarly, salt-dependent solubiliza-

tion of acrosin has been demonstrated in the hamster (Olson et al., 1988). By the association with the acrosomal matrix, acrosomal hydrolases could act at the appropriate site in proper timing during fertilization. This idea is supported by the nding that the release of acrosin during the acrosome reaction is slower than that of dipeptidyl peptidase II, which is not associated with the acrosomal matrix (Talbot and DiCarlantonio, 1985; Hardy et al., 1991).

Role of MC41 in the Secondary Binding of Sperm to the Zona Pellucida

The attachment of acrosome-intact sperm to the zona pellucida, referred to as primary binding, induces the acrosome reaction. It is followed by subsequent tight binding of acrosome-reacting or acrosome-reacted sperm to the zona pellucida, called secondary binding, which continues until the sperm head enters the zona pellucida, and is required for sperm penetration through it (Bleil and Wassarman, 1983). Since the anti-ZP2 antibody inhibits continued sperm binding to the zona pellucida (Bleil et al., 1988), ZP2 is considered a secondary ligand. Several candidate molecules for secondary sperm zona receptors have been reported. Acrosin and sp38 can bind to porcine ZP1, a molecule homolo-

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gous to mouse ZP2 (Mori et al., 1993). Since the anti-PH-20 antibody does not inhibit sperm binding to the zona pellucida in acrosome-intact sperm but in acrosome-reacted sperm, PH-20 is likely to be a secondary sperm zona receptor (Myles et al., 1987; Hunnicutt et al., 1996). As far as we know in the literature, there has been no report of a protein exhibiting specic afnity for mouse ZP2. In this study, Far Western blotting analysis provided evidence of the binding ability of MC41 to mouse ZP2, suggesting the involvement of MC41 in the secondary binding. The binding of MC41 to ZP2 was detected only in the presence of high-salt-soluble sperm proteins. Therefore, it is likely that MC41 indirectly binds to ZP2 via high-saltsoluble proteins. Alternatively, the binding needs complex formation of MC41 with the high-salt soluble proteins. Since the 75-kDa serine protease was contained in highsalt-soluble proteins, it is of interest to determine whether the 75-kDa serine protease functions in the binding of MC41 to ZP2. The 75-kDa serine protease remains to be further characterized. Since ZP2 undergoes limited proteolysis following articial activation of eggs in vitro, it was hypothesized that modication of ZP2, triggered by fertilization, may prevent further sperm penetration through zona pellucida (Bleil et al., 1981). In this context, it seems possible that sperm proteins with ZP2-binding activity cannot interact with ZP2 f, the processed form of ZP2. However, MC41 retained the ability to bind to ZP2 f. Therefore, the prevention of further sperm penetration through zona pellucida may not due to the loss of the ability of MC41 to bind to ZP2. However, it is not to be denied that MC41 has a physiological role in the spermZP2 binding. Another mechanism may affect spermzona interaction following fertilization, as previously suggested (Bleil et al., 1981). Since the inner acrosomal membrane is not exposed outside until the end of the acrosome reaction, it is likely that the molecules on hybrid vesicles are involved in the secondary sperm binding, especially before inner acrosomal membrane exposure. This study supports the idea of hybridvesicle-mediated spermzona binding because of the localization of MC41 on the surface of hybrid vesicles during the acrosome reaction, in addition to the binding ability of MC41 to ZP2. The idea is also supported by electron microscopic study of hamster sperm penetrating through the zona pellucida, showing that remnants of the acrosomal cap or hybrid vesicles consistently attach to the zona surface and appear to anchor acrosome-reacted sperm with the zona pellucida (Yanagimachi and Phillps, 1984). Still, it is believed that the molecules localized on the inner acrosomal membrane have a major role in the secondary binding. The main reason for this idea is that mouse ZP2-gold probe labels acrosome-reacted sperm heads preferentially, showing the high afnity of ZP2 for the inner acrosome membrane, but low afnity for the plasma membrane (Mortillo and Wassarman, 1991). However, considerable labeling of ZP2-gold probes is also shown on the outer acrosomal membrane of sperm. Thus, the possible role of

FIG. 6. Schematic diagram of the processes of spermzona binding. (A) Primary binding. Sperm attaches to zona surface to induce acrosome reaction due to the interaction of molecules on the sperm plasma membrane with ZP3. (B) Early stage of secondary binding. Sperm binds tightly to the zona pellucida by the interaction of molecules on hybrid vesicles with zona proteins. (C) Later stage of secondary binding. The inner acrosomal membrane-exposed sperm binds to the zona pellucida by the interaction of molecules on the inner acrosomal membrane with zona proteins. Mesh networks in the anterior acrosome and surrounding hybrid vesicles indicate acrosomal matrix proteins including MC41.

hybrid-vesicle-associated molecules in the secondary binding is undeniable. In view of the present results, secondary binding should be divided into two processes: binding of the hybrid-vesicle-accompanying sperm with the zona pellucida, and the subsequent interaction of the inner acrosome membrane-exposed sperm with the zona pellucida. Figure 6 illustrates the processes of spermzona binding. Primary binding induces the vesicular fusion of the plasma and outer acrosomal membranes, resulting in the release of acrosomal enzymes (Fig. 6A). As the vesiculation proceeds, most acrosomal enzymes are released, but some acrosomal matrix proteins remain around hybrid vesicles (Fig. 6B). These molecules on hybrid vesicles can function in the initial phase of secondary binding. At the end of the acrosome reaction, hybrid vesicles are dispersed from sperm head, and sequentially the inner acrosomal membrane is exposed outside, which enables the molecules on the inner acrosomal membrane to interact with zona molecules (Fig. 6C). Because of the localization on hybrid vesicles of acrosome-reacting sperm, acrosin and SP-10 as well as MC41 may function in the early phase of secondary binding (Yudin et al., 1999; Foster et al., 1994). Finally, the present study emphasized the importance of hybrid-vesiclemediated spermzona binding in the fertilization process.

ACKNOWLEDGMENTS
We thank Mr. Y. Fujii for his excellent technical assistance. This research was supported by grants from the Japanese Ministry of Education, Science and Culture.

Copyright 2001 by Academic Press. All rights of reproduction in any form reserved.

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