Sperm Proteasomes Are Responsible for the Acrosome Reaction And

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Developmental Biology 308 (2007) 222–231 www.elsevier.com/locate/ydbio

Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus ⁎ Naoto Yokota, Hitoshi Sawada

Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Toba, Mie 517-0004, Japan Received for publication 13 April 2007; revised 18 May 2007; accepted 21 May 2007 Available online 25 May 2007

Abstract

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG- 132 and MG-115, inhibited fertilization at 100 μM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization. © 2007 Elsevier Inc. All rights reserved.

Keywords: Proteasome; Sperm; Fertilization; Acrosome reaction; Vitelline layer; Lysin; Sea urchin

Introduction mammals, which is a formidable barrier to sperm–egg fusion. When sperm enter the jelly coat surrounding the vitelline layer Fertilization is an essential process for sexual reproduction of sea urchin eggs, they undergo the acrosome reaction, an and is precisely controlled by many gamete proteins. To accom- exocytosis of the acrosome. In many invertebrates including sea plish fertilization, sperm must penetrate through a proteinaceous urchins, this is followed by the formation of an acrosomal egg-coat called the vitelline coat (or vitelline layer or vitelline process. During this reaction, a lytic agent, lysin, located in the envelope) in marine invertebrates and the zona pellucida in acrosome is exposed and partially released, allowing sperm to penetrate through the vitelline coat. In mammals, it has long been believed that an acrosomal Abbreviations: AMC, 7-amino-4-methylcoumarin; ASW, artificial seawater trypsin-like protease, acrosin, is a zona-lysin (McRorie and (460 mM NaCl, 10 mM CaCl2, 50 mM MgCl2, 10 mM KCl, 50 mM Tris/HCl (pH 8.0)); Boc, t-butyloxycarbonyl; BSA, bovine serum albumin; DMSO, Williams, 1974). However, by using mice in which the acrosin dimethylsulfoxide; DTT, dithiothreitol; MCA, 4-methylcoumaryl-7-amide; gene was knocked out, it was revealed that acrosin is neither MFSW, Milllipore-filtered seawater; PBS, phosphate buffered saline; PBST, essential for sperm penetration of the zona pellucida nor PBS containing 0.1% Tween 20; PKA, protein kinase A; STI, soybean trypsin fertilization per se, although a delay was observed during the inhibitor; TBS, Tris-buffered saline; TBST, TBS containing 0.1% Tween 20; Z, benzyloxycarbonyl. fertilization process in this animal (Baba et al., 1994). It is ⁎ Corresponding author. Fax: +81 599 34 2456. currently proposed that acrosin is involved in the dispersal of E-mail address: [email protected] (H. Sawada). acrosomal contents during the acrosome reaction (Yamagata

0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.05.025 N. Yokota, H. Sawada / Developmental Biology 308 (2007) 222–231 223 et al., 1998b) and that the other sperm protease(s) must be zona- hibitor), chymostatin (chymotrypsin inhibitor) and fluorogenic substrates lysins in mammals (Yamagata et al., 1998a). (peptidyl-MCAs) were obtained from Peptide Institute (Osaka, Japan). Leupeptin (acetyl-Leu-Leu-argininal, a trypsin inhibitor) was kindly provided In order to identify the sperm proteases involved in fertil- by Dr. Tetsuya Someno of Nippon Kayaku Ltd. All the other reagents were ization we have utilized the solitary ascidian Halocynthia roretzi, purchased from Sigma Chemical Co., unless otherwise specified. Inhibitors, since large quantities of readily fertilizable gametes are easily except soybean trypsin inhibitor (STI), and fluorogenic substrates were obtained from this aquacultured species. We previously reported dissolved in DMSO to a final concentration of 10 mM, and stored at −20 °C that two sperm trypsin-like proteases, ascidian acrosin and until use. spermosin (Hoshi et al., 1981; Sawada et al., 1984b,c, 1996; Fertilization assay Sawada and Someno, 1996) and the sperm ubiquitin–proteasome system (Saitoh et al., 1993; Sakai et al., 2003, 2004; Sawada et “Dry” sperm were suspended in MFSW (about 0.5–1.0×105 sperm/ml) and al., 1983, 2002a,b) play key roles in the sperm binding to and preincubated with respective inhibitors or peptidyl–MCA substrates for 3 min, penetration of the vitelline coat in H. roretzi (see reviews: followed by the addition of 10 μl of egg suspension (about 150–200 eggs) to a Sawada, 2002; Sakai et al., 2004; Sawada et al., 2005). In final volume of 110 μl. Final concentration of DMSO was adjusted to 1%. To particular, Sawada et al. (2002a,b) demonstrated that the 70 kDa score percentage of fertilization, at least 150 eggs were examined at 5 min after insemination. Fertilization ratio was determined by counting the egg with a vitelline-coat main component, HrVC70, is ubiquitinated during fertilization envelope (Hoshi et al., 1979). In experiments where trypsin- fertilization, followed by the sperm proteasome-mediated inhibitors and -substrates inhibited elevation of the vitelline envelope (Hoshi et degradation (see reviews: Sawada, 2002; Sakai et al., 2004; al., 1979; Sawada et al., 1984a), the fertilization ratio was determined at 30– Sawada et al., 2005). Very significantly, it was recently reported 60 min after insemination by counting the percentage of cleaved eggs (Hoshi et that sperm proteasomes are also involved in mammalian al., 1979). To examine the effects of proteasome inhibitors on fertilization with acrosome reacted-sperm, sperm were diluted with MFSW containing egg jelly fertilization (Morales et al., 2003, 2004; Pizarro et al., 2004; (25 μg carbohydrate/ml) followed by standing for 2 min to induce the acrosome Sutovsky et al., 2004). In mammals, sperm proteasomes appear to reaction. The reacted sperm (100 μl) was further incubated with various inhi- be responsible not only for the acrosome reaction and sperm bitors for 3 min, followed by the addition of 10 μl of dejellied-egg suspension binding process to the zona pellucida (Pasten et al., 2005) but also (about 150–200 eggs) in a 96-well multidish. Sperm concentrations were – for penetration through the zona pellucida (Morales et al., 2003; adjusted to the minimum required for 80 100% fertilization. All the above assays were carried out at 20 °C. Sutovsky et al., 2004). These results led us to speculate that sperm proteasomes may function as lysins also in deuterostomes Assay of acrosome reaction other than chordates. In order to assess this issue, we here reappraise the identification of lysins in sea urchin fertilization. Assay of the acrosome reaction was carried out as described previously In sea urchins, purification and characterization of the pro- (Biermann et al., 2004; Vacquier and Hirohashi, 2004) with a slight mod- μ teasome from sperm was reported by Saitoh et al. (1991) and ification. Briefly, dry sperm were diluted with MFSW (1:200), a portion (200 l) of which was incubated with MG-132 for 5 min. The final concentration of Inaba et al. (1992). It was also reported that the sperm pro- DMSO in each assay was always adjusted to 1%. Then, 10 μl of egg jelly teasome may be involved in the acrosome reaction in sea (carbohydrate contents, 5 μg) or ionomycin (final concentration, 10 μM) was urchins (Matsumura and Aketa, 1989, 1991). However, the added to induce the acrosome reaction. After incubation for 3 min, 200 μl of ice- specific roles of sperm proteasomes in sea urchin fertilization by cold 4% paraformaldehyde in ASW was added and fixed for 1 h at 4 °C. Fixed using several proteasome-specific inhibitors have not yet been sperm were collected by centrifugation (1500×g for 15 min) and resuspended in 500 μl of ASW. To attach the sperm to a cover slip, 50 μl of sperm suspension studied. In this report, therefore, we systematically investigated was placed on a poly-L-lysine-coated cover slip. The sperm were permeabilized the effects of protease inhibitors, including proteasome with 0.05% (v/v) Triton X-100 and stained with PBS (pH 8.0) containing 0.2 inhibitors, on the acrosome reaction, sperm binding to and units Alexa Fluor 488 phalloidin, 1 mg/ml BSA and 20 mM glycine, for 2 h penetration of the vitelline layer, to unambiguously demonstrate under darkness. After washing, sperm with an acrosomal process were scored the roles of sperm proteasomes in sea urchin fertilization. under a fluorescence microscope using a 100×Plan Apo object lens (Nikon) and a filter cube B2A (Nikon).

Materials and methods Sperm binding assay

Animal and collection of gametes Binding of sperm to the vitelline layer of the dejellied eggs was carried out as described previously (Kato and Sugiyama, 1978). Briefly, dejellied eggs were The sea urchin, Pseudocentrotus depressus, was obtained near Sugashima fixed with 0.5% glutaraldehyde for 1 h, and then washed repeatedly with MFSW Marine Biological Laboratory, Mie prefecture, Japan. Gametes were obtained by (Hino et al., 1980). The fixed eggs were suspended in MFSW containing egg intracoelomic injection of 0.55 M KCl. “Dry” sperm was stored on ice until use, jelly (25 μg carbohydrates/ml) and 100 μM MG-132, followed by adding the while eggs were washed twice with Millipore-filtered seawater (MFSW) before sperm suspension (about 106 sperm/ml). After 2 min, the eggs were washed with experiments. Dejellied eggs and crude egg jelly were prepared as previously MFSW and fixed with 1% glutaraldehyde in MFSW for 30 min at room described (Wessel and Vacquier, 2004). Carbohydrate contents in egg-jelly temperature. Then, the eggs were washed with MFSW three times and sperm preparations were estimated by the phenol-sulfuric acid method using glucose as nuclei were stained with 0.5 μg/ml 4-diamidino-2-phenylindol (DAPI). The a standard (DuBois et al., 1956). number of sperm bound to a single egg was counted under a fluorescence microscope using a UV1A filter (Nikon). Chemicals Preparation, Western blotting, and gel filtration of acrosomal contents Alexa Fluor 488 phalloidin was obtained from Molecular Probes Inc. (Eugene, OR). MG-132 (Z-Leu-Leu-leucinal), MG-115 (Z-Leu-Leu-norvalinal), “Dry” sperm were washed twice with ASW by two cycles of centrifugation lactacystin, E-64-d (cell-permeable analog of E-64, a cysteine-protease in- (800×g, 5 min) to carefully remove the possibly contaminating coelomic cells, 224 N. Yokota, H. Sawada / Developmental Biology 308 (2007) 222–231 and induced to undergo the acrosome reaction with ionomycin at 10 μM. After fertilization using P. depressus. As shown in Table 1, incubation for 5 min, sperm were precipitated by centrifugation (1500×g, fertilization of P. depressus was strongly inhibited by MG- 30 min), and the resulting supernatant was further centrifuged at 17,000×g, for 30 min. The supernatant, thus prepared, included the acrosomal contents. The 132, and weakly by MG-115 and lactacystin. These acrosomal contents were subjected to SDS-PAGE in a mini-slab gel (12.5% gel), proteasome inhibitors affected fertilization in a concentra- followed by electronic transfer onto a PVDF membrane. After blocking with tion-dependent manner (Fig. 1). In contrast to the proteasomal TBS containing 3% skimmed milk, the membrane was incubated with anti- inhibitors, leupeptin, chymostatin, STI and E-64-d showed no α proteasome -subunits monoclonal antibody (Biomol International, PW8195), appreciable inhibition toward fertilization at 100 μM. It is which reacts with α1, α2, α3, α5, α6 and α7 subunits of the 20S proteasome, for 1 h at room temperature. After washing with TBST, the bands were detected by a known that MG-132 is capable of inhibiting the activities of horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham) and cysteine proteases, including cathepsin K, as well as the visualized by the ECL detection kit according to the manufacturer's protocol. proteasomal activity (Votta et al., 1997). However, it is Alternatively, the acrosomal contents were subjected to gel filtration using a thought that the effects of proteasome inhibitors on fertiliza- Superose 6 column (10×300 mm) equipped with ÄKTA purifier (Amersham tion are due to the inhibitory abilities toward the proteasomal Bioscience). Chromatography was carried out using TBS as an elution buffer at a flow rate of 0.5 ml/min, collecting 1 ml fractions. activities, since E-64-d, a potent inhibitor for cysteine proteases, showed no significant inhibition toward fertilization Assay of enzymatic activity (Table 1). To exclude the possibility that proteasome inhibitors affected A portion (150 μl) of the sample solution was mixed with 100 μl of 250 μM egg proteasome activity, high concentrations of sperm were fluorogenic peptide substrate dissolved in TBS. After incubation at 37 °C for added to the egg suspension, which had been incubated with 12 h, the enzymatic reactions were stopped by adding an equal volume of 2% 100 μM MG-132. In this experiment, no inhibitory effect on (w/v) SDS. The activity was determined by measuring the fluorescence intensity at 380 nm excitation and 460 nm emission. fertilization was observed (data not shown), confirming that it is the sperm proteasome, rather than egg proteasome, that is Isolation, SDS-PAGE, and Western blotting of vitelline envelope essential to sea urchin fertilization. Next, in order to address the issue whether proteasome in- Vitelline envelopes were isolated using DTT, according to the method hibitors affect the sperm motility, we examined the effect of described previously (Correa and Carroll, 1997; Epel et al., 1970). Dejellied MG-132 on sperm motility by microscopic observation. No eggs (settled volume, 4 ml) were suspended in 10 ml of VE isolation buffer significant difference in the ratio of motile sperm was observed (460 mM NaCl, 50 mM MgCl2, 10 mM KCl, 10 mM CaCl2, 20 mM DTT, 10 mM Tris–HCl (pH 9.0), 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml by microscopy in the presence and absence of 100 μM MG-132, leupeptin), followed by inverted mixing for 10 min. The egg suspension, thus which is dissolved in MFSW containing 1% DMSO (data not obtained, was centrifuged (400×g, 3 min), and the resulting supernatant was shown). Our results are not at variance with the results by Hoshi centrifuged again under the same conditions. The vitelline envelopes in the et al. (1979) that not only chymostatin but also all the protease supernatant were precipitated and repeatedly washed by centrifugation (5000×g, 5 min). The isolated preparation was subjected to SDS-PAGE (7.5% gel), inhibitors tested showed no inhibition toward sperm motility in followed by Western blotting using an FK2 monoclonal antibody (1:1000 sea urchins at the concentrations sufficient to inhibit fertilization dilution), which specifically reacts to multi- and mono-ubiquitinated proteins (Hoshi et al., 1979). These results led us to propose that the but not free ubiquitin (Fujimuro et al., 1994). The reacted bands were detected inhibitory effects on fertilization of proteasome inhibitors are by an ECL detection kit (Amersham) using an ATTO Light Capture model not due to the effects on sperm motility. AE6972. It is well known that there are two complexes in the pro- Immunocytochemistry teasome, i.e. the 26S and 20S proteasomes, and that the 20S proteasome, a cylindrical particle consisting of four stacked 7- Dejellied eggs were fixed with 50 mM MOPS/NaOH (pH 7.0), containing subunit rings (α1–7, β1–7, β1–7, α1–7), and a 19S regulatory 460 mM NaCl, 50 mM MgCl2, 10 mM KCl, 10 mM CaCl2, and 3% (w/v) particle (RP), consisting of about 18 subunits including multi- paraformaldehyde, for 30 min at room temperature. The fixed eggs were washed five times with MFSWand subjected to blocking with TBSTcontaining 3% (w/v) BSA. After incubation with FK2 monoclonal antibody (5 μg/ml) for 12 h at 4 °C, the eggs were washed five times with PBST, and incubated for 1 h with Table 1 rhodamine-conjugated anti-mouse IgG antibody (Chemicon) diluted with the Effects of protease inhibitors on fertilization of the sea urchin P. depressus buffer used for blocking. Following washing with TBST, the eggs were mounted μ on a cover slip and observed with a Nikon inverted fluorescent microscope using Inhibitor (100 M) Fertilization (% Control) a filter block G2A (Nikon). None 100 MG-132 4.4±1.8 MG-115 68.3±5.2 Results Lactacystin 77.0±10.1 E-64-d 97.4±4.2 Proteasome inhibitors inhibit sea urchin fertilization Leupeptin 103±3.8 Chymostatin 107±1.6 STI 104±3.1 It has been reported that proteasome inhibitors block μ fertilization in ascidians and mammals (Sawada et al., 1998, Sperm suspension (100 l) was incubated with the respective inhibitor for 3 min, followed by addition of the egg suspension (10 μl) up to a total volume of 110 μl 2002b; Sutovsky et al., 2004). Therefore, we first investigated in a multidish. Fertilization ratio was determined by counting the eggs as the effects of protease inhibitors, including proteasome fertilized that underwent either formation of a fertilization envelope or cleavage. inhibitors (MG-132, MG-115 and lactacystin), on sea urchin Data represent mean±S.D. (n=3). N. Yokota, H. Sawada / Developmental Biology 308 (2007) 222–231 225

rather than trypsin-like or chymotrypsin-like activity, of the proteasome plays a critical role in sea urchin fertilization.

MG-132 inhibits the egg jelly-induced acrosome reaction

After determining that the proteasome is involved in sea urchin fertilization, we explored which step(s) are blocked by proteasome inhibitors. It was proposed by Matsumura and Aketa (1989, 1991) that the proteasome is involved in the acrosome reaction of sea urchins, since chymostatin and Suc-Leu-Leu- Val-Tyr-MCA inhibit the acrosome reaction, albeit weakly (N200 μM). However, it has not been tested whether proteasome-specific inhibitors are capable of inhibiting the acrosome Fig. 1. Proteasome inhibitors block fertilization of the sea urchin P.depressus in a reaction. We investigated the effect of MG-132 on the acrosome concentration-dependent manner. Sperm suspension (100 μl) was incubated with reaction by counting the sperm forming an acrosomal process, various concentrations of respective proteasome inhibitors (final concentration: which was visualized by using fluorescent phalloidin according 10 μM, 50 μM, and 100 μM) for 3 min, followed by addition of a small volume (10 μl) of egg suspension. Data are mean±S.D. in three experiments. to the method described previously (see Fig. 3A) (Biermann et al., 2004; Vacquier and Hirohashi, 2004). As shown in Fig. 3B, the egg-jelly-induced acrosome reaction was inhibited by ubiquitin-recognizable subunit S5a, are capable of making up the 26S proteasome in an ATP-dependent manner, which can degrade the ubiquitin-conjugated proteins in an ATP-dependent fashion. It is also known that the 26S and 20S proteasomes possess three peptidase activities: chymotrypsin-like activity (preferred substrate, Suc-Leu-Leu-Val-Tyr-MCA; catalytic site, β5-subunit), trypsin-like activity (Boc-Leu-Arg-Arg-MCA; β2- subunit), and caspase-like activity (Z-Leu-Leu-Glu-MCA; β1- subunit) (Mayer et al., 2004). Therefore, in order to obtain insights into the catalytic centers of the proteasome participating in sea urchin fertilization, we examined the effects on fertilization of three peptide substrates for the proteasome. As shown in Fig. 2, Z-Leu-Leu-Glu-MCA and Boc-Leu-Arg-Arg-MCA inhibited fertilization in a concentration-dependent manner. In contrast, Suc-Leu-leu-Val-Tyr-MCA showed no appreciable inhibition at 100 μM. This suggests that caspase-like activity,

Fig. 3. Effects of the proteasome inhibitor MG-132 on the acrosome reaction. Fig. 2. Peptidyl-MCA substrates for the proteasome inhibited sea urchin fertil- (A) Whether sperm undergo the acrosome reaction was assayed by staining ization. A portion (10 μl) of intact egg suspension (about 150–200 eggs) was actin filaments with Alexa Fluor 488 phalloidin. Arrow heads indicate the added into 100 μl of sperm suspension, which had been incubated with acrosome-reacted sperm showing an acrosomal process. (B) Sperm were respective substrates for 3 min. Fertilization ratio was determined by counting incubated with various protease inhibitors (100 μM) for 3 min and then the eggs as fertilized that underwent either formation of a fertilization envelope induced to undergo the acrosome reaction with egg jelly (25 μM carbohydrate/ or cleavage. Suc-LLVY-MCA, Boc-LRR-MCA and Z-LLE-MCA are preferred ml) or ionomycin (10 μM). MG-132 inhibited the egg jelly-induced, but not substrates for chymotrypsin-like, trypsin-like and caspase-like activities of the ionomycin-induced, acrosome reaction. Data indicate mean±S.D. in three proteasome, respectively. Data represent mean±S.D. (n=3). experiments. 226 N. Yokota, H. Sawada / Developmental Biology 308 (2007) 222–231

100 μM MG-132, but the ionomycin (Ca2+ionophore)-induced acrosome reaction was not inhibited at all (Fig. 3B). Similar results were obtained in different batches of gametes (data not shown). These results suggest that the proteasome participates prior to the increase in intracellular Ca2+ concentration, during the acrosome reaction.

Proteasomal inhibitor also blocks fertilization with acrosome-reacted sperm

In ascidians (Sawada et al., 1998, 2002a,b) and mammals (Sutovsky et al., 2004), it is reported that the proteasome is Fig. 5. Effect of MG-132 on the binding of sperm to the vitelline layer. Sperm involved in sperm penetration of the proteinaceous egg-coat. and glutaraldehyde-fixed eggs were incubated in the presence of egg jelly (25 μg μ However, such a function of the sperm proteasome has not yet carbohydrate/ml) and MG-132 (100 M). After fixation, sperm were stained with DAPI. The number of bound sperm was counted by fluorescence micro- been proposed in sea urchins. In order to assess this issue, we scopy. Data are mean±S.D. (n=36). investigated the effect of MG-132 on the fertilization of dejellied eggs using acrosome-reacted sperm, to distinguish the inhibitory effect of MG-132 on the acrosome reaction. Since receptor, glutaraldehyde-fixed eggs were used in this study. As the fertilization ability of the acrosome-reacted sperm appears to shown in Fig. 5, MG-132 showed no significant inhibition decrease rapidly, dejellied eggs were used in this study instead toward the binding of sperm to the vitelline layer, suggesting of intact ones. Soon after inducing the acrosome reaction, sperm that the proteasome is not essential for sperm binding to the were incubated with MG-132, followed by mixing with vitelline envelope. Taken together, it is most plausible that the dejellied eggs. As shown in Fig. 4, the fertilization of dejellied sperm proteasome is involved not only in the acrosome reaction, eggs with reacted sperm was inhibited by MG-132 in a but also in sperm penetration of the vitelline layer, rather than concentration-dependent manner. In contrast, E-64-d showed no binding to the vitelline coat. inhibition at 100 μM. These results indicate that the sperm proteasome also participates in a fertilization process after the Acrosomal contents have the proteasome antigen and activity acrosome reaction. If sperm proteasomes function as lysins, they should be Effect of MG-132 on sperm binding to the vitelline layer partially secreted from the acrosome during the acrosome reaction. In order to assess this issue, we examined whether pro- We investigated whether the proteasome is involved in teasomes are detected in acrosomal contents by Western sperm binding to the vitelline coat, as reported in the ascidian blotting. We also investigated whether the proteasome activity H. roretzi (Takizawa et al., 1993). In sea urchins, it is currently is detected in acrosomal contents using three fluorogenic pep- believed that a sperm receptor on the vitelline envelope is either tide substrates for the proteasome. As shown in Fig. 6, the released or degraded by a trypsin-like protease released from proteasome was found to be present in the acrosome-reacted cortical granules upon fertilization (Vacquier and Moy, 1977; sperm supernatant by Western blotting, using a proteasome- Vacquier et al., 1973). To avoid degradation of the sperm specific monoclonal antibody. In contrast, no cross-reacted band was observed in a control experiment lacking the primary antibody. The acrosomal contents showed peptidase activities toward Suc-Leu-Leu-Val-Tyr-MCA, Boc-Leu-Arg-Arg-MCA and Z-Leu-Leu-Glu-MCA. MG-132 strongly inhibited the chy-

Fig. 4. Fertilization of dejellied eggs with acrosome-reacted sperm was inhibited Fig. 6. Evidence for the presence of the proteasome in acrosomal contents by by the proteasome inhibitor MG-132. Sperm were allowed to undergo the Western blotting. Acrosomal contents were subjected to SDS-PAGE, followed acrosome reaction with egg jelly (25 μg carbohydrate/ml) before incubation with by Western blotting using a monoclonal antibody specific for the proteasome α- 100 μM of MG-132. Following incubation with MG-132, sperm were added to subunits as a primary antibody (right panel) or without using the primary the dejellied egg suspension. Data represent mean±S.D. (n=3). antibody as a control (left panel). N. Yokota, H. Sawada / Developmental Biology 308 (2007) 222–231 227 motrypsin-like and caspase-like activities in the acrosomal contents (Fig. 7), while this inhibitor showed a weak inhibition to the trypsin-like activity (Fig. 7). Leupeptin was the strongest inhibitor against Boc-Leu-Arg-Arg-MCA activity among the inhibitors tested (Fig. 7). On the other hand, chymotrypsin-like and caspase-like activities were only slightly inhibited by leupeptin. Chymostatin was a moderate inhibitor toward the chymotrypsin-like and trypsin-like activities, but it showed no appreciable inhibition toward the caspase-like activity even at 100 μM(Fig. 7). E-64-d showed no significant inhibition toward the three peptidase activities (Fig. 7). Since several proteases could be released from the acrosome in addition to the proteasome, the acrosomal contents were

Fig. 8. Gel filtration of acrosomal contents. Acrosomal contents were fractionated by Superose 6 FPLC. Each fraction was incubated with 100 μMof fluorogenic substrates, Suc-Leu-Leu-Val-Tyr-MCA (for chymotrypsin-like activity), Boc-Leu-Arg-Arg-MCA (for trypsin-like activity), and Z-Leu-Leu- Glu-MCA (for caspase-like activity). The activity was determined by measuring the fluorescence intensity of AMC. The chymotrypsin-like and trypsin-like activities afforded three peaks, while the caspase-like activity gave two peaks. The elution positions of two molecular size markers (porcine thyroglobulin (669 kDa) and bovine serum albumin (66 kDa)) were indicated in the upper panel. Note that fractions 8 and 12 correspond to the elution positions of the 26S and 20S proteasomes, respectively, both of which possess chymotrypsin-like, trypsin-like, and caspase-like activities.

subjected to Superose 6 gel filtration to separate any such proteases. It is thought that the 26S proteasome (about 2500 kDa) and the 20S proteasome (700 kDa) are fractionated into fractions 8 and 12, respectively, under our experimental conditions (for the elution positions of the 26S and 20S proteasomes, see Sawada et al., 1993). As expected, three peptidase activities toward Suc-Leu-Leu-Val-Tyr-MCA, Boc-Leu-Arg- Arg-MCA and Z-Leu-Leu-Glu-MCA, residing on the 26S and 20S proteasome, were eluted in fractions 8 and 12 (see Fig. 8). In addition to the proteasome activities, chymotrypsin-like and trypsin-like activities were detected in fraction 16. These activities with a molecular mass of about 70 kDa are thought to be distinct from the proteasome on the basis of molecular size and enzymatic properties (Fig. 8). In order to exclude the possibility that the 70 kDa chymotrypsin-like protease is also involved in sea urchin fertilization, we compared the effects of Fig. 7. Acrosomal contents have proteasomal activity. Acrosomal contents were MG-132 on the chymotrypsin-like activities in fractions 12 and incubated with 100 μM of fluorogenic substrates in the presence or absence of 16. The chymotrypsin-like activity in the proteasome-contain- μ 100 M inhibitors for 12 h. The activity was determined by measuring fluo- ing fraction 12 was strongly inhibited by 100 μM MG-132, rescence intensity of AMC. Three kinds of proteasome activity were observed in the acrosomal contents. All the activities were inhibited by MG-132. Similar while the activity in the 70 kDa protease-containing fraction 16 results were also obtained by using two preparations of acrosomal contents was scarcely inhibited by MG-132 at the same concentration obtained from different individuals. (Table 2). These results indicate that the protease in acrosomal 228 N. Yokota, H. Sawada / Developmental Biology 308 (2007) 222–231

Table 2 Effects of MG-132 on chymotrypsin-like activities in acrosomal contents Inhibitor Chymotrypsin-like activity in Superose 6 fractions (% activity) Fraction 12 (20S proteasome) Fraction 16 (70 kDa protease) None 100 100 MG-132 30 87 Chymotrypsin-like activities in fractions 12 (20S proteasome) and 16 (70 kDa protease), which were separated from the acrosomal contents by Superose 6 gel filtration, were assayed in the presence and absence of 100 μM MG-132 by using Suc-Leu-Leu-Val-Tyr-MCA as a substrate. contents, which is involved in fertilization, is the proteasome rather than the 70 kDa chymotrypsin-like protease.

The vitelline envelope of unfertilized eggs is ubiquitinated

It is generally believed that the 26S proteasome degrades ubiquitinated proteins in an ATP-dependent fashion under physiological conditions, although several exceptional cases such as ornithine decarboxylase and CDK inhibitor p21CIP, both of Fig. 9. The vitelline envelope of unfertilized eggs is ubiquitinated. (A) Vitelline which are degraded by the proteasome in a ubiquitin-inde- envelopes were isolated from unfertilized eggs and subjected to SDS-PAGE pendent manner (Mayer et al., 2004), are known. When the (7.5% gel), followed by Western blotting with an FK2 monoclonal antibody, sperm proteasome functions as a lysin, it is assumed that the which reacts to mono- and multi-ubiquitinated proteins but not to free ubiquitin. vitelline envelope of the egg may be ubiquitinated before or (B and C) Immunocytochemistry of unfertilized eggs with FK2 monoclonal after sperm binding to the egg vitelline layer. In the ascidian, antibody (B) or control mouse IgG (C). H. roretzi, the vitelline coat of the egg appears to be ubiquitinated by a ubiquitin-conjugating enzyme complex released ubiquitinated vitelline-coat proteins by the sperm proteasome. from sperm during sperm reaction (Sawada et al., 2002a; Sakai In order to assess this issue, we investigated the effect on fertil- et al., 2003). In contrast, it is reported that the zona pellucida of ization of the addition of apyrase to deplete the extracellular the porcine ovum is ubiquitinated before contact with acrosome- ATP in the seawater surrounding the eggs. As shown in Fig. 10, reacted sperm (Sutovsky et al., 2004). To investigate whether the apyrase inhibited sea urchin fertilization in a dose-dependent sea urchin vitelline envelope is ubiquitinated, we carried out manner (Fig. 10), whereas BSA showed no inhibition even at Western blotting and indirect immunofluorescence microscopy. 1.0 mg/ml. Using heat-denatured apyrase drastically diminished To avoid possible contamination of cytoplasmic ubiquitinated its inhibitory ability toward fertilization (data not shown). These protein, the purified preparation of the vitelline envelope (Correa results indicate that extracellular ATP is required for fertilization and Carroll, 1997; Epel et al., 1970) was used for SDS-PAGE in the sea urchin, as it is in the case of the ascidian H. roretzi. analysis and Western blotting. As shown in Fig. 9A, smear bands with molecular masses more than 50 kDa were detected by the monoclonal antibody FK2, which is specific to the mono- and multi-ubiquitinated proteins but not free ubiquitin (Fujimuro et al., 1994). No reacted band was observed in a control experiment without using the primary antibody (data not shown). Immuno- cytochemistry of unfertilized eggs was also carried out using the FK2 monoclonal antibody. FK2-reactive proteins were mainly detected on the outer surface of the vitelline layer (Figs. 9B, C). Similar results were obtained by confocal fluorescence microscopy: only the vitelline layer was stained by fluorescence- labeled FK2 antibody (data not shown). These data suggest that the vitelline envelopes of sea urchin eggs are ubiquitinated prior to fertilization.

Extracellular ATP is required for sea urchin fertilization Fig. 10. Extracellular ATP is required for sea urchin fertilization. Sperm was mixed with various concentrations of apyrase (final volume, 100 μl), which deplete the extracellular ATP, followed by addition of a small volume (10 μl) of Extracellular ATP is necessary for fertilization in the asci- intact egg suspension. Fertilization ratio was determined as described in dian, H. roretzi (Sakai et al., 2003). In the case of sea urchins, Materials and methods. BSA was used as a control. Data represent mean±S.D. extracellular ATP may also be required for degradation of the in three experiments. N. Yokota, H. Sawada / Developmental Biology 308 (2007) 222–231 229

Although we cannot rule out the possibility that extracellular coat lysin, since the purified enzyme has a vitelline-envelope ATP is necessary for several fertilization processes other than lytic activity. However, since the activity is potently inhibited proteasome-mediated degradation, the present results support by low concentrations (less than 100 μM) of chymostatin and our hypothesis that the ubiquitinated vitelline-coat proteins may STI, it seems unlikely that such a chymostatin-sensitive be degraded in an ATP-dependent fashion by the sperm 26S protease is indispensable for sperm penetration through the proteasome, which is exposed and partially released during vitelline envelope in sea urchins under physiological conditions. acrosome reaction. Our results support the earlier observation that the proteasome may be involved in the acrosome reaction of sea urchin Discussion sperm (Matsumura and Aketa, 1989, 1991). In connection with this, it is interesting to note that PKA appears to be involved in It has been reported that the proteasome occurs in sea urchin the acrosome reaction of sea urchins (Su et al., 2005). Taking sperm (Saitoh et al., 1991; Inaba et al., 1992) and that the sperm into account that the regulatory subunits of PKA are known to proteasome may be involved in the acrosome reaction (Matsu- be degraded by the proteasome (Hegde et al., 1993), it is rea- mura and Aketa, 1989, 1991). However, the effects of pro- sonable to speculate that the sperm proteasome may play a role teasome-specific inhibitors on several fertilization processes in the degradation of the regulatory subunits of PKA resulting in have not been systematically investigated. In the present study, irreversible activation of PKA during the acrosome reaction of we have demonstrated that the proteasome is involved in sea sea urchins. In connection with a membrane fusion event, it is urchin fertilization at specific steps of the acrosome reaction and reported that the proteasome is involved in yeast vacuole penetration through the vitelline envelope by using proteasome- membrane fusion (Kleijnen et al., 2007), although the target for specific inhibitors. We also show that the sperm proteasome is the proteasome remains to be elucidated. Further studies are partially released by sea urchin sperm during the acrosome necessary to clarify the physiological substrate(s) for the protea- reaction and plays an extracellular role, most probably as a some during the acrosome reaction, in order to obtain insights lysin, in the degradation of ubiquitinated vitelline-coat proteins, on the signal transduction pathway leading to the acrosome similarly to ascidians (Sawada et al., 1998, 2002a)and reaction. mammals (Sutovsky et al., 2004). It was proposed that STI-sensitive protease is localized on the Our present results show that the sperm proteasome plays a surface of the acrosomal process of sea urchin sperm (Green and crucial role in the fertilization of echinoderms as well as mam- Summers, 1980). Our data also suggest that a ∼70 kDa trypsin- mals and protochordates. In addition to the inhibitors, synthetic like and also chymotrypsin-like proteases are partially released peptide substrates for the proteasome also inhibited fertilization. during acrosomal exocytosis (see Fig. 8). However, the occur- Z-Leu-Leu-Glu-MCA, a caspase substrate, showed the stron- rence of a certain protease in an acrosome does not necessarily gest inhibition. These results led us to propose the idea that a mean that the enzyme in question is involved in fertilization, caspase-like catalytic site (β1 subunit) (Mayer et al., 2004)of since not only STI but also chymostatin or leupeptin showed no the 20S proteasome may be critically involved in the degra- appreciable inhibition at 100 μM toward fertilization of sea dation of ubiquitinated vitelline-coat proteins. Such an unequal urchins. Although we cannot rule out the possibility that the contribution of three catalytic sites of the proteasome in the 70 kDa or other proteases are involved in some steps during degradation process has been reported (Tanaka et al., 2000; fertilization, it should be emphasized that the proteasome, in Kisselev et al., 2006). In connection with this, it is notable that particular its caspase-like catalytic site, must play a central role caspase-like activity of the proteasome contained in acrosomal in sperm penetration of the vitelline layer. Identification of the contents was potently inhibited by MG-132 but not by chy- ubiquitinated vitelline-coat proteins of sea urchin eggs as well as mostatin or leupeptin at 100 μM(Fig. 7), and that this inhibition examination of whether protostomes also utilize sperm protea- pattern coincided well with the inhibition pattern toward fer- somes as lysins is an intriguing issue remaining to be solved. tilization (Table 1). These results also support the idea that caspase-like catalytic center of the proteasome must play a Acknowledgments pivotal role in sea urchin fertilization. It is proposed that a chymotrypsin-like protease is a lysin in This study was supported in part by Grants-in-Aid for sea urchin fertilization, since two chymotrypsin inhibitors (chy- Scientific Research (B) (No. 18390022, 15390023) and for mostatin and tosyl-phenylalanyl-chloromethane) and p-nitro- Priority Areas (No. 17028024, 19044019) to HS from MEXT, phenyl-p′-guanidino-benzoate, but not the other protease Japan. We are grateful to Charles and Gretchen Lambert for inhibitors, including leupeptin, inhibited fertilization of the critical reading of this manuscript and valuable comments. We sea urchins Hemicentrotus pulcherrimus and Strongylocentro- also thank Mr. Masahiko Sunagawa for collection and culti- tus intermedius at concentrations higher than 200 μM(Hoshi et vation of sea urchins. al., 1979). Our data are not at variance with their results, since our preliminary data showed that chymostatin at concentrations References higher than 200 μM showed a significant inhibition toward fertilization (data not shown). A 19-kDa chymotrypsin-like Baba, T., Azuma, S., Kashiwabara, S., Toyoda, Y., 1994. Sperm from mice protease has been purified from sea urchin sperm (Yamada et carrying a targeted mutation of the acrosin gene can penetrate the oocyte al., 1982). It is proposed that this protease may be a vitelline- zona pellucida and effect fertilization. J. Biol. Chem. 269, 31845–31849. 230 N. Yokota, H. Sawada / Developmental Biology 308 (2007) 222–231

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