Journal of Reproduction and Development, Vol. 50, No. 5, 2004

—Original—

Synthesis, Processing, and Subcellular Localization of Mouse ADAM3 during Spermatogenesis and Epididymal Transport

Ekyune KIM1), Hitoshi NISHIMURA1)#, Shigeki IWASE1), Kazuo YAMAGATA1), Shin-ichi KASHIWABARA1) and Tadashi BABA1)

1)Graduate School of Life and Environmental Sciences and Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan #Present: Section of Molecular and Cellular Biology, University of California Davis, Davis, CA95616, USA

Abstract. To elucidate synthesis, processing, and subcellular localization of mouse ADAM3 (cyritestin) during spermatogenesis and epididymal sperm transport, we carried out immunoblotting and immunohistochemical analysis of testicular germ cells, and epididymal and vas deferens sperm, using affinity-purified anti-ADAM3 antibody. ADAM3 was initially synthesized as a 110-kDa precursor in round spermatids, and the precursor was then processed into a 42-kDa mature during the sperm transport into and/or once in the epididymis. The mature ADAM3 was localized on the anterior part of capacitated sperm heads and was rapidly removed from the head region during the calcium ionophore A23187-induced acrosome reaction. These results demonstrate that the mature form of ADAM3 is involved in the binding of sperm to the egg , not in the membrane fusion between sperm and egg. Key words: ADAM3, Spermatogenesis, Epididymis, Acrosome, Fertilization (J. Reprod. Dev. 50: 571–578, 2004)

family of ADAM transmembranous domain [5–7]. Although many ADAM containing a disintegrin and metalloprotease members are predominantly or exclusively domain has been implicated in diverse expressed in the testis, their roles in physiological processes such as fertilization, spermatogenesis and fertilization still remain to be neurogenesis, myogenesis, cancer, and elucidated. inflammation [1–3]. The members of the ADAM Several ADAM members including ADAM1 family have multi-functional domains consisting of (fertilin α), ADAM2 (fertilin β), and ADAM3 pro-, metalloprotease, disintegrin, cysteine-rich, (cyritestin) have been shown to play a key role(s) in epidermal growth factor-like, transmembrane, and fertilization. Fertilin is a heterodimeric protein cytoplasmic tail domains. The metalloprotease complex of ADAM1 and ADAM2 present on the domain exhibits a “sheddase” activity toward the sperm surface [8–10]. In the mouse, two different ectodomain of membranous precursor proteins [1, isoforms of ADAM1, termed ADAM1a and 4], while cell-to-cell adhesion is mediated by the ADAM1b, are produced in the testis [11, 12]. ADAM1a is localized within the endoplasmic Accepted for publication: June 11, 2004 Correspondence: T. Baba reticulum of testicular germ cells, whereas (e-mail: [email protected]) epididymal sperm contain only ADAM1b on the 572 KIM et al. cell surface [12]. Male mice lacking ADAM1a [13] Preparation of protein extracts and ADAM2 [14] are sterile because sperm are Testicular germ cells and epididymal sperm from defective in migrating from the into the ICR mice (3 to 5 months old) were suspended in a oviduct, and in binding to the egg zona pellucida. lysis buffer consisting of 20 mM Tris-HCl, pH 7.4, ADAM3-deficient mouse sperm are also incapable 1% Triton X-100, 150 mM NaCl, and 10% of binding to the zona pellucida, although the inhibitor cocktail (Sigma-Aldrich), kept on ice for sperm ascent into the oviduct is normal [15, 16]. 20 min, and centrifuged at 10,000 × g for 10 min at 4 Noteworthy is that the disruption of the functional C, as described previously [12, 13]. Protein ADAM1a, ADAM2, and ADAM3 genes results in concentration in the supernatant solution was the loss or severe reduction of ADAM3 in determined using a Coomassie protein assay epididymal sperm [13, 16]. Thus, ADAM3 may be reagent kit (Pierce). one of the proteins responsible for the binding of sperm to the zona pellucida. Immunoblot analysis Because the interaction of sperm with the zona Proteins were denatured by boiling for 5 min in pellucida is a regional process, it is important to the presence of 1% SDS and 1% 2-mercaptoethanol, know the localization of ADAM3 in sperm. Despite separated by SDS-polyacrylamide gel its importance, the ADAM3 localization is still electrophoresis (PAGE), and transferred onto controversial [17, 18]. This uncertainty may result Immobilon-P membranes (Millipore). After from the nature of the antibodies prepared. Indeed, blocking with 1% skim milk, the blots were a commercially available monoclonal antibody incubated with primary antibody for 2 h, and then against ADAM3 (7C1.2) is restricted to the use only with horseradish peroxidase-conjugated secondary for immunoblot analysis. We have previously antibody for 1 h. Immunoreactive proteins were prepared an affinity-purified anti-ADAM3 detected by an ECL Western blotting detection kit polyclonal antibody applicable to (Amersham Biosciences), as previously described immunoprecipitation analysis [13]. In this study, [23]. we thus re-examined synthesis, processing, and subcellular localization of mouse ADAM3 during Biotinylation of sperm surface proteins spermatogenesis and epididymal sperm transport, Cauda epididymal sperm (2.5 × 107/ml) were using the affinity-purified antibody. kept at room temperature for 30 min in phosphate- buffered saline (PBS) containing 1 mM sulfo-NHS- LC-biotin (Pierce), as previously described [13]. Materials and Methods The biotinylated sperm samples were washed twice with PBS and treated with the above lysis buffer. Materials Proteins were separated by SDS-PAGE under Affinity-purified polyclonal antibodies against reducing conditions followed by immunoblot ADAM1b, ADAM3, sp32, and TPAP were analysis. prepared as described previously [12, 13, 19, 20]. Monoclonal anti-ADAM2 (9D2.2) and anti-ADAM3 Immunostaining (7C1.2) antibodies were purchased from Chemicon. Mouse testicular tissues were snap-frozen and Monoclonal antibody MC101 [21] and rabbit anti- embedded in a TissueTek O.C.T compound (Sakura mouse AKAP82 antiserum [22] were generous gifts Finetechnical Co., Tokyo), as previously described from Drs. K. Toshimori and S. B. Moss, [24]. Sections (8 µm) were prepared by a Leica respectively. Horseradish peroxidase-conjugated CM3000 cryostat, mounted on silanized glass goat antibodies against mouse IgG (H + L) and slides, air-dried, and fixed in PBS containing 4% rabbit IgG (H + L) were purchased from Jackson paraformaldehyde at 4 C for 30 min. Slides were Immunoresearch Laboratories. ICR male mice then washed with PBS, blocked with 2% normal were obtained from Japan SLC Inc., Shizuoka. All goat serum, and incubated with affinity-purified animal experiments were carried out according to anti-ADAM3 antibody in PBS containing 0.05% the Guide for the Care and Use of Laboratory bovine serum albumin at 4 C overnight. After Animals in University of Tsukuba. washing with PBS, the slides were treated with 0.3% hydrogen peroxide to remove endogenous LOCALIZATION OF ADAM3 573 peroxidase activity, washed with PBS, and 2.5 ml each of 2.20 and 2.05 M sucrose, and incubated with horseradish peroxidase-conjugated ultracentrifuged at 100,000 × g for 16 h at 4 C. ARH goat anti-rabbit IgG. The slides were stained with and ART fractions corresponding to the heads and 3,3’-diaminobenzidine as a chromogen, tails of acrosome-reacted sperm were recovered counterstained with 2.5% methyl green, and from the bottom of the centrifuge tube and the 2.05 viewed under an Olympus BX50 microscope. For M sucrose layer, respectively, and were indirect immunofluorescence microscopy, sperm precipitated by centrifugation at 100,000 × g. The were capacitated in modified Krebs-Ringer purities of these two fractions were microscopically bicarbonate solution (TYH medium) containing more than 95%. glucose, sodium pyruvate, bovine serum albumin, and antibiotics [25] under 5% CO2 in air at 37 C for 2 h, put on slides, and fixed in PBS containing 4% Results and Discussion paraformaldehyde at room temperature for 15 min. The slides were then washed with PBS, blocked To examine synthesis and processing profiles of with PBS containing 3% normal goat serum and ADAM3 during spermatogenesis and epididymal 0.05% Tween-20, and incubated with primary sperm transport, we carried out immunoblot antibody at 4 C overnight. The sperm samples analysis of proteins from purified populations of were washed, and incubated with goat anti-mouse testicular germ cells, and from epididymal and vas and anti-rabbit IgGs labeled with Alexa Fluor 488 deferens sperm, using affinity-purified anti- and 568 (Molecular Probes, Eugene, OR), ADAM3 antibody (Fig. 1A). ADAM3 was initially respectively, as previously described [26]. After synthesized as a 110-kDa precursor in postmeiotic washing with PBS containing 0.05% Tween-20, the round spermatids. No processing of the precursor slides were observed under an Olympus IX-70 occurred in elongating spermatids. Sperm from fluoromicroscope. caput, corpus, and cauda epididymides, and vas deferens contained a 42-kDa protein corresponding Subcellular fractionation of acrosome-reacted to the mature form of ADAM3. Consistent with epididymal sperm previous results reported by Linder et al. [17], Subcellular components of cauda epididymal epididymal sperm lacked the 110-kDa ADAM3 sperm after the acrosome reaction were prepared as precursor (Fig. 1A). However, when monoclonal previously described [24, 27]. The acrosome anti-ADAM3 (7C1.2) antibody was used as a probe, reaction of sperm (5 × 107 sperm/ml) was induced a 50-kDa protein as well as the 42-kDa mature by incubation with 10 µg/ml calcium ionophore ADAM3 was found only in caput and corpus A23187 in TYH medium under 5% CO2 in air at 37 epididymal sperm (data not shown). The 50-kDa C for 60 min. The sperm samples were centrifuged protein may correspond to an intermediate form of at 800 × g for 10 min to remove sperm, and the ADAM3 that is then converted into the mature supernatant solution was ultracentrifuged at protein. Thus, the ADAM3 precursor is processed 100,000× g for 90 min at 4 C. The resulting into the 42-kDa mature protein possibly via the 50- supernatant was used as a source of soluble kDa intermediate during the sperm transport into proteins released by the A23187-induced acrosome and/or once in the epididymis. reaction, including acrosomal components (SPA We next examined whether ADAM3 is localized fraction). The precipitate obtained by on the surface of acrosome-intact sperm (Fig. 1B). ultracentrifugation was washed with PBS, re- Biotinylation of surface proteins on cauda suspended in PBS, and used as a source of epididymal sperm resulted in slow migration of membranous proteins on the plasma and outer ADAM1b, ADAM2, and ADAM3 on SDS-PAGE. acrosomal membranes fused during the acrosome Two acrosomal proteins, a proacrosin-binding reaction (MPA fraction). Acrosome-reacted sperm protein, sp32 [19], and a 155-kDa protein (termed were washed three times with PBS, and sonicated MC101) recognized by monoclonal antibody as previously described [28]. To a 2.5-ml MC101 [21], were not biotinylated. Therefore, these suspension, an equal volume of 1.8 M sucrose was three ADAMs including ADAM3 are localized on added, and the mixture was put onto a the cell surface of cauda epididymal sperm. discontinuous sucrose gradient solution containing Immunohistochemical analysis of testicular 574 KIM et al.

Fig. 1. Synthesis and processing of ADAM3 precursor during spermatogenesis and sperm maturation. (A) Immunoblot analysis of protein extracts from testicular germ cells (TGC) and from epididymal and vas deferens sperm. Proteins in Triton X-100 extracts from pachytene spermatocytes (PS), round spermatids (RS), and elongating spermatids (ES) were separated by SDS-PAGE under reducing conditions, and subjected to immunoblot analysis using affinity- purified anti-ADAM3 antibody. Caput, corpus, and cauda epididymal sperm, and vas deferens sperm (Vas) were also examined. Arrows indicate the precursor and mature forms of ADAM3. (B) Biotinylation of sperm surface proteins. Proteins on the surface of cauda epididymal sperm were untreated (–) or biotinylated (+), separated by SDS-PAGE, and then analyzed by immunoblotting using antibodies against ADAM3, ADAM1b, and sp32, and monoclonal antibodies 9D2.2 (anti-ADAM2 antibody) and MC101.

sections indicated that ADAM3 is localized in that the immunoreactive signal is restricted to the round and elongating spermatids (Fig. 2). No equatorial region of sperm, and the staining pattern significant signal for ADAM3 was detected in is unchanged after the induction of the acrosome spermatogonia, spermatocytes, and Sertoli cells. reaction. In the present study, our affinity-purified These data are consistent with the results obtained anti-ADAM3 antibody gave immunoreactive by immunoblot analysis (Fig. 1A). Monoclonal signals on the anterior part of the head of antibody 7C1.2 commercially available gave no capacitated, acrosome-intact cauda epididymal immunoreactive signal. sperm, including the apical tip of the sperm head The information on the localization of ADAM3 in (panel a in Fig. 3). The localization of ADAM3 on sperm is essential for understanding the functional cauda epididymal sperm was essentially similar to role of ADAM3 in sperm/egg interaction. Linder et those on sperm from caput and corpus al. [17] first reported that an antibody against the epididymides, and vas deferens (data not shown). cytoplasmic tail domain of ADAM3 recognizes this The staining patterns with monoclonal antibody protein in the acrosomal region of acrosome-intact MC101 and 4’-6-diamidino-2-phenylindole (DAPI) sperm, and on the inner acrosomal membrane of revealed the absence of ADAM3 in the acrosome acrosome-reacted sperm. However, indirect and nucleus of acrosome-intact sperm (panels b-d). immunofluorescence analysis using another Unexpectedly, the immunoreactive signals for antibody against the (residues 453–475) ADAM3 rapidly disappeared from the sperm head of ADAM3 prepared by Yuan et al. [18] indicated during the calcium ionophore A23187-induced LOCALIZATION OF ADAM3 575

Fig. 2. Immunohistochemical analysis of testicular sections. Sections of mouse testis were probed by monoclonal antibody 7C1.2 (anti-ADAM3 antibody, see a and c) or affinity-purified anti-ADAM3 antibody (b and d), and observed under a microscope (magnification, × 200 for a and b, and × 400 for c and d). No significant signal was detected when pre-immune serum was used as the probe.

Fig. 3. Indirect immunofluorescent analysis of capacitated epididymal sperm. The acrosome reaction of capacitated cauda epididymal sperm was induced by calcium ionophore A23187 for 60 min. Acrosome-intact (AI) and reacted (AR) sperm were probed by affinity-purified anti-ADAM3 antibody (a and e) and monoclonal antibody MC101 (b and f). Sperm were also stained with 4’-6-diamidino-2-phenylindole, DAPI (c and g). Arrows indicate acrosome-reacting sperm. Note that when affinity-purified anti-testis-specific poly(A) polymerase, TPAP, antibody and Alexa Fluor 568- conjugated secondary antibody were used as controls, no significant staining was observed (not shown). Moreover, only the staining patterns of ADAM3 on cauda epididymal sperm are shown here, because the localization of ADAM3 was similar among capacitated, acrosome-intact sperm from caput, corpus, and cauda epididymides, and vas deferens. 576 KIM et al.

[22], in the flagellum fibrous sheath, were used as controls (Fig. 4). ADAM3 as well as two acrosomal proteins, sp32 and MC101, was found in the SPA (soluble proteins released by the acrosome reaction including acrosomal components) fraction. The localization of ADAM1b and ADAM2 on acrosome-reacted sperm was totally different from that of ADAM3; ADAM1b and ADAM2 were still present in the ARH (acrosome-reacted sperm heads) fraction. These data are consistent with the fact that ADAM3 is removed from the sperm head during the acrosome reaction (Fig. 3). However, it is uncertain at present that the molecular size of the mature form of ADAM3 (42 kDa) remains unchanged (Fig. 4) after the removal from the sperm heads. As noted above, ADAM3-deficient mouse sperm show a severe defect in binding to the egg zona pellucida, despite normal membrane fusion between sperm and egg [15, 16]. Our data Fig. 4. Subcellular localization of ADAM3 in cauda epididymal sperm after the acrosome reaction. presented here strongly support the functional role Subcellular fractions from acrosome-reacted of ADAM3 in the sperm/egg interaction. Notably, epididymal sperm were analyzed by the loss of ADAM3 from the surface of sperm head immunoblotting using antibodies against ADAM3, during the acrosome reaction (Figs. 3 and 4) ADAM1b, sp32, and AKAP82, and monoclonal antibodies 9D2.2 (anti-ADAM2 antibody) and demonstrates the involvement of ADAM3 not in MC101. SPA, soluble proteins released by A23187- the membrane fusion between sperm and egg, but induced acrosome reaction, including acrosomal in the binding of sperm to the egg zona pellucida. components; MPA, membranous proteins on plasma and outer acrosomal membranes fused during the acrosome reaction; ARH, proteins in acrosome- reacted sperm heads; ART, proteins in acrosome- Acknowledgements reacted sperm tails. The contributions of E. K. and H. N. to this study are equal. This work was partly supported by Grant-in-Aids for Scientific Research on Priority acrosome reaction (panels e-h). We occasionally Areas, for Scientific Researches (A) and (B), for observed acrosome-reacting sperm where Exploratory Research, for Young Scientists (B) from acrosomal protein MC101 was being dispersed the Japan Society for the Promotion of Science from the acrosome (arrows in panels f and h). Even (JSPS) and the Ministry of Education, Culture, on the head of acrosome-reacting sperm, most Sports, Science and Technology of Japan (MEXT), ADAM3 had already disappeared. Thus, ADAM3 and by the 21st Century COE Program from MEXT. is localized on the anterior part of the sperm head, S. I. was supported by a Research Fellowship for and is removed during the acrosome reaction. Young Scientists from JSPS. We thank Drs. K. To obtain more definitive data concerning the Toshimori (Chiba University) and S. B. Moss localization of ADAM3, proteins in four subcellular (University of Pennsylvania) for kind gifts of fractions (SPA, MPA, ARH, and ART fractions) of monoclonal antibody MC101 and anti-AKAP82 cauda epididymal sperm after the A23187-induced antibody, respectively, and Drs. M. Okabe (Osaka acrosome reaction were examined by immunoblot University), G. L. Gerton (University of analysis (Fig. 4). Antibodies against sp32, MC101, Pennsylvania), and R. Yanagimachi (University of and a protein kinase A anchor protein, AKAP82 Hawaii) for helpful discussion. LOCALIZATION OF ADAM3 577

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