Haprin, a Novel Haploid Germ Cell-specific RING Finger Protein Involved in the Acrosome Reaction*

The acrosome reaction (i.e. the exocytosis of the sperm vesicle) is a prerequisite for fertilization, but its molecular mechanism is largely unknown. We have identified a cDNA clone for a gene named haprin, which encodes a haploid germ cell-specific RING finger protein. This protein is a novel member of the RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family that has roles in several cellular processes, such as exocytosis. It is transcribed exclusively in testicular germ cells after meiotic division. Western blot and immunohistochemical analyses showed the molecular weight of Haprin protein to be Mr ∼82,000. It was localized in the acrosomal region of elongated spermatids and mature sperm and was not present in acrosome-reacted sperm. The specific antibody against the RING finger domain of Haprin inhibited the acrosome reaction in permeabilized sperm. These results indicated that the novel RBCC protein Haprin plays a key role in the acrosome reaction and fertilization.

The acrosome reaction is the sperm exocytotic event that is required for mammalian fertilization. An abnormal acrosome reaction could result in male infertility (1,2). The acrosome, a large Golgi-derived vesicle located in the anterior head of the sperm, contains enzymes to digest the zona pellucida coating around the egg (3). The acrosome membrane consists of the outer acrosomal membrane, which underlies the plasma membrane, and the inner acrosomal membrane, which overlies the nucleus. The acrosome reaction involves multiple fusions between the outer acrosomal membrane and the plasma membrane, and, consequently, the inner acrosomal membrane is exposed and able to bind the plasma membrane of the egg (4). As seen in other regulated exocytosis systems, the acrosome reaction is triggered by external signal transduction and an observed increase in Ca 2ϩ levels (4). In addition, recent studies have shown that the molecular machinery components of membrane fusion, which were initially identified in synaptic exocytosis, also exist in sperm (5,6). However, the molecular basis of the acrosome-specific mechanism remains largely unknown.
In testis, the acrosome develops during spermiogenesis, which is the process of morphological change in postmeiotic haploid germ cells for the production of sperm. Previously, we isolated cDNA clones that are specifically expressed in mouse haploid germ cells from the subtracted cDNA library (7). Many of these haploid germ cell-specific genes are thought to be involved in the differentiation process of spermiogenesis and/or in sperm function (7)(8)(9)(10)(11)(12)(13)(14)(15). We report here the characterization of a novel RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family gene named haprin (from haploid germ cell-specific RBCC protein), which was cloned from a subtracted cDNA library. Several studies have shown that the RBCC motif family is required in protein-protein interactions and that it is implicated in several cellular processes (16). Haprin protein is expressed specifically in testis and is localized in the acrosomal region of testicular germ cells and mature sperm. In order to investigate the role of Haprin in the acrosome reaction, streptolysin O (SLO) 1 was used to permeabilize sperm (17,18). SLO is a bacterial toxin that binds cholesterol in the plasma membrane, aggregates, and forms stable, relativity large pores, while leaving the inner organelle membranes intact (19). The acrosome reaction of permeabilized sperm is induced by Ca 2ϩ and can be stimulated or inhibited by the addition of specific peptides or antibodies prior to the Ca 2ϩ addition (17). In this study, an anti-Haprin antibody specific to the N-terminal RING finger motif inhibited the acrosome reaction, but an antibody to the C-terminal region did not, suggesting that Haprin exists outside of the acrosome in the sperm cytoplasm and has an important role in the acrosome reaction at RING finger motifs through an association with itself or with other molecules.

EXPERIMENTAL PROCEDURES
Construction of the Subtracted cDNA Library, Screening, and DNA Sequencing-A haploid germ cell-specific cDNA library in the pAP3neo vector (Takara, Shiga, Japan) was generated by subtracting the mRNA of a 17-day-old testis from an adult (35-day-old) mouse testis cDNA library, as described previously (20). Plasmid DNAs of randomly selected clones from the subtracted cDNA library were screened by Northern blot analysis using testicular RNA from both 17-and 35-day-old mice. We designated the testis-specific cDNA sequences as TISP (transcription increased in spermiogenesis) (7). One of these clones, TISP36, was used as a probe to screen an adult mouse testis cDNA library in order to isolate its complete sequence. An adult mouse testicular pAP3neo cDNA library in Escherichia coli MC1061A cells (20) was diluted to seed a nitrocellulose filter on an LB plate at a final concentration of 1 ϫ 10 5 colony-forming units. After incubation at 37°C, grown colonies were transferred to two nylon replica filters, and the filters were sequentially soaked at room temperature as follows: 5 min in 0.5 M NaOH and 1.5 M NaCl, 5 min in 0.5 M Tris-HCl (pH 7.4) and 1.5 M NaCl, and 5 min in 2ϫ SSC. After baking at 80°C for 2 h, the filters were washed, and bacterial debris was removed. A [␣-32 P]dCTP-labeled probe was prepared from a 1.0-kb EcoRI-NotI fragment of the TISP36 cDNA using a BcaBest random primer kit (Takara). Filters were hy-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Northern Blot Analysis-Freshly removed organs of adult mice (C57BL/6 strain) were homogenized in RNAzol B (Tel-Test, Inc., Tokyo, Japan). Germ and other somatic cells of the testes were prepared as described in our previous report (21). Briefly, seminiferous tubules were obtained and placed in 20 mM Hepes buffer containing 0.1% collagenase. The tubule suspension was left standing to sediment tubules. The supernatant containing separated cells was used as the Leydig cell fraction. The remaining tubules were cut into small fragments in PBS, and the suspension was again left standing. The supernatant was used as the germ cell fraction, and sedimented tubules containing mainly Sertoli cells were used as the Sertoli cell fraction. Total RNAs were extracted according to the manufacturer's recommendations (Tel-Test) and quantified by optical density measurements. RNA samples containing 2.2 M formaldehyde were subjected to electrophoresis on a 1.1% agarose gel containing 0.66 M formaldehyde (22). RNAs were transferred to a nitrocellulose filter in 20ϫ SSC. Hybridization was performed with 32 P-labeled cDNA prepared with the BcaBest random primer kit at 42°C for 16 h in a solution containing 4ϫ SSC, 5ϫ Denhardt's solution, 0.2% SDS, 12 g/ml denatured salmon sperm DNA, and 50% formamide. Filters were washed twice in 0.1ϫ SSC and 0.1% SDS at 60°C. Band signals were detected with a Fuji image analyzer (Fuji Film, Tokyo, Japan).
Antibodies-Two synthetic peptides (for the N-terminal region of Haprin, INRPGWKRNSLTPR at residues 96 -109; and for the C-terminal region, QLEEAITAKYLEYEEDV at residues 713-729) were designed from the deduced amino acid sequence of Haprin and were purified by HPLC for the immunization of Japanese white rabbits (MBL, Nagoya, Japan). Polyclonal antisera were obtained by injection of each peptide conjugated to keyhole limpet hemocyanin, followed by booster injections at 3-week intervals. Both anti-Haprin antisera rec-ognized a common band of the same molecular weight M r ϭ 82,000 when examined by Western blot analysis. The antiserum raised to the N-terminal region of Haprin gave a clearer result and was primarily used in the following experiments.
Immunohistochemical Analysis-Testes were immersed in O.T.C. compound embedding medium (Tissue-Tek, Sakura, Tokyo, Japan) and frozen at Ϫ20°C. Sections of 10-m thickness were prepared using a cryomicrotome (HM 500 OM) (Microm, Walldorf, Germany) and were fixed with 70% methanol at Ϫ20°C for 5 min. Samples were permeabilized in PBS containing 0.1% Triton X 100 for 5 min. Each section was incubated with 10% normal goat serum in PBS for 1 h at room temperature. After a wash, sections were reacted with anti-Haprin rabbit antiserum diluted at a ratio of 1:300 and then reacted with anti-rabbit Ig goat serum conjugated with fluorescein isothiocyanate (FITC) (Amersham Biosciences) diluted at 1:500 in PBS. Sections were counterstained with DAPI (Nacalai Tesque). The slides were washed and examined under a fluorescent microscope.
Mature sperm were taken from the cauda epididymis and vas deferens and were spotted onto Micro Slide Glass (Matsunami Glass, Kishiwada, Japan). Sperm samples were fixed with 70% methanol at Ϫ20°C for 5 min and reacted with anti-Haprin antiserum (1:300) after blocking with 10% normal goat and donkey sera in PBS. The samples were then reacted with anti-rabbit Ig antiserum conjugated with FITC or Texas Red (Amersham Biosciences). The slides were counterstained with DAPI and/or 20 g/ml FITC-conjugated peanut agglutinin (FITC-PNA) (Sigma) and examined under a fluorescent microscope.
Sperm Induction of the Acrosome Reaction-Mature sperm from the cauda epididymis and vas deferens were incubated in TYH medium (119 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.0 mM MgSO 4 , 25 mM NaHCO 3 , 5.6 mM glucose, 0.5 mM sodium pyruvate, and 4 mg/ml bovine serum albumin) (23) at 37°C in a humidified incubator with 5% CO 2 , 95% air to allow capacitation. After 15 min, highly motile sperm were taken from the upper part of the medium, and the calcium ionophore A23187 (Sigma) was added at final concentration of 10 M to induce the acrosome reaction. After an additional 15-min incubation, sperm were spotted onto microscope glass and examined using immu- nofluorescence. The acrosome status was evaluated by staining with FITC-PNA, which binds the outer acrosomal membrane and therefore would not stain acrosome-reacted sperm (24).
Subcellular Fractionation of Sperm-Subcellular fractionation of sperm was performed according to the previously described method (25,26) with minor modifications. All buffers contained a protease inhibitor mixture (Sigma). Sperm from cauda epididymis were suspended in ice-cold TN buffer (20 mM Tris-HCl, pH 7.0, 130 mM NaCl) and sonicated briefly for 15 s on ice. Cell lysate was centrifuged at 10,000 ϫ g for 10 min. The supernatant was further ultracentrifuged at 100,000 ϫ g for 2 h at 4°C using SW41 rotor (Beckman). The precipitant was used as the membrane fraction. The proteins in the supernatant were precipitated by addition of trichloroacetic acid at a final concentration of 10% and then washed with acetone and air-dried. Lectin blotting with biotinylated PNA (Vector Laboratory, Burlingame, CA) was performed to confirm the membrane fractionation (25). The membrane fraction was extracted with 4% Triton X-100, 1.5 M NaCl, 2 M urea, or 100 mM Na 2 CO 3 (pH 11.5) as described elsewhere (26,27). Proteins released by the calcium ionophore, A23187-induced acrosome reaction were also separated into the membrane and soluble fractions. The samples were solubilized in SDS sample buffer and subjected to Western blot analysis.
Sperm Permeabilization with SLO and the Acrosome Reaction Assay-Sperm were incubated in TYH medium under capacitation conditions for 30 min. Highly motile sperm were recovered, washed, and resuspended in PBS, and then SLO permeabilization was performed as described by Diaz et al. (17). After washing with PBS, permeabilized sperm were resuspended in ice-cold sucrose buffer (20 mM HEPES-K, pH 7, 250 mM sucrose, 0.5 mM EGTA, 2 mM dithiothreitol, 1.5 mM MgCl 2 , and 50 mM KCl) and incubated at 37°C. After 15 min, anti-Haprin antibody or preimmune serum was added, and the incubation was continued for 15 min. CaCl 2 was added at a final concentration of 500 M to stimulate the acrosome reaction. Both negative (only perme-abilization; no stimulant and no antisera) and positive (calcium addition in permeabilized sperm) controls were included in all experiments. Sperm were spotted onto slides and fixed in methanol at Ϫ20°C for 5 min. The acrosome status was evaluated by staining with FITC-PNA to identify acrosome-reacted sperm (24). More than 200 sperm were counted in each experimental condition, and data were reported as the percentage of acrosome-reacted sperm after subtraction of the negative control (the number of sperm that reacted solely due to SLO treatment, ϳ36%).
Statistical Analysis-Differences between experimental and control conditions were tested by one-way analysis of variance and Fisher's protected least significant difference tests. Percentages were transformed to the arc-sin before analysis. Significant differences (p Ͻ 0.05) are discussed here.

RESULTS
Cloning and Sequencing of Haprin cDNA-From a subtracted cDNA library of mouse testis, we isolated a new haploid germ cell-specific cDNA clone of 851 bp, designated TISP36 (DDBJ accession number AB046301; Fig. 1) (7). Because this clone did not have a full-length open reading frame, as judged by the size of the band obtained from Northern blot analysis (3.0 kb), we rescreened an adult mouse testicular cDNA library in pAP3neo using TISP36 as a probe (2.2 kb). We also performed cap site hunting to determine the first methionine codon of the cDNA (Fig. 1). We identified an open reading frame consisting of 2324 nucleotides, which encoded 729 deduced amino acids (DDBJ accession number AB103063; Figs. 1 and 2). A stop codon was present 24 bp upstream from the first ATG (data not shown). The deduced amino acid sequence of the cDNA contains a RING finger domain followed by two B-box type zinc-binding domains and an ␣-helical coiled-coil domain, which is known as a RBCC tripartite motif. In the C-terminal region, fibronectin type III (28) and SPRY (Spla kinase and ryanodine receptor) (29) domains were present (Fig. 2, A and  B). We named this novel gene haprin.
Messenger RNA Expression of Haprin in Mouse Organs-Northern blot analysis revealed that haprin expressed specifically in the testis as a single transcript of 3.0 kb but was not detectable in any of the other organs observed, including brain, heart, intestine, kidney, liver, lung, muscle, ovary, and spleen (Fig. 3A). In testis, the transcript was detected in the germ cell fraction and was not present in the somatic cell fractions (e.g. Sertoli or Leydig cells) (Fig. 3B). To identify the developmental expression pattern of haprin, prepubertal mouse testes were examined. The transcript was not found in testis until the fourth week postpartum (29 days), when elongated spermatids appeared, and it then increased with age (Fig. 3C).
The Expression of Haprin Protein Observed by Western Blot and Immunofluorescence Analysis-Western blot analysis with anti-Haprin rabbit antiserum against the N-terminal region (Fig. 2B) showed a single band with a molecular mass of 82 kDa exclusively in testis extracts (Fig. 4A); no signal was detected with preimmune rabbit serum (data not shown). The positive signal of the Western blot analysis in testis was first detected at the age of 4 weeks (Fig. 4B), which is consistent with the Northern blot analysis. These results indicated that the expression of Haprin protein was precisely regulated in haploid male germ cell development.
Immunofluorescence analysis of Haprin in adult mouse testis showed that the positive signal was detected predominantly in elongated spermatids, at the late steps (steps 9 -16) of haploid germ cell development (Fig. 5, C-F); this was consistent with the Northern blot and Western blot analyses. The subcellular localization of Haprin was restricted to the cytoplasmic, acrosomal region (Fig. 5F). No signal was detected in immature germ cells, such as spermatogonia and spermatocytes, or in somatic cells, such as Sertoli and Leydig cells.
Haprin Protein in Intact and Acrosome-reacted Sperm-In sperm, the localization of Haprin protein was limited to the cytoplasmic, acrosomal region (Fig. 6). When the acrosome reaction was induced by a calcium ionophore, Haprin was no longer detectable; the only sperm with Haprin protein in the cytoplasmic, acrosomal region were the remaining few that retained an acrosome and PNA binding (Fig. 6). Western blot analysis of the Haprin protein in sperm from the epididymis and vas deferens showed a band of the same size (82 kDa) in the testis (Fig. 7A). After subcellular fractionation, this band was also found in both the membrane and soluble fractions, containing plasma and acrosomal membranes and containing cytosolic and acrosomal components, respectively (Fig. 7A). From the membrane fraction, Haprin was partly extracted with 2 M urea, 1.5 M NaCl, or pH 11.5 conditions to the soluble fraction but not with 4% Triton X-100 (Fig. 7B). After the  acrosome reaction, Haprin was found in the soluble fraction of the supernatant of the acrosome-reacted sperm (Fig. 7C), indicating that the Haprin protein is released after the acrosome reaction.
The Biological Role of Haprin Protein in the Acrosome Reaction-To elucidate the function of Haprin in sperm, we studied the effect of antibody on the acrosome reaction. After permeabilization with SLO (17,18), anti-Haprin antibody was introduced into the sperm to investigate the effect on the acrosome reaction induced by Ca 2ϩ . The status of the acrosome was evaluated with FITC-PNA staining (24). Incubation with the antibody against the N-terminal region of Haprin protein, which contains the RING finger motif, strongly inhibited the acrosome reaction, as determined by the significant number of acrosome-intact sperm remaining after the induction with Ca 2ϩ . In contrast, the antibody raised against the C-terminal region of Haprin protein (residues 713-729) did not exhibit an effect on the acrosome reaction in permeabilized sperm (Fig. 8). These results indicated that the Haprin protein plays an important role in the acrosome reaction by association with itself and/or with other protein(s) at the RING finger region. DISCUSSION In this study, we identified a novel RBCC motif protein termed Haprin. The full-length cDNA of the haprin gene was isolated from a testicular cDNA library in pAP3neo using the probe TISP36 (7), which was cloned from a subtracted cDNA library of the mouse testis condensed with the cDNAs specifically expressed in haploid spermatids (30). The expression of  the haprin gene was detected specifically in haploid testicular germ cells and was precisely regulated during the development of male germ cells. Immunofluorescence studies of the mouse testis showed that the Haprin signal is localized to the acrosomal region of elongated spermatid. Although acrosome formation begins in the early steps of round spermatids (3), haprin gene expression starts at the late steps of spermatid differentiation, and some potential acrosome reaction modulators, such as rab3A, may be produced at the late steps of differentiation (31). Haprin protein is also observed in the cytoplasmic, acrosomal region of mature sperm (Figs. 6 and 7). No positive immunostaining signal was detected in intact sperm fixed on a slide glass (data not shown), but one was demonstrated in detergent-permeabilized samples, suggesting that Haprin was not on the cell surface of the sperm head. Western blot analysis showed that the Haprin protein is localized both to the soluble fraction enriched by cytosolic and acrosomal components and to the membrane fraction enriched by plasma and acrosomal membrane. The membrane-associated Haprin protein could further be extracted by urea, high salt, or alkali pH and easily released to the soluble fraction (Fig. 7), indicating that this protein is peripherally associated with membranes (26). The resistance to extraction by Triton X-100 suggests that this protein is associated with cytoskeleton (26). It has been demonstrated that an actin network is located between the plasma and outer acrosomal membrane and plays an important role in the acrosome reaction (32). Our results indicate that the Haprin protein is in the cytosolic and cytoskeletal compartment of the sperm head and would act as a regulatory element for the acrosomal exocytosis process (Fig. 9).
The anti-Haprin antibodies were able to pass through the pores made by SLO in the sperm plasma membrane, whereas the acrosome itself and the outer acrosomal membrane remained intact (17,18). The antibody against the N-terminal region, but not the antibody against the C-terminal region of Haprin, inhibited the acrosome reaction of sperm (Fig. 8). Thus, the inhibition of Haprin by the antibody was specific to the epitope of this antibody within the RING finger domain of Haprin's RBCC motif. The RBCC motif is believed to provide an essential scaffold for homo/hetero-oligomerization by interacting with other proteins in order to form the regulatory complexes required for cellular processes (16,33). Thus, the antibodies against the RING finger domain of Haprin may have interfered with Haprin homo-oligomerization and/or interactions with other proteins required for the acrosome reaction (Fig. 9).
It is important to identify the protein(s) with which Haprin interacts to further characterize the physiological function of Haprin in spermatogenesis. The Haprin protein contains a RING finger type zinc finger domain, two B-box type zinc finger domains, and an ␣-helical coiled-coil domain, which are the characteristic features of the RBCC motif family of proteins. The RBCC family, or TRIM (tripartite motif) family (34), of proteins contains a large number of proteins that have been implicated in many cellular processes, including transcription, signal transduction, vesicle transport (16,33,35), and vesicle exocytosis. Specifically, ADP-ribosylation factor domain protein 1 is localized to lysosomes and the Golgi apparatus and is involved in the regulation of vesicle transport (36); brain-expressed RING finger protein interacts with myosin-V to mediate vesicle transport (35); and Spring protein regulates synaptic exocytosis via interactions with SNAP-25 (37). The C-terminal region varies among RBCC family members, and shared C-terminal domains may serve common functions (38,39). The functional roles of the fibronectin type III and SPRY domains of Haprin are not yet clear but may associated with the cytoskeleton.
Over the past few years, several studies have reported that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex components and their regulatory proteins exist in the acrosomal region of mature sperm of humans and mice (31, 40 -43). The SNARE complex is the general model of intracellular membrane fusion machinery, which was originally demonstrated in synaptic exocytosis. Proper vesicle-plasma membrane fusion requires a specific complex formation between the vesicle-associated and plasma membrane-associated SNAREs. Considering the functions of other RBCC motif proteins, Haprin may have a role in the formation of a SNARE complex in sperm. In synaptic exocytosis, Spring interacts with SNAP-25, a component of the SNARE complex (37). Because the fibronectin type III and SPRY domains in the C terminus of Haprin are also present in Spring protein (37), the role of Haprin in acrosomal exocytosis may be similar to that of Spring in synaptic exocytosis. However, SNAP- 25 has not yet been observed in the mouse sperm.
In summary, we have identified a novel RBCC motif protein, Haprin, which is exclusively expressed in testis during late spermiogenesis and plays an important role in the acrosome reaction. This is the first report of the localization and functional analysis of the testis-specific RBCC motif protein, Haprin. Further studies are in progress to identify and characterize proteins interacting with Haprin and to knock out the haprin gene.