Molecular Human Reproduction, Vol. 8, No. 6, 531-539,
June 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Molecular cloning and characterization of the human orthologue of male germ cell-specific actin capping protein 
3 (cp3)
1 Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University and 2 Department of Urology, Osaka University Medical School, Osaka, Japan
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Abstract |
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We report here the molecular cloning and characterization of a novel human actin capping protein
3 (cp3) cDNA, an orthologue of the mouse male germ cell-specific cp3, and the organization of the human cp3 genomic structure. The entire coding region of the human cp3 cDNA showed 82.1% similarity with the mouse cp3. The predicted amino acid sequence was 91.3% identical to the mouse protein and the actin-binding motif in the C-terminal region is highly conserved among species. The mRNA of the human cp3 gene was found to be exclusively expressed in the testis. Western blot analysis detected a 33 kDa protein in human testis and sperm. Immunohistochemistry showed that the main localization of human CP3 protein was in the neck region of ejaculated sperm, with moderate and faint signals also detected in the tail and postacrosome region respectively. Furthermore the localization of CP3 coincided with the species-specific distribution of actin in human sperm. The human cp3 gene was mapped to chromosome 12p12 by computer database cloning of human genomic DNA and was proven to be intronless. CP3 may play a physiologically important role in sperm architecture as well as in fertility of the human male.
actin/CP3/Gsg3/sperm/testis
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Introduction |
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Actin is a major cytoskeletal protein involved in several cellular processes, including the maintenance and remodelling of cell shape, cell migration and interaction with the extracellular matrix (Cooper, 1991
; Symons and Mitchison, 1991). In cells, actin is found either in a monomeric (G-actin) or in a polymerized form (F-actin) and actin-regulatory proteins govern the dynamics of these two forms to construct the actin cytoskeleton during physiological events in the cells (Hartwig et al., 1991).
Actin capping protein (CapZ) (Casella et al., 1987) is one of the actin regulatory proteins and the most evolutionarily conserved of all barbed end capping proteins, being expressed in virtually all mammalian cell types (Witke et al., 2001). The binding of CapZ to actin filaments stabilizes the length of the filaments by inhibiting the addition and loss of actin monomers. CapZ binds weakly to actin monomers but clearly nucleates actin polymerization from monomers. The capping activity of CapZ is greatest in the absence of Ca2+ in the intracellular ionic conditions and is reversibly inhibited by phosphatidylinositol 4,5-bisphosphate (Heiss and Cooper, 1991; Schafer et al., 1996), suggesting that CapZ plays dynamic and essential roles in regulating the actin cytoskeleton.
CapZ is a heterodimer composed of CP and CPß which bind very tightly and require each other for actin-binding activity in vitro and stability in vivo (Amatruda et al., 1992; Hug et al., 1992). In humans, two genes have been identified, referred to as CP1 on chromosome 1 and CP2 on chromosome 7 (Hart et al., 1997a). However, only a single gene for the ß subunit has been identified. This gene is on human chromosome 1 and produces two isoforms, ß1 and ß2, by alternative splicing (Schafer et al., 1994). A decrease in CapZ hinders cell motility in Dictyostelium (Hug et al., 1995) and loss of CapZ has near lethal effects in yeast (Amatruda et al., 1990).
Previously, we cloned a new capping protein subunit gene from a subtracted cDNA library of mouse testis; it was named mouse germ cell-specific gene 3 (gsg3) (Tanaka et al., 1994), and later called capping protein 3 (cp3) (Hart et al., 1997a). Genomic analysis has revealed that mouse cp3 (mcp3) is an intronless gene on chromosome 6 (Matsui et al., 1997; Yoshimura et al., 1999). The expression of cp3 is haploid germ cell-specific and CP3 protein expression coincides with the position of the developing acrosome in the rat testis (Hurst et al., 1998). Furthermore, CP3 exhibits dynamic changes of distribution in both the head and tail of bovine sperm during epididymal maturation and the acrosome reaction (Howes et al., 2001). Therefore, it has been suggested that CP3 is one of the actin regulators, which may play a critical role in spermatogenesis as well as sperm function.
We report here the isolation of the human orthologue of cp3 (hcp3), and its genomic organization and protein expression in human testis and sperm. The highly conserved structure of the gene and the species-specific distribution of the protein in mature sperm suggest that CP3 plays an important physiological role in spermatogenesis and in male fertility.
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Materials and methods |
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Cloning of hcp
3 cDNA and PCR cloning of hcp3 genomic DNABefore cloning of the human orthologue of mcp
3 cDNA (DDBJ accession number: D87471) (Tanaka et al., 1994), a computer-assisted homology search of the DNA Data Bank of Japan (DDBJ), GenBank, EMBL, Swiss-Prot and Protein Identification Resource (PIR) data banks was performed. We found a human genomic sequence homologous to the mcp3 in the chromosome 12 working draft sequences of the human genome resources from GenBank (NCBI accession number: NT_028330). Comparison of this human genomic sequence with mcp3 cDNA revealed a putative open reading frame (ORF) of human cp3 (hcp3). To isolate this novel ORF, a primer hCAPA (5'-CAGGAGGCTCAGACCTTGCCAGAC-3') consisting of the 24 nucleotides upstream of the first methionine and a primer hCAPB (5'-TGGCTAAGTGAGAGACATATCTCTAC-3') consisting of 24 nucleotides from the 3' region of the hcp3 genome were made for PCR cloning of the hcp3 gene.
PCR was carried out in a reaction buffer (50 µl) containing 4 µg of human genomic DNA, 0.2 µmol/l primer hCAPA and hCAPB, 2.5 mmol/l each of dGTP, dATP, dCTP and dTTP, 20 mmol/l Tris–HCl (pH 8.0), 100 mmol/l KCl, 2.5 mmol/l MgCl2 and 2.5 IU of LA Taq polymerase (TaKaRa, Shiga, Japan). The Gene Amp PCR System 9700 (PE Applied Biosystems, CA, USA) was used to run PCR samples under the following conditions: one cycle of denaturation at 96°C for 3 min and 35 cycles of successive denaturation at 96°C for 45 s, annealing at 65°C for 45 s and extension at 72°C for 90 s, followed by a final extension at 72°C for 7 min. The PCR product was cleaned by SUPRECTM–PCR (TaKaRa), subcloned into the pT7Blue(R) vector (Novagen, WI, USA) and used as a probe for the screening of the hcp3 cDNA. The probe was radiolabelled with -P32 dCTP using the BcaBest Random Primer DNA Labeling Kit (TaKaRa).
The cDNA of hcp3 was isolated from a human testis cDNA library constructed with the plasmid vector pAP3neo (Tanaka et al., 1997). More than 2x106 E. coli containing recombinant plasmids were screened by hybridization at 65°C with the probe as mentioned above and washed twice in 2x saline sodium citrate (0.15 mol/l NaCl, 0.015 mol/l sodium citrate, pH 7.6) at 60°C for 1 h each. Hybridized colonies were isolated and purified by limiting dilution and rescreening. Four independent positive clones were isolated and the cDNA inserts were subcloned into pBluescript II KS+ (Stratagene, CA, USA) to obtain the complete cDNA sequence.
Human genomic DNA was isolated from human whole blood of healthy male volunteers with DNAZOL BD Reagent (Gibco BRL, OH, USA) according to the manufacturer's instructions. The donors gave permission for their blood to be used for the analysis of genomic DNA in this research.
Dideoxy-chain-termination sequencing reactions were performed with fluorescent dye-labelled primers and thermal cycle sequencing kits purchased from Applied Biosystems (CA, USA). The reaction products were analysed with the ABI-PRISM® 310 Genetic Analyzer (Applied Biosystems).
Northern blot analysis
To examine tissue-specific expression of hcp3, a human multiple tissue Northern blot purchased from Clontech (CA, USA) was hybridized with 0.1 µg of the radiolabelled full-length hcp3 cDNA according to the manufacturer's instructions.
Human tissue and sperm samples
Human testis fragments were obtained with consent from a fertile patient of middle age castrated for the treatment of prostate cancer and stored at –80°C until use.
Human semen was obtained by masturbation after 2–3 days abstinence from fertile male volunteers and placed in phosphate-buffered saline (PBS; 150 mmol/l NaCl, 5 mmol/l KCl, 3.2 mmol/l Na2HPO4, 0.8 mmol/l KH2PO4, pH 7.3). After liquefaction, the semen was gently suspended into PBS to release sperm. After 
10 min, the PBS was centrifuged and the pellet was resuspended in either lysis buffer for Western blot analysis or PBS for immunostaining of sperm.
Preparation of antiserum
Production of antiserum was described in our previous report (Tanaka et al., 1999). Briefly, the full-length ORF of mcp3 cDNA was subcloned into the pGEX-1 vector (Smith and Johnson, 1988). Glutathione S-transferase fusion protein was produced in E. coli by isopropyl-ß-D-thiogalactopyranoside induction and purified with glutathione–agarose beads. Polyclonal antiserum was raised by injection of the above antigens followed by booster injections at 3 week intervals, several times in total, into Japanese White rabbits.
Construction of expression vector of hcp3 and transfection into cultured cells
The expression vector carrying hcp3 was constructed by PCR cloning of amplified hcp3 cDNA into pEGFP-C1 (Clontech). PCR of the full-length hcp3 cDNA coding region was performed using a linker (BglII) oligonucleotide primer for the 5'-region of hcp3 cDNA (5'-GCAGATCTACACTTAGCGTGCTGAGCAG-3') and a linker (KpnI) oligonucleotide primer of the 3'-region (5'-GCGGTACCTTATATTATCCAGTTGCAC-3'). Amplification products were then digested with BglII and KpnI and ligated at the BglII–KpnI site of the mammalian expression vector pEGFP-C1 (Clontech). The resultant clone expressed the hCP3 protein fused with enhanced green fluorescent protein (EGFP) (EGFP–hCP3).
Human embryonic kidney (HEK)-293 cells were transfected with expression vectors pEGFP-C1 and pEGFP–hCP3 using LipofectAmine Plus reagent (Gibco BRL, Life Technologies Oriental, Tokyo, Japan). Transfection was performed according to the manufacturer's procedures. Twenty-four hours after transfection, cells were observed with a fluorescent microscope and harvested for Western blot analysis.
Western blot analysis
Human testis and sperm were homogenized with a lysis buffer containing 10 mmol/l Na2HPO4 (pH 7.2), 160 mmol/l NaCl, 1% Triton X-100, 1% deoxycholic acid, 0.3% sodium dodecyl sulphate (SDS) and 2 mmol/l phenyl methyl sulphonyl fluoride (Wako, Osaka, Japan) on ice. HEK-293 cells transfected with pEGFP–hCP3 were also sonicated in the lysis buffer. After centrifugation, protein concentrations of each supernatant were estimated by a Bradford Protein Assay (BioRad, CA, USA). Each extract containing 50 µg of protein was subjected to SDS–polyacrylamide gel electrophoresis, followed by electroblotting to polyvinylidenedifluoride membrane filters (Millipore, MA, USA). The filters were blocked with 5% non-fat dry milk for 30 min and washed for 15 min with Tris-buffered saline (TBS)–Tween (T) (TBS: 50 mmol/l Tris–HCl pH 7.5, 150 mmol/l NaCl; T: 0.05% Tween-20). The filters were then reacted with polyclonal anti-CP3 rabbit antiserum (1:100 dilution) in TBS for 1 h at 25°C and washed in TBS–T three times for 5 min each. Finally, the filters were incubated with anti-rabbit immunoglobulins (Igs) conjugated with peroxidase (1:500) (Amersham Pharmacia Biotech, Tokyo, Japan) for 1 h at 25°C. After further washing, reactive bands were visualized by development with a peroxidase staining kit (Wako).
Western blotting with anti-EGFP monoclonal antibody (Tanaka et al., 1999) was also performed as a control for the cross-reactivity of anti-mCP3 with EGFP–hCP3 fusion protein.
Immunostaining of human sperm
Human sperm samples were spotted on Superfrost microslide glass with a silane-coating (Matsunami Glass Ind., Ltd, Osaka, Japan) and treated with 4% paraformaldehyde on ice for 20 min. For indirect immunofluorescent staining, the slides were blocked with 5% non-fat dry milk for 1 h and incubated with anti-CP3 rabbit antiserum, diluted 1:500, in PBS for 16 h at 4°C. After washing, the slides were blocked with 5% normal donkey serum for 30 min and treated with fluorescein-linked anti-rabbit donkey Igs (Amersham), diluted 1:300, for 2 h at room temperature, then washed with PBS and observed under a fluorescent microscope (Olympus BX50, Tokyo, Japan).
Co-localization of CP3 with actin proteins was accomplished by incubating human sperm with anti-CP3 rabbit antiserum and anti-actin goat polyclonal antibody (Santa Cruz Biotechnology, CA, USA), diluted 1:500, for 16 h at 4°C. Then samples were washed in PBS, blocked with casein blocking solution (Nacalai Tesque Inc., Kyoto, Japan) and 5% normal donkey and bovine serum for 30 min, then treated with fluorescein-linked anti-rabbit Igs donkey antibody (Amersham) and rhodamine-linked anti-goat Igs bovine antibody (Santa Cruz Biotechnology), diluted 1:300, for 2 h at room temperature.
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Results |
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Isolation and characterization of hcp
3 cDNA and mRNA expression in various organsA human testis cDNA library was screened with a putative ORF sequence of hcp
3 amplified by PCR from human genomic DNA (see Materials and methods). Twenty-five positive clones were independently isolated. Four clones having the longest cDNA insert, 1.2 kb, were sequenced. They all had the same nucleotide sequences and a single uninterrupted ORF which was identical to the putative ORF obtained by genomic PCR. The complete nucleotide sequence and its deduced amino acid sequence are shown in Figure 1 (DDBJ accession number: AB053259). A stop codon is located at nucleotide 45 upstream of the ATG sequence, which we assumed to be the translation initiation codon. The hcp3 cDNA consisted of 1072 nucleotides with an ORF of 897 nucleotides encoding 299 putative amino acid residues. No obvious polyadenylation signal was found in the human cDNA, as in the mouse cDNA (DDBJ accession number: D087471). The entire coding region of the hcp3 cDNA and its deduced amino acid sequence showed 82.1 and 91.3% identity with mcp3 respectively. The carboxyl-terminal sequence of 50 amino acids in CP, which is presumably required for binding to actin (Casella and Torres, 1994), is also conserved in both human and mouse cp3. The protein motif K-X-(L/M)-R-R-X-L-P-(I/V)-(N/T)-R designated as the `F-actin capping protein subunit signature 2' (residues 254–264) (Sizonenko et al., 1996) and a putative S100 protein binding site (residues 266–273) (Ivanenkov et al., 1996; Kilby et al., 1997) were also found in this region.
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We examined the expression of the hcp
3 gene in various organs by Northern blot analysis with the full-length hcp3 cDNA as a probe. As shown in Figure 2, the hcp3 gene was expressed as a 1.2 kb mRNA in the testis, but not in the other organs we examined.
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Expression of hCP
3 protein in testis and spermTo check the cross-reactivity of anti-mcp
3 rabbit antiserum with the hCP3 protein, Western blot analysis of HEK-293 cells transfected with recombinant EGFP–hCP3 fusion protein was performed. The expression vector pEGFP-C1 alone, without hcp3 cDNA, was used as a control. While the antiserum did not react with EGFP protein only (30 kDa) (Figure 3A, left blots), it specifically detected the EGFP–hCP3 fusion protein (60 kDa) without any extra band (Figure 3A, right blots). Using this antiserum, one positive signal with a molecular weight of 33 kDa was observed in the human testis and sperm. There was no reaction in controls probed with preimmune serum (Figure 3B). When the localization of hCP3 protein in ejaculated and fixed sperm was examined by indirect immunofluorescent staining, there was strong fluorescence in the neck region, moderate and patchy fluorescence along the tail and weak fluorescence in the postacrosomal region (Figure 4). The distribution of CP3 in human sperm (Figure 4B and E) corresponded to the immunofluorescent staining of actin protein (Figure 4C and F), as demonstrated by the double immunostaining (Figure 4G). No significant labelling was detected in controls exposed to preimmune rabbit serum, normal goat serum or bovine serum in the indirect immunofluorescent staining (data not shown). These results were also confirmed in the three independent donor sperm samples.
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Genomic localization and construction of hcp
3On searching the DDBJ, GenBank, EMBL, Swiss-Prot and PIR data banks for a human genomic clone having sequence homology with mcp
3 cDNA, we found one match (NCBI accession number: NT_028330) in the 12p12 region, and this included the hcp3 ORF as an intronless gene (Figure 5A). To confirm the localization of the hcp3 gene in this clone, we performed PCR with human genomic DNA as a template (Figure 5B) using one set of oligonucleotide pairs (hCAPA and hCAPB in Figure 5A) encompassing both the 5' and the 3' ends of the hcp3 ORF. The size of each PCR product obtained by agarose gel electrophoresis was consistent with that of the putative fragment from the genomic DNA sequence (Figure 5B) and the sequence of each product was also consistent with that of the cDNA sequence. Furthermore, Southern blot analysis of human genome digested with BamHI with a radiolabelled mcp3 cDNA gave an 11 kb single DNA band compatible with the restriction map of the human genome of cp3 (Yoshimura et al., 1999). Taken together, hcp3 was found to be an intronless gene localized to12p12.
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In the 5'-flanking region of the hcp
3 gene, neither a TATA box nor GC rich motifs were found, but two consensus sequences of cyclic AMP response elements (CREs) (nucleotide positions –99 to –92 and –69 to –62) existed in the same positions as in the mouse genome (Figure 5C
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Discussion |
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The architecture of sperm actually begins at the end of meiosis with the birth of spermatids. These haploid, round cells of
10 µm in diameter have an organization similar to that of any undifferentiated somatic cell. Early spermatids contain a rudimentary cytoskeleton made of microtubules and actin filaments (Weber and Russell, 1987). Intermediate filament (IF) proteins such as keratin and nuclear lamin are also present in spermatids (Moss et al., 1993; Escalier et al., 1997) and recent studies have identified male germ cell-specific IF-like proteins (Eddy et al., 1991; Catalano et al., 2001). Depending on the species, the process of spermatid differentiation into testicular sperm takes 2–3 weeks. This metamorphosis gives rise to filiform cells, 60 µm long in humans and up to 200–250 µm in rodents, with a small head and long flagellum (tail). The head is composed of a condensed nucleus, a giant lysosome (acrosome) and a compartmentalized perinuclear cytoskeleton. The flagellum is bound to the head with a connecting piece (neck) composed of the proximal centriole, remnants of the distal centriole and pericentriolar components (Fawcett, 1975; Knobil, 1988). During the processes of sperm morphogenesis, two major cytoskeletal elements, actin filaments and microtubules, are involved in cytoplasmic translocation and organelle redistribution, resulting in spectacular morphological changes of the germ cells. While the role of microtubules is actually well defined, the actin organization inside the spermatogenic cells remains controversial (Camatini et al., 1992). However, it is generally accepted that the actin localization of germ cells changes in a stage- and species-specific manner, and filamentous actin does not seem to exist in mature sperm except in rodents (Flaherty et al., 1986).
To understand the control of actin in spermatogenic cells, several actin regulatory proteins expressed in testicular germ cells have been cloned and investigated. They are classified according to their effect on actin: the actin sequestering protein [thymosin ß10 (Lin and Morrison-Bogorad, 1991); profilin-3 (Hu et al., 2001)], actin capping protein [CP3 (Tanaka et al., 1994); CPß3 (von Bülow et al., 1997)], actin severing protein and depolymerizing protein [scinderin (Pelletier et al., 1999); destrin (Howes et al. 2001)] and actin crosslinking protein [ß-spectrin (Virtanen et al., 1984); ß-actinin and tropomyosin (Yagi and Paranko, 1992); fascin (Holthuis et al., 1994); scruin (Sanders et al., 1996) and espin (Chen et al., 1999)]. Individual actions of these proteins in developing spermatogenic cells as well as in mature sperm may appear conflicting among themselves, but taken together they show a logical progression of related events designed to meet specific physiological requirements (Pelletier et al., 1999). Among these testis-specific actin-regulatory proteins, we have previously isolated the cp3 gene from a subtracted cDNA library of mouse testis (Tanaka et al., 1994). The full-length mcp3 cDNA and deduced amino acid sequence has significant similarity with the somatic cell-type actin CP (Cooper, 1991). The cp3 gene is exclusively expressed in haploid germ cells and its protein, CP3, is retained in mature sperm, suggesting that CP3 is a novel actin capping protein subunit which regulates morphological changes in developing spermatids and mature sperm (Hurst et al., 1998).
The hCP3 protein sequence exhibits low identity with two other human somatic CP proteins (Hart et al., 1997b): 33.6% with CP1 and 33.3% with CP2. In spite of a substantial amount of divergent evolution and no evidence of whether CP3 forms a heterodimer with a CPß subunit or binds actin, hCP3 remains a candidate for a molecule regulating the actin cytoskeleton in germ cells, since the putative actin binding motif (residues 254–273) in the carboxyl terminal region is highly conserved among species, and since co-localization of CP3 with actin in human sperm has been observed. The sequence divergence and expression pattern of cp3 are consistent with the observation that the most diverged isoforms in vertebrates are narrowly expressed (Hastings, 1996).
The whole structure of hcp3 is the same as that of the mouse genome; it lacks introns and is considered to be made by a retrotransposon, a class of genes created by reverse transcription of a mature mRNA and insertion of the cDNA copy into the genome (Vanin et al., 1985; Valentin et al., 2000). These two genes lack both a TATA box and a GC-rich motif in the putative promoter of the 5'-flanking region, but show high similarity. There are two CREs in both the mouse and human gene regions. It has been suggested that CP3 is a potential CREMtau target gene (Nantel and Sassone-Corsi, 1996).
The human and mouse gemomic DNAs of cp3 do not contain the typical polyadenylation signal, AATAAA, or variants thereof (Beaudoing et al., 2000). This unusual finding is not uncommon in haploid germ cell-specific molecules, such as acrosin, proacrosin-binding protein sp32 and zona pellucida-binding protein sp38 (Baba et al., 1994; Mori et al., 1995). In recent studies, a novel isoform of poly(A) polymerase specifically expressed in testis was identified (Kashiwabara et al., 2000; Lee et al., 2000). Interestingly this testis-specific poly(A) polymerase (TPAP) exhibits polyadenylation activity against germ cell specific mRNA even if it does not contain a typical polyadenylation signal or upstream cytoplasmic polyadenylation element (Hake and Richter, 1994; Simon and Richter, 1994). The mechanism by which TPAP extends the poly(A) tail of mRNA in haploid germ cells is not fully elucidated. However, it is possible that the cp3 gene utilizes this new poly(A) polymerase so that the mRNA of cp3 is actively transcribed and accumulated in haploid germ cells with the onset of chromatin condensation at mid-spermatogenesis when gene transcription ceases (Monesi, 1965; Kierszenbaum and Tres, 1975).
The hCP3 protein in testis and sperm could be specifically recognized with antiserum specific for mCP3 recombinant protein, because of the high degree of sequence identity between human and mouse CP3 (91.3%). Strikingly, the main localization of hCP3 protein in sperm is at the neck, the connecting piece where the centrioles are associated. Moderate and faint fluorescence was also noted in the tail and postacrosome of the sperm respectively. The distribution of actin in human sperm seems to coincide with that of CP3 on dual-fluorescent staining. In the mature human sperm, actin has been found to be localized to the connecting piece in the neck region and external surface of the fibrous sheath in the tail by immunogold electron microscopy (Flaherty et al., 1988; Escalier et al., 1997). Furthermore, most of the sperm actin is presumed to be G-actin (Camatini et al., 1986; Castellani-Ceresa et al., 1986; Flaherty et al., 1986). This suggests a possible link between G-actin and CP3. Since the physiological role of actin in the pericentriolar and periaxonemal cytoskeleton remains to be clarified, further investigation of CP3 may provide insight into this issue.
CP3 protein is reported to be present mainly in the acrosomal region of rat testicular sperm (Hurst et al., 1998) and in the anterior part of acrosome of bull ejaculated sperm (Howes et al., 2001). The distribution of CP3 protein in sperm seems to vary according to the status of maturation or acrosome reaction (Howes et al., 2001). In the present study, we also detected CP3 in the acrosomal region of human ejaculated sperm, but the main localization was to the neck region. This discrepancy may be explained by a species difference.
In conclusion, we have isolated a human orthologue of mcp3 expressed specifically in the testis and demonstrated that cp3 is a single and intronless gene. The physiological role of CP3 in the cytoskeleton of sperm would be of interest in human reproduction biology.
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Acknowledgements |
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We are grateful to our colleagues for providing valuable information, advice and technical assistance: Mrs Hiromi Nishimura, Mr Hiroshi Ohta, Dr Junji Tsuchida, Dr Akira Tohda and Dr Akira Tsujimura. This work was supported by Health Science Research Grants (H13-GENOMU-009) from Ministry of Health Labour and Welfare.
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3 To whom correspondence should be addressed at: Research Institute for Microbial Diseases, Osaka University, 3–1 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: nishimun{at}biken.osaka-u.ac.jp
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Submitted on November 22, 2001; accepted on March 6, 2002.
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