Molecular Human Reproduction, Vol. 9, No. 5, 237-243,
May 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Molecular cloning and characterization of the human orthologue of the oppo 1 gene encoding a sperm tail protein
Submitted on December 3, 2002; accepted on January 23, 2003
1 Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 and 2 Department of Urology, Osaka University Medical School, Osaka, Japan
3 To whom correspondence should be addressed. e-mail: nishimun{at}biken.osaka-u.ac.jp
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ABSTRACT |
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We report here the molecular cloning and characterization of a human orthologue of oppo 1, a mouse gene encoding a male germ-cell-specific sperm tail protein, and the organization of its genomic structure. The mRNA of the human oppo 1 gene (h-oppo 1) was expressed exclusively in the testis, and the 30 kDa protein encoded by the mRNA was detected in human testis and sperm. Immunohistochemical analyses showed that human OPPO 1 protein was localized in the flagellae of ejaculated sperm. A human genomic DNA database search indicated that the h-oppo 1 gene mapped to chromosome 17. The genomic structure of h-oppo 1 showed differences in exon/intron usage, the sequence of the 5'-flanking region, and the first intron was rich in Alu repeats as compared with the mouse oppo 1 gene. Comparison of the two genomic sequences indicated that human oppo 1 has evolved independently, resulting in substantial differences in the genomic structure after the human–mouse split, whereas the sequence of the basic functional unit of the oppo 1 gene seems to have been relatively well conserved.
Key words: Alu repeat/oppo 1/outer dense fibres/sperm/spermiogenesis
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Introduction |
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Spermatogenesis is composed of three major events: proliferation and differentiation of spermatogonia, meiotic events of spermatocytes at prophase, and marked morphological changes during differentiation from the haploid round spermatids to mature sperm cells. During haploid germ cell differentiation, known as spermiogenesis, many different molecules involved in chromatin condensation and development of the acrosome and tail are expressed specifically in haploid germ cells.
Previously, we have isolated various cDNA clones that are expressed specifically in mouse haploid germ cells by subtracted cDNA library construction (Tanaka et al., 1994; Iguchi et al., 1999). The mouse oppo 1 gene encoding a novel sperm tail protein was cloned from this cDNA library (Nakamura et al., 2002). On Northern blot analysis, Western blot analysis, and immunohistochemical analysis, the expression of mouse oppo 1 was detected specifically in the haploid germ cells of the testis, but not in somatic tissues. Furthermore, the oppo 1 gene product is localized in the outer dense fibres (ODFs) of the tails of mature sperm (Nakamura et al., 2002), which is a complex structure involved in generating and regulating the beat of the flagellum (Baltz et al., 1990; Lindemann, 1996). In addition to the axoneme and its associated proteins, the flagellum consists of two cytoskeletal components; the fibrous sheath (FS) in the principal piece, and the ODFs in the middle and principal pieces of the sperm tail. There have been several previous reports concerning the component proteins of ODF (Olson and Sammons, 1980; Vera et al., 1984; Oko, 1988; Kim et al., 1999; Petersen et al., 1999), but the precise functions of the ODFs have yet to be determined.
Here, we report the isolation and characterization of a human oppo 1 orthologue, and compare the structures of the human and mouse genomic DNAs. The evolutionarily conserved expression pattern suggests that OPPO 1 protein should have some important role(s) in the sperm tail.
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Materials and methods |
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Cloning of human oppo 1 (h-oppo 1) cDNA
Before cloning the human orthologue of mouse oppo 1 cDNA (DDBJ accession number: AB074438) (Nakamura et al., 2002), a computer-assisted homology search of mouse oppo 1 cDNA was performed in the DNA Data Bank of Japan (DDBJ), GenBank, EMBL, Swiss-Prot and Protein Identification Resource (PIR) databases. The search identified a cDNA sequence homologous to the mouse oppo 1 from a human testis EST cDNA library (NCBI accession number: AI125365). A 26-mer oligonucleotide based on this sequence (5'-GGAATGTATCCATCCCCATAGGCTGG-3') was synthesized for use as a probe. The cDNA of h-oppo 1 was isolated from a human testis cDNA library constructed with the plasmid vector pAP3neo (Tanaka et al., 1997). Aliquots of more than 2x106 Escherichia coli containing recombinant plasmids were screened by hybridization with the [
-32P]dCTP-labelled probe prepared using a BcaBEST Random Primer Kit (Takara, Shiga, Japan). Hybridization was performed for 20 h at 60°C, followed by washing 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 time. Hybridization-positive colonies were isolated and purified by limiting dilution and rescreening. Four independent positive clones were isolated, and the cDNA inserts of these clones were sequenced. Dideoxy chain-termination sequencing reactions were performed with fluorescent dye-labelled primers and thermal cycle sequencing kits purchased from Applied Biosystems (Foster City, CA, USA). The reaction products were analysed using an ABI-PRISM® 310 Genetic Analyzer (Applied Biosystems).
Determination of the transcriptional start site
To identify the transcriptional start site, the cap-site hunting method was performed using Cap Site cDNA® dT (Nippon Gene, Toyama, Japan) in accordance with the manufacturer’s instructions. The primers used for the first PCR were 5'-GATGCTAGCTGCGAGTCAAGTC-3' (1RDT; Nippon Gene) and 5'-CTCCTAGAGAGGTCCAGCCATTTC-3' (h-oppo 1-specific primer; positions 488–511). The primers used for nested PCR were 5'-CGAGTCA AGTCGACGAAGTGC-3' (2RDT; Nippon Gene) and 5'-GAAGGCCATG ACCAATAGTAGG-3' (h-oppo 1-specific primer; positions 461–482). Cycling conditions were as follows: 96°C for 1 min, followed by 35 cycles of denaturation at 96°C for 45 s, annealing at 65°C for 45 s, and extension at 72°C for 60 s. The PCR products were cloned into the pT7Blue vector (Novagen, Madison, WI, USA) and sequenced.
RT–PCR analysis
To examine the tissue-specific expression of h-oppo 1, we carried out RT–PCR analysis using a Rapid-ScanTM gene expression panel containing cDNAs from 24 different human tissues (Origene Technologies, Rockville, MD, USA). The h-oppo 1-specific 23-mer primers, 5'-GGATGCAGAGTACTCTGGGAATG-3' and 5'-ACATGTGTATCCTTCTGCTCAGG-3' (Figure 1A), were used to amplify a fragment of 768 bp. Cycling conditions were as follows: 96°C for 1 min, followed by 35 cycles of denaturation at 96°C for 45 s, annealing at 62°C for 45 s, and extension at 72°C for 60 s. As a control, actin was also amplified according to the manufacturer’s protocol.
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Genomic cloning of h-oppo 1
Human genomic DNA was isolated from human whole blood, obtained from healthy male volunteers, with DNAZOL BD Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s instructions. The volunteers gave their written consent for use of their blood for the analysis of genomic DNA in this study.
PCR cloning of h-oppo 1 genomic DNA was performed using the following 24-mer primers: A1, 5'-TCCCCCATCTCACTGTGCCCCAGG-3'; A2, 5'-AA TACATCCAGGGTGCAGATACAG-3'; B1, 5'-GGGCCAGTCACTCTGTG TGTGGTG-3'; B2, 5'-TTTCTCCCTTCCTCTCCCTCCTCC-3'; C1, 5'-GC CCACGTTATGTCCATGGGGCTC-3'; C2, 5'-CTTGGGCCCAGCAGTTTG AGGCTG-3'; D1, 5'-CCACAGACACTCTATTCAGAGACA-3'; D2, 5'-TA ATGGGGAATAGCATGAGGGTGG-3'. PCR was carried out in a reaction buffer (50 µl) containing 4 µg of human genomic DNA, 0.2 µmol/l primer, 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 U LA Taq polymerase (Takara, Shiga, Japan). A Gene Amp PCR System 9700 (PE Applied Biosystems) was used for amplification of PCR samples. Cycling conditions were as follows: 96°C for 1 min, followed by 35 cycles of denaturation at 96°C for 45 s, annealing at 63°C (A1–A2 and D1–D2 primer sets), 65°C (B1–B2), or 70°C (C1–C2) for 45 s, and extension at 72°C for 60 s. The DNA sequences were determined by direct sequencing using the same primers as used for PCR.
Human tissue and sperm samples
Human testis fragments were obtained with informed consent from a fertile middle-aged patient castrated for treatment of prostate cancer, and stored at –80°C until use. Other human protein samples (liver, lung, ovary and smooth muscle) were purchased from Clontech (BD Biosciences Clontech, Palo Alto, CA, USA). 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 samples were gently suspended in PBS to release sperm. After 10 min, the PBS/semen samples were centrifuged, at 500 g for 5 min and the pellets were resuspended in either lysis buffer (see below) for Western blot analysis or in PBS for immunostaining of sperm.
Western blot analysis
Human testis and sperm were homogenized in 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. After centrifugation, protein concentrations of each supernatant were estimated using a Bradford Protein Assay kit (BioRad, Hercules, CA, USA). Protein samples of other organs, purchased from Clontech, were also used. Each sample containing 50 µg of protein was subjected to SDS–polyacrylamide gel electrophoresis, followed by electroblotting onto polyvinylidene difluoride membrane filters (Millipore, Bedford, MA, USA). The filters were blocked with 5% non-fat dried 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-OPPO 1 rabbit antiserum (1:100 dilution) in TBS for 1 h at 25°C, and washed three times in TBS–T for 5 min each time. Finally, the filters were incubated with polyclonal peroxidase-conjugated anti-rabbit immunoglobulin (Ig) antibody (Amersham Pharmacia Biotech, Tokyo, Japan), diluted 1:500, for 1 h at 25°C. After further washing, reactive bands were visualized by development with a peroxidase staining kit (Wako).
Immunofluorescence microscopy of human sperm
Human sperm samples were spotted onto silane-coated Superfrost microslide glasses (Matsunami Glass Ind., Ltd, Osaka, Japan), and treated with 4% paraformaldehyde on ice for 10 min. For indirect immunofluorescent staining, the slides were blocked with 5% non-fat dried milk for 1 h, and incubated with anti-OPPO 1 rabbit antiserum, diluted 1:100, 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 polyclonal fluorescein-conjugated anti-rabbit donkey Ig antibody (Amersham), diluted 1:300, incubated for 2 h at room temperature, then washed with PBS and observed under a fluorescence microscope (Olympus BX50; Olympus, Tokyo, Japan).
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Results |
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Isolation and characterization of the h-oppo 1 cDNA
A human testis cDNA library was screened with a putative human oppo 1 cDNA sequence obtained from the human testis EST database (see Materials and methods). Four positive clones were isolated independently. Cap-site hunting was also performed to determine the transcriptional start site of the human oppo 1 gene. The three clones with the longest cDNA insert, 1.1 kb, were sequenced. They all had the same nucleotide sequence, and a single uninterrupted open reading frame. The complete nucleotide and deduced amino acid sequences are shown in Figure 1A and B (DDBJ accession number: AB081120). The h-oppo 1 cDNA consisted of 1149 nucleotides, with an open reading frame of 771 nucleotides encoding a putative product of 257 amino acid residues. A stop codon was found at nucleotide 39 upstream of the ATG sequence, assumed to be the translational initiation codon. A polyadenylation signal, AATAAA, was detected 20 nucleotides upstream of the poly(A) tract, as in the mouse oppo 1 cDNA (DDBJ accession number: AB074438). The entire h-oppo 1 cDNA and deduced amino acid sequences showed 55.9 and 39.6% identity with those of mouse oppo 1 (DDBJ accession number: AB074438) respectively.
Expression of h-oppo 1 mRNA and protein and immunofluorescent staining of human sperm
We examined the expression of the human oppo 1 gene in various organs by RT–PCR analysis using h-oppo 1-specific primers (Figure 1A). The h-oppo 1 gene was shown to be expressed in the testis as a single band of 768 bp, but no expression was found in the other organs examined (Figure 2A). We have confirmed the cross-reactivity of anti-mouse OPPO 1 rabbit antiserum with the recombinant h-OPPO 1 protein (data not shown). Western blot analysis showed one positive band with a molecular weight of 30 kDa in the human testis and sperm using anti-mouse oppo 1 antiserum (Figure 2B). Immunofluorescent staining of ejaculated sperm with the antiserum showed that the expression of h-OPPO 1 protein was restricted to a region from the neck to the middle and principal pieces of the tail of the sperm (Figure 3).
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Chromosome localization and genomic construction of the h-oppo 1 gene
By searching the DDBJ, EMBL, Swiss-Prot, GenBank and PIR databases for a human genomic clone with sequence homology to h-oppo 1 cDNA, we found a genomic clone (accession number: NT_010718), which mapped to chromosome 17. To confirm the construction of the h-oppo 1 gene, we performed PCR with human genomic DNA as a template and four pairs of oligonucleotide primers to amplify DNA fragments (A–D) overlapping by more than 100 nucleotides at the join of each fragment. These primers encompassed the whole h-oppo 1 cDNA sequence (Figure 4A). The sizes of the PCR products were consistent with those of the putative fragments predicted from the genomic DNA sequence (Figure 4B).
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In comparison with the mouse oppo 1 genomic sequence (chromosome 11; accession number: AL603662) shown in Figure 4A, the human oppo 1 gene showed some differences in exon/intron structure. Exon 1 of the human gene extended over exons 1 and 2, including intron 1, of the mouse homologue. Furthermore, the human gene lacked an exon corresponding to exon 3 (170 bp in human) of the mouse homologue. This region of the human gene contained multiple stop codons (data not shown), and no putative transcript containing this region (768 bp + 170 bp = 938 bp) was detected on RT–PCR analysis (Figure 2A), although the same splicing donor and acceptor sequences remained at both ends (Figure 4A). The whole sequence of h-oppo 1 cDNA and the predicted amino acid sequence showed relatively low levels of identity, 55.9 and 39.6% respectively, with those of the mouse. In contrast, the partial amino acid sequences in conserved regions (e.g. residues 53–151 and 152–196 of h-OPPO 1; Figure 1B) showed higher levels of identity (59.6 and 77.8% respectively) with those of the mouse.
No TATA box, GC-rich promoter motifs, or cAMP-responsive promoter elements were found in the upstream region of the h-oppo 1 gene (data not shown). The 5'-flanking region and the first intron of the h-oppo 1 gene were rich in Alu repeats. In the first intron of 
4.8 kb, six Alu sequences were found, including one in the reverse orientation. Also, two tail-to-tail Alu sequences were found 84 nucleotides upstream of the putative transcriptional initiation site (Figure 4A). These results indicated that the h-oppo 1 gene evolved and changed after the human–mouse split in association with Alu insertion, although some important regions required for the functions of oppo 1 were conserved in both the mouse and human genomes.
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Discussion |
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In this study, we demonstrated that human oppo 1 is expressed exclusively in the testis, and that the gene products are localized in the sperm tail. The human oppo 1 gene (h-oppo 1) was mapped to chromosome 17, which is syntenic to a region of mouse chromosome 11 (Gregory et al., 2002). Although the homology of h-oppo 1 to the mouse orthologue is relatively low in overall amino acid sequence (39.6%), it is higher in some limited regions (59.6 or 77.8%) due to differences in exon/intron usage between these two species. Exon 1 of h-oppo 1 is not only split into exons 1 and 2, but also partially eliminated as intron 1 in the mouse, and the counterpart of mouse exon 3 is not used in the human gene (Figure 4). Considering these differences, the degrees of sequence homology and conservation of protein structure of these two genes are substantial.
The 5'-flanking region of the h-oppo 1 gene contains two Alu repeats. Alu repeats are found in primate genomes, reaching a copy number of nearly 1x106 per haploid genome (Jelinek and Schmid, 1982; Britten, 1994). Alu repeats increase their copy number by a process of retroposition (Weiner et al., 1986) and possess transcriptional regulatory elements (Englander and Howard, 1995; Britten, 1996; Piedrafita et al., 1996), although evidence for the functions of these sequences in vivo is limited (Wu et al., 1990; Thorey et al., 1993). In Alu repeats, CpG sites are almost fully methylated in somatic tissue. However, a subset of Alu repeats are completely hypomethylated in mature sperm and other male germ line cells in the testis (Hellmann-Blumberg et al., 1993; Kochanek et al., 1993; Rubin et al., 1994). Although the DNA methylation status of the h-oppo 1 gene was not examined, the methylation status of Alu repeats in the 5'-flanking region of the h-oppo 1 gene may be associated with male germ-cell-specific expression in spermatogenesis.
In the first intron, the region corresponding to mouse exon 3 showed 75% identity to the genomic sequence of the mouse homologue, with multiple in-frame stop codons (data not shown), and was skipped in the human mRNA. As Alu insertion into introns has been shown to cause exon skipping (Wallace et al., 1991; Miki et al., 1996; Oldridge et al., 1999), the differences in exon/intron usage between the human and mouse orthologues may be due to the Alu insertion just upstream of this region. Comparison of the genomic structures of human and mouse genes indicates that the human oppo 1 gene has evolved even after the human–mouse split some 80 million years ago, while retaining the basic function of oppo 1 protein. The insertion of Alu elements, and the functions of these elements, may have been responsible for the human-specific evolution of the h-oppo 1 gene.
Male infertility affects up to 10% of couples (Dessars and Cochaux, 1999). Sperm motility is one of the major determinants of fertility in men, and asthenospermia, poor sperm motility, is considered a common cause of human male infertility (Martin-Du Pan et al., 1997). Structural abnormalities of sperm flagellae could affect sperm motility (Hancock and de Kretser, 1992; Coutade et al., 1998). Identification of genes involved in human sperm tail structure is thus required for candidate genes involved in male infertility to be identified.
Previously, mouse OPPO 1 was shown to be associated with ODF of sperm flagellae (Nakamura et al., 2002). Although the precise function of the ODF remains to be elucidated, it appears to be involved in the morphogenesis of the sperm tail and/or in sperm motility. The ODF and FS are specialized cytoskeletal structures of the mammalian sperm tail that have no counterparts in somatic cells. Proteins comprising the ODF or FS are unique, and lack homology with previously described cytoskeletal proteins (Si and Okuno, 1993; Carrera et al., 1994). The putative amino acid sequences of human and mouse oppo 1 have relatively well-conserved regions, which may be elements important for the functions of OPPO 1 (Figure 4A). Defects in the functions of this protein may cause impairment of sperm movement, such as asthenospermia or sperm immotility, or malformation of sperm tail structure resulting in male infertility. Further studies are currently in progress in our laboratory to elucidate the molecular function of OPPO 1 in the sperm tail, and to screen for mutations or polymorphisms in the h-oppo 1 gene in infertile men.
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