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Regular Article |
a Department of Urology, Osaka University Medical School, Osaka, Japan
b Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
c Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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33 kDa by Western blot analysis, and was localized to occupy the flagella from the connecting piece through the principal piece. We named this newly isolated gene oppo 1, and we suggest that it plays an important role in sperm tail structure and/or sperm movement.
sperm, sperm maturation, sperm motility and transport, spermatid, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, we cloned many genes specifically expressed in haploid germ cells from a subtracted cDNA library [1, 2] that was generated by subtracting the mRNA from 17-day-old mouse testes from the cDNA of 35-day-old mouse testes. Detailed analyses of mRNA expression revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in haploid germ cells and were developmentally controlled. Some encoded novel proteins remain to be characterized; the others encoded putative proteins whose functions could be inferred by computer-assisted domain analysis.
In this paper, we report the isolation of another gene. We called this gene oppo 1, and characterized its protein product, which is localized in the sperm tail. We speculate that it is involved in sperm tail structure and/or sperm motility.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The strategy used to prepare the subtracted cDNA library has been described by Tanaka et al. [1]. A library was generated by subtracting the mRNA from 17-day-old mouse testis, just before the start of spermiogenesis, from the cDNA of 35-day-old mouse testis, which contains many differentiated germ cells [2]. Plasmid DNA of each clone randomly selected from the subtracted cDNA library was screened by Northern blot analysis using testes mRNA taken from 17- and 35-day-old mice. We called the clones expressed exclusively in 35-day-old testis "transcript increased in spermiogenesis (TISP)" clones. One of these, TISP-62, showed no homology with any reported sequence tags. To obtain a full-length cDNA of the isolated clone, a 
gt10 phage library of mouse testis was initially screened under high-stringency hybridization conditions. A 32P-labeled probe was prepared with a BcaBest random primer kit (Takara, Shiga, Japan) using the TISP-62 cDNA fragment. Two independent clones containing an 1.1-kilobase (kb) insert cDNA were isolated. Then, a pAP3neo cDNA library of mouse testis was screened using this 1.1-kb cDNA fragment [1] as a 32P-labeled probe under high-stringency hybridization conditions. Four independent clones with cDNA inserts of about 1.2 kb were isolated, subcloned into pBluescript SK II (-) (Stratagene, La Jolla, CA), and sequenced.
Northern Blot Analysis
Freshly removed organs of an adult mouse (C57BL/6 strain) were homogenized in Trizol reagent (Gibco-BRL, Grand Island, NY). Germ and other somatic testes cells were prepared as described in our previous report [3]. Total RNA was extracted according to the manufacturer's recommendations and quantified by optical density measurement.
Samples of RNA containing 2.2 M formaldehyde were electrophoresed in a 1.0% agarose gel containing 0.66 M formaldehyde. The RNA was transferred to a nitrocellulose membrane filter in 20x saline-sodium citrate (SSC; 1x SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate). After baking for 2 h at 80°C, the membrane was preincubated in a solution containing 50% formamide, 4x SSC, 5x Denhardt solution, 0.2% SDS, and 120 µg/ml of denatured sonicated salmon sperm DNA at 42°C for 12 h. Hybridization was performed with the 32P-labeled full-length oppo 1 cDNA probe prepared using a BcaBest random primer kit (Takara) and the same preincubation conditions for 24 h. The membrane was washed twice with a solution of 0.2x SSC and 0.1% SDS at 55°C for 30 min. Band signals were detected with an Image Analyzer (Fuji Film, Tokyo, Japan).
Dideoxy-chain-termination sequencing reactions were performed with fluorescent dye-labeled primers and thermal cycle sequencing kits purchased from Li-Cor (Li-Cor, Lincoln, NE). The reaction products were analyzed using a Model 4000 (Li-Cor). The DDBJ, GenBank, EMBL, Swiss-Prot, and PIR data banks were searched for sequences homologous to the isolated cDNA or the deduced amino acid sequence.
In Situ Hybridization
Antisense digoxigenin (DIG)-labeled RNA was used for in situ hybridization. Testes were fixed in 4% paraformaldehyde and embedded in methyl methacrylate resin. Thin sections (4 µm) were collected on APS-coated Superfrost microslide glass (Matsunami Glass, Osaka, Japan). An oppo 1 probe was generated from a 442-base pair (bp) HincII-NotI cDNA fragment containing the 3'-untranslated region cloned into pBluescript SK II (-). An antisense probe was generated by transcription of a HincII digest with T7 RNA polymerase, and a sense probe was generated by transcription of a NotI digest with T3 RNA polymerase. The probes were labeled with DIG-UTP (Boehringer Mannheim, Mannheim, Germany). In situ hybridization was performed using the TSA Plus DNP System (NEN Life Science Products, Inc., Boston, MA). After hybridization, the bound probe was detected by incubation with anti-DIG-Fab fragments conjugated with peroxidase (Boehringer Mannheim), followed by a color reaction involving 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Dojindo, Kumamoto, Japan). Sections were counterstained with 1% methyl green stain solution (Muto Pure Chemicals, Ltd., Tokyo, Japan) and examined under a microscope.
Preparation of Antiserum
A partial oppo 1 cDNA fragment was subcloned into the NcoI and BamHI sites of pET30a expression vector (Novagen, Madison, WI). The protein histidine-tagged N-terminus was expressed in Escherichia coli BL21 by induction with isopropyl-ß-D-thiogalactopyranoside, purified with the Ni+ Spin kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, and then used to raise polyclonal antiserum in 3 rabbits together with GERBU Adjuvant 100 (GERBU Biotechnik, Gaiberg, Germany). Each rabbit serum detected the antigen by Western blotting. Antibodies were purified from 1 serum by using Affi-gel Protein A MAPS II Kit (BioRad, Hercules, CA) and used for immunohistochemical analysis.
Western Blot Analysis
Protein samples of various mouse tissues (C57BL/6 strain) were prepared with RIPA buffer (1% NP-40, 0.1% [w/v] sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl pH 7.6). After centrifugation, aliquots of the samples (70 µg per lane) were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride filters (Millipore, Bedford, MA), and then blocked with TBS-T (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 50 mM KCl, 0.05% (w/v) Tween 20) containing 5% skim milk and 10% (v/v) normal rat serum. After incubation in blocking solution in PBS, pH 7.2 (Nacalai, Kyoto, Japan), the filters were reacted overnight with rabbit antiserum in TBS-T (1:500) at 4°C. Then, the filters were treated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Ig) G and developed with a peroxidase staining kit (Wako, Osaka, Japan).
Subcellular Fractionation of Mouse Sperm
Epididymal sperm from 10 mice were fractionated according to a modified protocol described previously [4]. The protein concentrations of all fractions (soluble membrane [M1, M2, and M3], ODF, and fibrous sheath and head (FS/H) fractions) were estimated using the Bradford Protein Assay (Nacalai). About 20 µg of each fraction was separated by 12% SDS-PAGE. Western blotting was performed as described previously. Control antibodies for the membrane (CD46) [5], FS/H (anti-AKAP82) [6], and ODF (anti-ODF-1) [7] fractions were used at 500x dilution to verifying the extracted proteins.
Immunohistochemistry
The testis was fixed in Bouin solution, embedded in paraffin, and sectioned at 7 µm. After deparaffinization with xylene, the sections were treated with TBS-T for 30 min and then blocked with 5% skim milk and subsequently with 1% donkey serum for 30 min at room temperature. Then, the sections were incubated with rabbit antiserum (1:500 in TBS-T) overnight at 4°C. The antibodies were visualized using fluorescein isothiocyanate (FITC)-conjugated secondary antibody (anti-rabbit IgG; Amersham Pharmacia Biotech, Tokyo, Japan). To determine the subcellular localization of OPPO 1 protein, mature sperm were collected from the epididymis of an adult mouse and spotted on a Superfrost microslide glass (Matsunami Glass). Sperm samples were fixed in Bouin solution for 10 min at room temperature and then blocked with 5% skim milk and 1% donkey serum for 30 min each at room temperature and then incubated with anti-OPPO 1 rabbit antiserum in TBS-T (1:500) overnight at 4°C. Then, the samples were treated with FITC-conjugated secondary antibody for 2 h at room temperature. Samples were examined under a fluorescent microscope.
To visualize individual fibers of the ODF, mature sperm were treated with a solution containing 10 mM Tris-HCl (pH 8.0), 30 mM ß-mercaptoethanol, 0.2 mM PMSF, 0.05% cetyltrimethylammonium bromide (CTAB), and 1 M sucrose for 3 h at room temperature. The sample was spotted on microslide glass (Matsunami Glass) and dried, and then blocked with 5% skim milk and subsequently with 1% donkey serum for 30 min at room temperature; under reducing conditions, a cationic detergent removes all of the tail structures except for the ODFs, which are released from their tight native form [8]. Finally, the samples were incubated with anti-OPPO 1 antiserum, anti-SHIPPO 1 antiserum [9], or anti-ODF-2 rabbit polyclonal antibody (1:500 in TBS-T) overnight at 4°C. The antibodies were visualized with FITC-conjugated secondary antibody (anti-rabbit IgG; Amersham Pharmacia Biotech).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To elucidate the mechanism of spermatogenesis, we prepared a cDNA library of adult mouse testis subtracted using the mRNA from immature testis (17 days of age) [2]. Haploid-specific cDNA clones are concentrated in the library because haploid germ cells develop after the age of 17 days. Clones randomly selected from the subtracted cDNA library were screened by Northern blot analysis using mRNA of testes taken from 17- and 35-day-old mice [2]. One of the clones, TISP-62, was specifically expressed in haploid germ cells. To obtain the complete cDNA clone, 2 x 106 colonies from a gt10 mouse testis cDNA library were screened with the 32P-labeled 0.7-kb EcoRI-NotI fragment of the TISP-62 cDNA, and 2 positive clones containing a 1.1-kb cDNA insert were independently isolated and sequenced. The clones had almost the same nucleotide sequences. Using this 1.1-kb cDNA fragment, 2 x 106 colonies of the pAP3neo mouse testis cDNA library [2] were again screened, and 30 positive clones were independently isolated. The 4 clones with the longest cDNA insert, 1.2 kb, were sequenced. They all had the same nucleotide sequence and contained 1 long open reading frame that started with a putative start codon at nucleotide 1 and terminated with a putative stop codon at nucleotide 870. The complete nucleotide and deduced amino acid sequences (DDBJ accession no. AB074438) are shown in Figure 1. A stop codon was located 42 nucleotides upstream from the ATG sequence at position 1, which we assumed to be the translation initiation codon of an 870-bp cDNA. The cDNA encoded 290 putative amino acids and contained a 3' untranslated region of 130 nucleotides with a poly(A)+ tail of about 11 bases, following a consensus AATAAA polyadenylation signal at position 974–979. We did not find any nucleotide sequences having high homology with this cDNA in a computer search of the DDBJ, GenBank, and EMBL databases. Next, we analyzed the whole putative amino acid sequence using the Blocks motif database [10, 11]. This showed that 22 amino acids of the deduced sequence contained the consensus motif of Salmonella flagella basal body rod protein (Fig. 1). We named this newly isolated gene oppo 1, from the Japanese word for tail.
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Northern Blot Analysis and In Situ Hybridization of oppo 1 in the Testis
Northern blotting of mRNA from various organs exclusively identified a 1.2-kb transcript expressed in the testis (Fig. 2A). In fractionated mouse testicular cells (i.e., germ, Sertoli, and Leydig cells) [3], the oppo 1 gene was exclusively expressed in germ cells (Fig. 2B). During male germ cell development, no transcripts were detected in the neonatal mouse testis until 8 days of age. The transcript was detected beginning at the age of 11 days, and a significant signal was present on Northern blotting by 17 days (Fig. 2C). These results indicate that the oppo 1 gene is expressed in germ cells only in a developmentally regulated manner. To determine the developmental stages of the germ cells expressing oppo 1 mRNA, in situ hybridization analysis was performed. Specific staining detected the antisense probe exclusively in spermatocytes and spermatids from the late pachytene stage to terminal elongated spermatids in the seminiferous tubules (Fig. 3, A–C).
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Western Blot and Immunohistochemical Analyses of OPPO 1 Protein
Western blot analysis revealed a protein band with a molecular weight of approximately 33 kDa exclusively in the testis and sperm (Fig. 4, A and B). In chronologic observations of testicular development, OPPO 1 was detected first in the 3-wk-old mouse testis (Fig. 4C). During germ cell differentiation in adult mouse testis, the OPPO 1 protein was first detected in the flagella of elongated spermatids in an immunohistochemical examination of testis (Fig. 5). A positive signal was also observed in the tail of epididymal sperm (Fig. 6). These observations were in good agreement with the results of Western blot analysis, indicating that OPPO 1 is a novel differentiation-associated molecule specifically expressed after differentiating haploid spermatids reach the elongated step.
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Western Blot Analysis and Immunocytochemical Staining of Subcellularly Fractionated Sperm
To examine the localization of OPPO 1 in sperm further (i.e., the membrane/cytoplasm, axoneme, FS, and ODF distribution), subcellular fractionation of sperm proteins and Western blot analysis was performed [4]. The fidelity of the subcellular fractionation of sperm protein was guaranteed by the specific blotting of marker proteins in each fraction. The strongest 33-kDa band of OPPO 1 protein was seen in the ODF fraction, and some was found in the fibrous sheath and head (FS/H) fraction (Fig. 7). To confirm these observations, sperm treated with 0.05% CTAB to visualize individual ODFs were immunostained. In these extracted sperm, the fibers were labeled with anti-OPPO 1 antiserum and anti-ODF-2 antibody [12] (another major ODF protein), which is better than anti-ODF-1 antibody for immunohistochemical observation, whereas the SHIPPO 1 [9] signal was progressively lost in the same fraction (Fig. 8). Combined, these results demonstrated that OPPO 1 is associated with ODFs, and the association is stronger than that of SHIPPO 1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A significant signal was detected from the age of 11 days through to adulthood (Fig. 2). In situ hybridization demonstrated specific staining in spermatocytes and spermatids from late pachytene to the terminal elongated spermatids in the seminiferous tubules (Fig. 3). The storage of transcripts and translational control would be common phenomena in the late stage of spermiogenesis in transcriptionally inactive elongating spermatids [14]. Nucleotide sequence analysis of cDNA and a computer-assisted homology search found no homologous sequence registered in cDNA banks, but a motif database search indicated that the predicted amino acid sequence of OPPO 1 protein has a 22-amino acid sequence common to the flagella basal body rod protein of Salmonella (Fig. 1). The flagellum is the organelle of motility for Salmonella typhimurium and many other bacterial species. Its known structural features are the basal body, hook, hook-associated proteins, and helical filament. The assembly of the bacterial flagellum in Salmonella is considered to have 3 distinct stages: formation of the basal body (which functions as a transmembrane motor), the hook (which serves as a flexible linker), and the filament (which serves as the propeller) [15–17]. Although there are large differences between the flagella of bacteria and those of mammals, we speculate these proteins have very important roles in the formation and function of the flagellum. OPPO 1 is a new flagella protein that may be concerned with flagella basal body formation in mammals.
Western blot analysis and immunohistochemical staining showed that OPPO 1 protein is expressed in haploid spermatids slightly after the beginning of mRNA transcription (Figs. 4 and 5). These results indicate that the expression of OPPO 1 protein is controlled at both the transcriptional and translational levels. OPPO 1 protein is localized in the tail region of sperm and was recovered in the FS/H and ODF fractions of the mouse sperm tail (Figs. 6 and 7). The sperm tail was separated into 4 regions: the connecting, middle, principal, and end pieces. The ODF are sperm tail-specific cytoskeletal structures [18]. They consist of 9 fibers, which surround the outer side of the axoneme, accompanying the tubulin doublets in the middle and principal piece of the sperm tail. At its anterior end, the ODF make close contact with the connecting piece and extend posteriorly for varying lengths into the principal piece. The FS surrounding the ODF is localized at the principal piece.
Apart from the axoneme and its associated proteins, the sperm tail consists mostly of ODF and FS proteins, which are the most abundant proteins, and specialized cytoskeletal structures of the mammalian sperm tail. The component proteins are expressed in spermatids and are recruited to the flagellum for assembly during the terminal stage of spermiogenesis; this last step of sperm formation finishes in the seminiferous epithelium. Although the exact functions of the ODF and FS are not clear, the ODF might maintain the passive elastic recoil of the sperm tail, and the FS might give elastic rigidity to the sperm tail or define the shape of its beat by placing a constraint on its plane of bending [19]. In any case, OPPO 1 occupies the flagella from the connecting piece through the principal piece (Fig. 6). This widespread localization consistent with the Western blotting pattern of fractionated sperm suggests that OPPO 1 is associated with ODF and that it plays an important role in the morphogenesis of the sperm tail and/or in sperm tail motility.
Impaired spermatogenesis accounts for approximately 90% of all male infertility, although its etiology remains mostly unknown. For normal fertilization, it is very important to ensure the normal movement of sperm. Asthenospermia (abnormal motility of sperm) is defined as a loss or reduction of motile sperm to less than 50%–60% of the total ejaculated sperm, or decreased linear and progressive movement of sperm. Poor sperm motility is often associated with morphologic abnormalities. Although the majority of men with asthenospermia have accompanying defects in sperm production or morphology, isolated asthenospermia also occurs in as many as 20% of subfertile men. We have shown that the oppo 1 gene is expressed abundantly at meiosis until spermiogenesis. The gene product OPPO 1 was localized in the ODF of mouse sperm flagella and thus was a component of sperm flagella. We found a putative human homologue with oppo 1 in a computer search of the DDBJ, GenBank, and EMBL databases. Now, we will try to identify the entire sequence of human oppo 1. It likely plays important roles in sperm morphogenesis and function and might also give a helpful clue to an understanding of the etiology of asthenospermia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Y. Nishimune, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp 
Accepted: January 11, 2002.
Received: November 27, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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