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a Department of Urology, Osaka University Medical School, Suita City, Osaka 565-0871, Japan
b Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Suita City, Osaka 565-0871, Japan
c Graduate School of Integrated Science and Department of Biology, Yokohama City University, Yokohama City, Kanagawa 236-0027, Japan
d Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical College, Miyazaki, Miyazaki 889-1692, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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spermatid, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have raised testis-specific polyclonal antibodies [9, 10], isolated many germ cell-specific antigen cDNAs from an expression library of mouse testicular cDNAs, and carried out chromosome mapping [11, 12]. Here we report one of such cDNAs, mapped to the middle region of chromosome-X and termed halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) cDNA. The halap-X cDNA encoded an acidic protein specifically expressed in round spermatids through to elongated spermatids but not in immature spermatogonia, spermatocytes, and sperm or other somatic cells. The Halap-X protein was present in the nucleoplasm of round spermatids, gradually decreased as spermatid matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular germ cells isolated from adult mice were used as an antigen source for production of an anti-mouse germ cell antiserum [9, 10]. Rabbits were immunized extensively with the antigens using complete Freund's adjuvant. The obtained rabbit antiserum was injected into the abdominal cavity of castrated adult male mice to remove nonspecific anti-mouse antibodies (in vivo absorption). Three hours after the antiserum injection, the mice were sacrificed by bleeding. To eliminate remaining nonspecific anti-mouse antibodies, the antiserum was mixed with a homogenate of mouse liver for an hour at 4°C, centrifuged for 10 min at 12 000 x g. The clear supernatant called testis-specific polyclonal antibody was used as the primary antiserum for screening and cloning of germ cell-specific antigen genes (gsg) [11]. All procedures involving animals were conformed to established guidelines for animal use and care [13].
Library Construction and Screening
Total RNA was extracted from the testis by guanidine thiocyanate followed by CsCl centrifugation, and poly(A)+ RNA was selected by oligo(dT)-cellulose chromatography. The cDNA was synthesized by a modification of the method of Gubler and Hoffman [14], ligated with 
gt11 arms and packed into lambda coat proteins, using Gigapack System (Stratagene, La Jolla, CA). The unamplified library was plated at a density of 7.5 x 102 plaque-forming units/140-cm2 Petri dish with Escherichia coli strain Y1090 as its host. After incubation at 42°C and induction with isopropyl-D-thiogalactoside, induced proteins were transferred to nitrocellulose filters that were then incubated with 5% skim milk in Tris-buffered saline (20 mM Tris-HCl, pH 7.6, 137 mM NaCl), followed by screening with the anti-mouse germ cell antiserum. Binding of the anti-serum was detected using goat anti-rabbit Igs coupled with horseradish peroxidase (HRP; Dako Japan, Kyoto, Japan). Positive clones were identified after screening 1 x 106 plaques from the expression library by the antiserum. The phage DNAs isolated were digested with EcoRI, and sizes of their cDNA inserts were checked. The cDNA inserts were further subcloned into the EcoRI site of pUC19 or Bluescript SKII+, and the nucleotide sequences were determined by the dideoxy-termination method with a Taq dye terminator cycle sequencing kit (applied by Biosystems, Foster City, CA). The sequence data of isolated cDNA or amino acids were searched for homology by using the GenBank, EMBL, DDBJ, Swiss-Prot, and PIR data banks.
RNA Extraction and Northern Blot Analysis
Freshly removed mouse organs (C57BL/6 strain) were homogenized in 4.0 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% Sarcosyl, 0.1 M ß-mercaptoethanol. Total RNA was prepared by the method of Chomczynski and Sacchi [15]. The RNA was quantified by optical density measurement, separated by electrophoresis on a 1% formaldehyde gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). After baking for 2 h at 80°C, the nitrocellulose membrane was prehybridized at 42°C for 2 h in a solution containing 50% formamide, 4x saline-sodium citrate (SSC), 5x Denhardt's solution, 0.2% SDS, and 120 mg/ml denatured sonicated salmon sperm DNA, and then hybridized with 32P-labeled cDNA probes, prepared by the random hexamer method, under the same conditions for 24 h. The membrane was washed with 0.1x SSC, 0.1% SDS at 55°C for 30 min.
Immunization and Preparation of Anti-Halap-X Polyclonal Antibody
A fragment of the halap-X DNA (Fig. 1, 540 base pairs [bp] from nucleotide [nt] positions 463–1002) was further subcloned into a pGEX-1 vector to express it as a glutathione S-transferase (GST) fusion protein [16]. The fusion protein was produced in E. coli by isopropyl ß-D-thiogalactopyranoside induction and purified with glutathione-agarose beads. Polyclonal antiserum was prepared by injection of the Halap-X-GST fusion protein into rabbits.
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Immunochemical Identification of Halap-X
Freshly removed mouse organs were dissected, washed twice in cold PBS, and homogenized at 4°C in three volumes of lysis buffer containing 100 mM Tris-HCl (pH 7.6), 0.05% Triton X-100, and 0.1 mM PMSF. These homogenates were centrifuged at 10 000 x g for 30 min at 4°C to remove insoluble debris. Protein concentration was determined by the method of Bradford [17], using BSA as a standard. Each supernatant was analyzed SDS-PAGE as reported by Laemmli [18] or by two-dimensional gel electrophoresis according to the procedure of O'Farrell [19]. After electrophoresis, proteins were transferred to Immobilon P filters (Millipore, Bedford, MA) according to the basic procedure of Towbin et al. [20]. Briefly, gels were incubated in transfer buffer (20 mM Tris-HCl, 150 mM glycine, pH 8.3, with 20% methanol) for 30 min after electrophoresis. Samples were transferred electrophoretically by blotting onto Immobilon P filters. The blotted filters were soaked in blocking solution (TBST: 10% [w/v] low-fat milk in Tris-buffered saline [pH 7.4] with 0.05% Tween-20) for an hour prior to incubation with the anti-Halap-X antiserum. Peroxidase-conjugated swine anti-rabbit immunoglobulins (IgGs; Dako Japan) were used as the secondary antibody and incubated at room temperature in blocking solution. Reactive antigens were detected by the color reaction with diaminobenzidine (DAB) in 50 mM Tris-HCl (pH 7.5) plus 0.3% H2O2.
Immunohistochemistry
Mouse testes were fixed in Bouin's solution, embedded in paraffin, and sectioned at 6 µm. After deparaffinization with xylene, sections were treated with 0.3% H2O2 in PBS, and then reacted with the anti-Halap-X polyclonal antibody for 16 h at 4°C, followed by incubation with HRP-conjugated swine anti-rabbit IgGs (Dako Japan) for an hour at room temperature. Sections were washed with TBST in between the above incubations. Bound peroxidase was visualized with DAB in 50 mM Tris-HCl (pH 7.5) plus 0.3% H2O2. Morphological identification of spermatogenic cells was based on the criteria of Oakberg [21].
Immunoelectronmicroscopy
In this study, cryostat sections were prepared as reported previously [22]. In brief, while adult male mice were anesthetized with diethyl ether, the testes were fixed with intracardiac perfusion with periodate-lysine-2% paraformaldehyde (PLP) in 0.1 M phosphate buffer (PB, pH 7.4). Then the testes were removed and further immersed in the same fixatives for at least 4 h at 4°C. The testes were washed in PB containing sucrose, and quickly frozen in OCT compound (Miles, Elkhart, IN). Samples cut into 6-µm-thick sections were prepared on a Leitz cryostat (model 1720), placed on silane-coated glass slides, and dried. The PLP-fixed cryosections were blocked with normal goat serum and incubated with anti-Halap-X polyclonal antibody for 12 h at 4°C. After washing, the sections were incubated with the HRP-conjugated Fab's fragment of goat anti-rabbit IgGs (Protos Immunoresearch, San Francisco, CA) at a dilution of 1:50 for 2 h, fixed briefly with 0.5% glutaraldehyde, reacted with a DAB-H2O2 solution, and postfixed with 1% osmium tetroxide. Control sections were processed in parallel without the primary antibody. The sections were dehydrated through a gradient ethanol series and propylene oxide and embedded in Epon 812. Samples were cut into ultrasections on an LKB Ultrotome (model 2088), and examined at an accelerating voltage of 75 kV without counterstaining using a Hitachi H-7100 transmission electron microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With anti-germ cell-specific polyclonal antiserum raised in rabbits [11, 12], 15 cDNA clones have been independently isolated by screening 1 x 106 plaques from an unamplified gt11 mouse testis cDNA library. Among them, one positive clone contained an insert of about 0.9 kilobases (kb) in length. The insert operationally designated as Tsg (testis-specific gene) a-8 [12] appeared to lack about 100 bp from the 5' terminal of the mRNA, as judged by Northern blotting. To obtain the full-length positive clone cDNA, we have screened the mouse testis cDNA library in pAP3 neo vector [23] using the Tsg a-8 insert as a probe. One of positive clones contained an insert cDNA of 1080 bp with an open reading frame of 714 bp (nt positions 250–963), encoding 238 deduced amino acids (Fig. 1). Because, at the 5' region, a stop codon was located 150 nt upstream of an ATG codon in the same reading frame (nt 100–102), the ATG sequence at nt positions 250–252 was likely the translation initiation codon for the open reading frame. The cDNA contained a 5'-untranslated region of 249 nt and also a 3'-untranslated region of 117 nt with a poly(A)+ tail of 7 bases following two consensus AATAAA polyadenylation signals at nt positions 1026–1031 and 1054–1059. The deduced protein had a basic region at the NH2 terminus (amino acid residues 1–78), including a nuclear localization signal (NLS) at amino acid positions 64–70, showing a predicted isoelectric point of pH 10.5. In contrast, the rest of the amino acid sequence consisted of acidic amino acid residues including two types of acidic repetitive sequences, showing acidic pI of 2.9 (Fig. 2B). One type repetitive sequence was a set of the nominal repeat sequence A-A-A-A-A-P-E-A-A-A-(S)-(P)-(E)-(S)-(S) occurring at five sites at amino acid residues 79–93, 94–108, 109–123, 124–138, and 139–148, and the other was a set of the nominal repeat sequence P-A-A-P-E-A occurring at four sites at amino acid residues 153–158, 171–176, 180–185, and 189–194 (Fig. 2A). These two types of repeats were rich in alanine and included one typical acidic amino acid, i.e., glutamic acid (E). The predicted isoelectric point of the whole deduced protein was acidic and calculated to be pI 4.5. Computer-assisted sequence analyses revealed no homologous sequence had ever been registered. By analysis of a panel of cDNA samples from interspecific backcross mice, the cDNA was located on the X-chromosome [12]. Thus, we designated this novel cDNA halap-X, standing for haploid-specific alanine-rich acidic protein located on chromosome-X. The sequence of halap-X has been deposited in GenBank under accession no. AB032764.
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Expression of the halap-X mRNA in Various Organs and Testis at Different Developmental Stages of Germ Cells
The expression of halap-X cDNA was investigated in various mouse organs by Northern blotting. The halap-X transcript of 1.1 kb was exclusively detected in the testis (Fig. 3A). During male germ cell development, significant signals were detected in 18-day-old testes at the time when haploid germ cells started to differentiate, and then hereafter increased through to the adult testes (Fig. 3B). These results indicated that the mRNA expression occurred specifically in the testis, and that its expression was precisely timed for the development of male germ cells to haploid spermatids.
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Western Blot Analysis of Halap-X Protein
Western blot analysis with anti-Halap-X antiserum showed that a protein of 55 kDa was specifically present in mouse testicular extracts. No such positive signals were detected in other organs, including the liver, stomach, intestine, brain, kidney, heart, lung, and spleen, neither in the sperm of the vas deferens or epididymis (Fig. 4A). In the testes, such positive signals became apparent from the 23-day-old through to the adult (Fig. 4B). Two-dimensional gel electrophoresis of an adult testicular lysate showed that Halap-X had an isoelectric point of 
4.3–4.7 (Fig. 4C), consistent with the calculated pI of 4.5.
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Immunohistochemistry
Immunohistochemical examination of the adult mouse testis using the anti-Halap-X polyclonal antibody revealed that haploid germ cells were stained positive, whereas somatic cells such as Leydig or Sertoli cells were negative. Expression of the Halap-X protein was first detected in the nuclei of round spermatids at step 1 (Fig. 5C) and became stronger as round spermatids matured (Fig. 5, D–F). Then both the nuclei and cytoplasm became positively stained at step 8 (Fig. 5G). Hereafter, however, the positive staining in the nucleus decreased gradually in concert with a gradual increase in the cytoplasmic signal (Fig. 5, H–J), as morphogenesis proceeded further. The positive stainings in both the nucleus and cytoplasm, however, faded out at the terminal stage of spermiogenesis (Fig. 5K), resulting in negative staining in the sperm (data not shown). Germ cells located within the first layer of the seminiferous epithelium (spermatogonia, leptotene, zygotene, and pachytene spermatocytes) were not stained at all (Fig. 5B). These results were reproducible in all sections (approximately 20 sections were evaluated in 5 mice). These observations were in good agreement with the results of Western blot analysis, indicating that Halap-X is a novel differentiation-associated molecule specifically expressed in the early to middle stages of spermiogenesis.
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Immunoelectronmicroscopy
By immunoelectronmicroscopy, we have carried out further studies of detailed localization of the Halap-X protein in spermatids as well as of its fading-out mechanism during spermiogenesis. Strong immunoreactivity to the protein was detected in the nucleoplasm from step 1 round spermatids throughout to step 8 spermatids (Fig. 6A). However, the reactivity gradually decreased hereafter as spermatids elongated (Fig. 6B) and became hardly detectable at around step 13 spermatids; and further, the immunoreactivity in the nucleoplasm started to disappear particularly at the anteriormost region (Fig. 6B). Concurrently, some of vesicular structures in the elongating spermatid cytoplasm that had been negative at the round spermatid stage (up to step 8) became positive (Fig. 6B). Because immunoreactive intensity in the cytoplasm was not so strong (Fig. 6B) as compared with that of immunohistochemical images (Fig. 5, H–J), we were unable to detect any image of the immunoreactive materials crossing from the nucleoplasm to the cytoplasm.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Halap-X amino acid sequence contained two types of nominal repeat sequences, A-A-A-A-A-P-E-A-A-A-S-(P)-E-S-S and P-A-A-P-E-A (Fig. 2A). These alanine-rich repeat sequences may confer a characteristic and functional structure on Halap-X. The calculated molecular mass of the deduced polypeptide was about 30 kDa, significantly smaller than the 55 kDa estimated by Western blotting. Such differences between the observed and predicted molecular mass have been documented for a number of polypeptides including the centromere autoantigen CENP-B [25], SS-A/Ro [26], amphibian nucleoplasmin [27], calmegin [28], and meichroacidin [11]. Although it is not yet clear why these proteins migrate aberrantly in SDS-polyacrylamide gels, highly negatively charged proteins or their highly negatively charged domains are believed to interfere with SDS binding, thereby influencing their mobility in SDS gels. Overestimation of the molecular size of Halap-X by SDS-PAGE might be due to its highly acidic nature. In fact, two-dimensional gel electrophoresis showed that Halap-X was highly acidic (pI 4.3–4.7) (Fig. 4C). The acidic nature of this protein may also facilitate specific association with some basic chromosomal proteins in haploid cell nuclei.
The halap-X cDNA was located in the middle region of chromosome-X by interspecific backcross mouse mapping, and the corresponding human homologues were at Xq28 and Xq21.1–Xq21.3 [12]. However, in contrast with the genetic analysis of the human Y-chromosome, the X-chromosome has rarely been investigated for male infertility. Further, little is known about the molecular mechanism of sex chromosome inactivation during spermatogenesis, except that participation of the Xist gene in X-chromosome inactivation is well documented. The Xist RNA is present in spermatogenic cells from type A spermatogonia through to spermatids [29], though X-chromosome inactivation is probably confined to the stage of primary spermatocytes, a midpoint between the stages of type A spermatogonia and spermatids. However, in spite of the presence of Xist RNA in spermatids, postmeiotic transcription activities of the X-chromosomal genes Ube1X and mHR6A were reported [30]. Furthermore, intriguingly, the products of the X-chromosomal genes Ube1X and mHR6A expressed in spermatids are both involved in the ubiquitin system. Nucleosome destabilization induced by histone ubiquitination may play a facilitating role in histone-to-protamine replacement [31]. It is possible that Halap-X could also be involved in such replacement events.
During spermatid development, the nucleus undergoes elongation and subsequent condensation. Its final nuclear shaping may partly depend on chromatin condensation during spermatogenesis as well as on a precise organization of DNA within the nucleus. The development from spermatids to mature spermatozoa is accomplished by a series of structural and chemical modifications. Gradual replacement of lysine-rich histones by transition proteins and then by protamines results in compaction of chromatins in the sperm nucleus [31, 32]. This process is termed sperm chromatin condensation [33]. As mentioned above, Halap-X protein has characteristics of the N-terminal basic region and the carboxy-terminal acidic repetitive regions (Fig. 2B). These characteristics may contribute to sperm chromatin condensation by interacting with both DNA and basic nucleoproteins. This way, Halap-X might be involved in a DNA condensation process such as replacing basic proteins bound to DNA and/or its transportation through the nuclear membrane to the cytoplasm forming complexes with the basic proteins.
Abnormality of the nuclear chromatin is probably one of the causes for morphological aberrations of the sperm head. A relationship between abnormal chromatin and male infertility has been reported [34]. Mutations in the human homologue of halap-X cDNA might be involved in male infertility, in the case of sperm head abnormality. Further studies are now in progress to isolate and characterize the human homologue of halap-X cDNA. Furthermore, an antibody against Halap-X could be useful as a unique immunological probe for identifying specific germ cells among a variety of testicular cells to diagnose male infertility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Yoshitake Nishimune, Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita City, Osaka 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp 
Accepted: May 9, 2000.
Received: December 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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