Biomedical subjects

Yoshitake Nishimune

Publications and source records attributed to Yoshitake Nishimune.

53 records · Page 3Linked to original sources

PubMed2002

Type XXVI collagen, a new member of the collagen family, is specifically expressed in the testis and ovary.

HSP47 is a collagen-specific molecular chaperone that specifically recognizes and binds to the triple helical domain of various types of collagens. Here we report the cloning of the entire coding region of a novel collagen-like protein by yeast two-hybrid screening of a 17.5-day whole mouse embryo cDNA library using HSP47 as a bait. The cDNA of this protein and its deduced amino acid sequence are 2,690 bp and 438 amino acids long, respectively. The protein contains two clusters of Gly-X-Y collagenous repeats and three noncollagenous domains. Northern blot analysis showed that its mRNA is specifically expressed in the testis and ovary in adult tissues and that expression in these tissues is highest in the neonate. Biochemical characterization of this protein revealed that its proline residues are hydroxylated, it undergoes N-linked glycosylation, it forms trimers, and it is secreted in vitro. Immunohistochemical studies showed that the myoid cells and the pre-theca cells synthesized it in the testis and ovary, respectively, resulting in the accumulation of this protein in the extracellular spaces of these organs. These observations suggest that this protein is a new member of the collagen protein family. We thus designated this protein as type XXVI collagen.

Aging↗

PubMed2002

Nectin couples cell-cell adhesion and the actin scaffold at heterotypic testicular junctions.

Actin-based cell-cell adherens junctions (AJs) are crucial not only for mechanical adhesion but also for cell morphogenesis and differentiation. While organization of homotypic AJs is attributed mostly to classic cadherins, the adhesive mechanism of heterotypic AJs in more complex tissues remains to be clarified. Nectin, a member of a family of immunoglobulin-like adhesion molecules at various AJs, is a possible organizer of heterotypic AJs because of its unique heterophilic trans-interaction property. Recently, nectin-2 (-/-) mice have been shown to exhibit the defective sperm morphogenesis and the male-specific infertility, but the role of nectin in testicular AJs has not been investigated. We show here the heterotypic trans-interaction between nectin-2 in Sertoli cells and nectin-3 in spermatids at Sertoli-spermatid junctions (SspJs), heterotypic AJs in testes. Moreover, each nectin-based adhesive membrane domain exhibits one-to-one colocalization with each actin bundle underlying SspJs. Inactivation of the mouse nectin-2 gene causes not only impaired adhesion but also loss of the junctional actin scaffold at SspJs, resulting in aberrant morphogenesis and positioning of spermatids. Localization of afadin, an adaptor protein of nectin with the actin cytoskeleton, is also nectin-2 dependent at SspJs. These results indicate that the nectin-afadin system plays essential roles in coupling cell-cell adhesion and the cortical actin scaffold at SspJs and in subsequent sperm morphogenesis.

Actins↗

PubMed2002

Spermatogonia-dependent expression of testicular genes in mice.

Spermatogenesis is initiated by the interaction of germ cells and somatic cells in seminiferous tubules. We used cDNA microarrays and representational difference analysis to identify genes that are expressed in the testis of the jsd/jsd mutant mouse, which contains only type A spermatogonial germ cells and Sertoli cells, but not in the testis of the W/W(v) mutant mouse, where Sertoli cells but few germ cells are present. We isolated 20 known genes and 4 novel genes, including 2 genes encoding lipocalin family members (prostaglandin D synthetase and 24p3) and 2 tumor suppressors (protein tyrosine phosphatase TD14 and Sui1). All 24 of these jsd/jsd-derived genes were highly expressed in the cryptorchid testis as well as in the jsd/jsd testis. This indicates that their selective expression is not directly caused by the as-yet-uncharacterized jsd gene product, but is rather correlated to the cessation of spermatogonial differentiation. In situ hybridization analysis and flow cytometric sorting followed by reverse transcriptase-PCR revealed that these genes are expressed in both the spermatogonial germ cells and the somatic cells in the developing gonads and adult testes. As the mRNAs of these jsd/jsd-derived genes were barely detectable in the W/W(v) testis, we propose that early spermatogonial germ cells regulate the expression of a group of testicular genes.

Animals↗

PubMed2002

Induction of instability of p34(cdc2) expression by treatment with cisplatin (CDDP) in mouse teratocarcinoma F9 cells.

p34(cdc2) is a protein kinase that plays an important role in the control of the cell cycle and regulation of activity of the tumor suppressor gene. Previously, we demonstrated that cisplatin (CDDP) induced growth suppression resulting in differentiation of teratocarcinoma F9 cells. In the present study, we investigated the mechanism of cell cycle retardation by CDDP in F9 cells, focusing on p34(cdc2). After the induction of differentiation with CDDP, F9 cells were arrested at the late S+G2/M. After treatment with CDDP, the level of the expression of cdc2 mRNA did not change. However, the half-life of p34(cdc2) was greatly reduced, thus, the level of p34(cdc2) protein was decreased. These findings suggest that the cell cycle arrest by CDDP is due partly to the induced instability of p34(cdc2) in F9 cells.

Animals↗

PubMed2002

Homeostatic regulation of germinal stem cell proliferation by the GDNF/FSH pathway.

Stem cell regulatory mechanisms are difficult to study because self-renewal and production of differentiated progeny, which are both strictly controlled, occur simultaneously in these cells. To focus on the self-renewal mechanism alone, we investigated the behavior of germinal stem cells (GSCs) in progeny-deficient testes with defective GSC differentiation. In these testes, we found that the proliferation of undifferentiated spermatogonia, some of which are GSCs, was accelerated by high concentrations of glial cell line-derived neurotrophic factor (GDNF). Furthermore, we found that follicle-stimulating hormone (FSH) stimulation via homeostatic control was one of the major regulators of GDNF concentration. These results suggest that in mammalian testes, GSC proliferation and population size are regulated homeostatically by the GDNF/FSH pathway.

Animals↗

PubMed2002

Use of stepwise subtraction to comprehensively isolate mouse genes whose transcription is up-regulated during spermiogenesis.

We report the isolation of 153 mouse genes whose expression is dramatically up-regulated during spermiogenesis. We used a novel variation of the subtractive hybridization technique called stepwise subtraction, wherein the subtraction process is systematically repeated in a stepwise manner. We named the genes thus identified as TISP genes (transcript induced in spermiogenesis). The transcription of 80 of these TISP genes is almost completely specific to the testis. This transcription is abruptly turned on after 17 days of age, when the mice enter puberty and spermiogenesis is initiated. Considering that the most advanced cells present at these stages of spermatogenesis are the spermatids, it is likely that we could isolate most of the spermatid-specific genes. DNA sequencing revealed that about half the TISP genes are novel and uncharacterized genes, confirming the utility of the stepwise subtraction approach for gene discovery.

Animals↗

PubMed2002

Cloning and characterization of a human orthologue of testis-specific succinyl CoA: 3-oxo acid CoA transferase (Scot-t) cDNA.

Succinyl CoA: 3-oxo acid CoA transferase (scot; EC 2.8.3.5) is a key enzyme for metabolism of ketone bodies. Previously, we have cloned a testis-specific succinyl CoA: 3-oxo acid CoA transferase (scot-t) cDNA from a subtracted cDNA library of mouse testis and demonstrated its expression to be specific to late haploid male germ cells. In this study, the human orthologue of mouse scot-t was cloned and characterized. The entire coding region of the mRNA and the deduced amino acid sequence of human Scot-t (h-Scot-t) showed 75.4 and 75.8% identity with mouse scot-t respectively. The mRNA was exclusively expressed in the testis, and the protein was localized to the midpiece of ejaculated spermatozoa where mitochondria exist. We showed that the h-Scot-t gene was intronless by using a polymerase chain reaction technique and that a non-functional pseudogene, 98% similar to h-Scot-t, was also located on the human genome (1p33-34.3). Furthermore, the genomic structure of the actual h-Scot-t transcription unit was found to be located in an intron (1p34.1-35.3) of the bone morphogenetic protein 8 (BMP8) gene. In conclusion, we have demonstrated that h-Scot-t is a single intronless gene specifically expressed in the testis.

Amino Acid Sequence↗

PubMed2002

Cloning and characterization of the human tektin-t gene.

Tektin-t is a part of the tektin protein family, the members of which form filamentous polymers in the walls of ciliary and flagellar microtubules. In mice, tektin-t protein has been localized to the tail of mature sperm, suggesting that it has a role in the formation of sperm flagella and/or sperm motility. In the present study, we have cloned a human orthologue of mouse haploid germ cell-specific tektin-t. The cloned human cDNA and the predicted amino acid sequence showed 82 and 83% identity with mouse tektin-t respectively. Included were a sequence conserved in the tektin B1 family, the TEKTIN2 motif, and the consensus sequence in the tektin protein family composed of nona-peptide. Northern blot analysis of mRNA isolated from various human organs showed that the transcript was approximately 1.7 kb in length and strongly expressed in the testis. Human (h-)tektin-t protein, having a molecular weight of 54 kDa, was exclusively expressed in the testis, whereas two additional stronger bands of 46 and 56 kDa existed in sperm. The h-tektin-t localized specifically to the principal piece of flagella and to the post-acrosomal region. The h-tektin-t gene was assigned to chromosome 1 by a radiation hybrid mapping technique.

Amino Acid Sequence↗

PubMed2002

Molecular cloning and characterization of the human orthologue of male germ cell-specific actin capping protein alpha3 (cpalpha3).

We report here the molecular cloning and characterization of a novel human actin capping protein alpha3 (cpalpha3) cDNA, an orthologue of the mouse male germ cell-specific cpalpha3, and the organization of the human cpalpha3 genomic structure. The entire coding region of the human cpalpha3 cDNA showed 82.1% similarity with the mouse cpalpha3. 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 cpalpha3 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 CPalpha3 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 CPalpha3 coincided with the species-specific distribution of actin in human sperm. The human cpalpha3 gene was mapped to chromosome 12p12 by computer database cloning of human genomic DNA and was proven to be intronless. CPalpha3 may play a physiologically important role in sperm architecture as well as in fertility of the human male.

Actins↗

PubMed2002

Electroporated transgene-rescued spermatogenesis in infertile mutant mice with a sertoli cell defect.

The molecular basis of most human male infertility arising from spermatogenesis disruption is poorly understood because of a lack of useful investigation systems. To study the roles of the supporting Sertoli cells in mammalian spermatogenesis, we improved an electroporation technique for seminiferous tubules in vivo. Because Sertoli cells barely proliferate in mature testis, linear transgenes are not incorporated into the genome and quickly degrade. However, circular expression vector is stably expressed in Sertoli cells for a long period. By electrotransformation of a complete cDNA, we rescued defective spermatogenesis in infertile Sl(17H)/Sl(17H) mutant mice with partial dysfunction of stem cell factor in Sertoli cells. Application of this gene transfer system will facilitate both the understanding of spermatogenesis and the development of new gene therapies for human male infertility.

Actins↗

PubMed2002

Restoration of spermatogenesis and fertility in azoospermic mutant mice by suppression and reelevation of testosterone followed by intracytoplasmic sperm injection.

Advances in assisted reproduction techniques such as in vitro fertilization and intracytoplasmic sperm injection have made paternity possible for many patients with male infertility. However, at least some sperm or spermatids are required for these techniques to be successful, and patients incapable of producing spermatids cannot be helped. Male mice homozygous for the mutant juvenile spermatogonial depletion (jsd) gene show spermatogonial arrest and an elevated intratesticular testosterone level like many other experimental infertility models such as those with iradiation- or chemotherapy-induced testicular damage. In this category of infertile males, suppression of the testosterone level induces spermatogonial differentiation to the stage of spermatocytes but no further. In the present study with jsd mutant mice, we induced spermatogenesis first to spermatocytes and then to elongated spermatids by suppression of testosterone levels with a GnRH antagonist, Nal-Glu, at a dose of 2500 microg kg(-1) day(-1) for 4 wk and then withdrawal of Nal-Glu. Spermatids were seen in the cross-sections of seminiferous tubules in all mice treated by administration and subsequent withdrawal of Nal-Glu. Four weeks after withdrawal of Nal-Glu, some of the germ cells differentiated into elongated spermatids. Supplementation with testosterone and Nal-Glu after 4 wk of treatment with Nal-Glu alone also induced spermatogenesis similar to the induction by withdrawal of Nal-Glu. Thus, we ascribe the restoration of the differentiation of spermatocytes to spermatids to reelevation of the testosterone level. Furthermore, we successfully rescued male sterility in jsd mice by subsequent intracytoplasmic sperm injection using the elongated spermatids induced by the programmed hormone therapy.

Animals↗

PubMed2002

Molecular cloning and characterization of a complementary DNA encoding sperm tail protein SHIPPO 1.

Formation of the tail in developing sperm is a complex process involving the organization of the axoneme, transport of periaxonemal proteins from the cytoplasm to the tail, and assembly of the outer dense fibers and fibrous sheath. Although detailed morphological descriptions of these events are available, the molecular mechanisms remain to be fully elucidated. We have isolated a new gene, named shippo 1, from a haploid germ cell-specific cDNA library of mouse testis, and also its human orthologue (h-shippo 1). The isolated cDNA is 1.2 kilobases long, carrying a 762-base pair open reading frame that encodes SHIPPO 1, a sperm protein predicted to consist of 254 amino acids. The amino acid sequence includes 6 Pro-Gly-Pro repeats, which are also present in the human orthologue protein (hSHIPPO 1) as well as in 2 other newly reported proteins of Drosophila melanogaster. Transcription of shippo 1 is exclusively observed in haploid germ cells. Antibody raised against SHIPPO 1 identified a testis-specific M(r) 32 x 10(-3) band in Western blot analysis. The protein was further localized in the flagella of the elongated spermatids and along the entire length of the tail in mature sperm. SHIPPO 1 in sperm is resistant to treatment with nonionic detergents and coextracted with the cytoskeletal core proteins of the mouse sperm tail.

Amino Acid Sequence↗

PubMed2002

Development of the stages of the cycle in mouse seminiferous epithelium after transplantation of green fluorescent protein-labeled spermatogonial stem cells.

To study the mechanism of male germ cell differentiation, testicular germ cells carrying green fluorescent protein (GFP) as a transgene marker were transplanted into infertile mouse testis. Fluorescence-positive seminiferous tubule segments colonized with GFP-labeled donor germ cells were isolated and measured, and differentiated germ cells were analyzed in living squashed preparations. Cell associations in normal stages of the seminiferous epithelial cycle were also studied and used as a reference. Two months after transplantation, the average length of the colonies was 1.3 mm. The cell associations of transplanted colonies were consistent with those of normal stages of the cycle. However, stages of the cycle were not necessarily identical in different colonies. Three months after transplantation, the average length of transplanted colonies was 3.4 mm, and the cell association in every portion of a colony was similar to that of the corresponding stage of the cycle. Even in long fused colonies made by transplantation of a higher concentration of male germ cells, the cell association patterns in various regions of a single colony were similar and consistent with those of some of the normal stages of the cycle. Development of different stages inside the colony was observed by 6 mo after transplantation. These results indicate that the commencement of spermatogonial stem cell differentiation occurs randomly to develop different stages of the cycle in different colonies. Then, each colony shows one single stage of the cycle for a long time, even if it becomes a very large colony or fuses with other colonies. These observations indicate the existence of some kind of synchronization mechanism. By 6 mo, however, normal development of the stages of the cycle appeared in seminiferous tubules.

Animals↗

PubMed2002

Molecular cloning and characterization of oppo 1: a haploid germ cell-specific complementary DNA encoding sperm tail protein.

We isolated a cDNA clone specifically expressed during spermatogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1085 nucleotides and had an open reading frame of 870 nucleotides encoding a putative protein of 290 amino acid residues. Northern blot analysis revealed a 1.2-kilobase mRNA exclusively expressed in the testis in adult mice; the mRNA was first detected late pachytene stage, and expression increased as the animals matured. The protein encoded by the mRNA had a molecular weight of approximately 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.

Amino Acid Sequence↗

PubMedDate not supplied

Identification and characterization of an antigen recognized by monoclonal antibody TRA 54 in mouse epididymis and vas deferens.

Spermatozoa in testicular fluid are known to have weak forward motility and cannot fertilize eggs. The epididymis is known to participate in sperm maturation leading fertilization, but little is known about the specific epididymal molecules involved in the modification of sperm. In this study, we characterized the new pattern of expression of an antigen previously identified in testicular germ cells by monoclonal antibody (mAb) TRA 54. This antigen is expressed in epididymal and vas deferens epithelial cells in mice older than 24 days but not during younger developmental stages. Evaluation by immunohistochemistry shows that antigen expression is limited to the cytoplasm of a specific cell population of epithelia along the epididymal regions and vas deferens of adult mice. The molecules synthesized and released by epididymal and vas deferens epithelia into their lumen seem to bind on spermatozoa moving down through the ducts. Immunoblot analysis showed that the molecules recognized by mAb TRA 54 in testis and epididymis were similar and share a common epitope involving carbohydrate domains. Interestingly, the antigens identified in epididymal and vas deferens epithelial cells were expressed independently of testicular germ cells and are produced in an androgen-dependent manner. Finally, the molecules recognized by mAb TRA 54 seem to play an important role in spermatogenesis, as well as in epididymal function related to spermatozoa maturation and ability to fertilize.

Androgens↗

PubMedDate not supplied

Identification of human HAPRIN potentially involved in the acrosome reaction.

The acrosome reaction in sperm is an exocytotic event required for fertilization. Previously, we isolated a novel haploid-germ-cell-specific gene in the mouse; this gene, named haprin, encodes the RING-finger, B-box-type zinc finger and coiled-coil domain (RBCC) motif protein and may be involved in the acrosome reaction. Here we report the molecular cloning and characterization of a human haprin ortholog. The deduced amino acid sequence of human haprin had 91% identity with the mouse ortholog. Transcripts of human haprin were detected exclusively in the testes. Western blot and immunocytochemical analyses detected HAPRIN protein in the testes and sperm. The protein was localized in the acrosomal region of sperm and disappeared after the acrosome reaction. Our results indicate that the function of HAPRIN is highly conserved in humans and mice and that the protein could play an important role in the regulation of the acrosome reaction.

Acrosome Reaction↗

PubMedDate not supplied

Single-nucleotide polymorphisms and mutation analyses of the TNP1 and TNP2 genes of fertile and infertile human male populations.

Previously, we examined the relationship between protamine gene variations and human male infertility. In this study, we show specific variability in the transition nuclear protein genes (TNPs) of sterile male patients. Transition nuclear proteins (TPs) are major nuclear proteins that replace nuclear histones, leading to eventual substitution by protamines during human spermiogenesis. Analysis of the human TNP1 and TNP2 gene sequences in 282 sterile male patients and 270 (TNP1) and 266 (TNP2) proven-fertile male volunteers revealed 5 amino acid substitution-causing single nucleotide polymorphisms (SNPs) in the open-reading frame of the TNP2 gene. On the other hand, a deletion of 15 nucleotides, which encompassed the recognition site for the cAMP response element (CRE) transcription factor, was found in the 5'-promoter region of the TNP1 gene in infertile men. This deletion reduces TNP1 expression and may cause human male infertility.

Amino Acid Sequence↗