Biomedical subjects

Hiromitsu Tanaka

Publications and source records attributed to Hiromitsu Tanaka.

44 records · Page 3Linked to original sources

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

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

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↗