Takashi W Ijiri1, Takahiro Nagase2, Yoichi Matsuda1,3. 1. Laboratory of Cytogenetics, Division of Bioscience, Graduate School of Environmental Earth Science and. 2. Department of Human Gene Research, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan. 3. Laboratory of Animal Cytogenetics, Center for Advanced Science and Technology, Hokkaido University, Sapporo, and.
Abstract
Background and Aims: Isolation and analysis of spermatogenesis-specific genes provide important information for elucidating the mechanisms of human infertility. The aim of the present study was to suggest an effective strategy for the comprehensive isolation of novel genes associated with spermatogenesis in mice. Methods: To isolate novel testis-specific genes associated with meiosis in mice, we constructed a mouse pachytene spermatocyte-enriched cDNA library by the centrifugal elutriation method, and sequenced 120 cDNA clones isolated from the cDNA library. A basic local alignment search tool (BLAST) search was carried out on the cDNA clones to find novel genes and then a detailed expression analysis was carried out by Northern blot hybridization and in situ hybridization. Results: Of the 120 cDNA clones, 35 clones (29%) were novel and 18 clones (15%) were expressed only in the testis. The expression patterns of seven novel testis-specific clones were examined on the testis sections. Three clones were expressed in spermatocytes and other germ cells, and two clones were exclusively expressed in spermatocytes. Amino acid sequences of seven novel testis-specific clones were deduced from their nucleotide sequences, suggesting that two of them contain known functional repeat structures. Conclusions: This method provides a powerful strategy to isolate novel testis-specific genes efficiently. (Reprod Med Biol 2005; 4: 231-237).
Background and Aims: Isolation and analysis of spermatogenesis-specific genes provide important information for elucidating the mechanisms of humaninfertility. The aim of the present study was to suggest an effective strategy for the comprehensive isolation of novel genes associated with spermatogenesis in mice. Methods: To isolate novel testis-specific genes associated with meiosis in mice, we constructed a mouse pachytene spermatocyte-enriched cDNA library by the centrifugal elutriation method, and sequenced 120 cDNA clones isolated from the cDNA library. A basic local alignment search tool (BLAST) search was carried out on the cDNA clones to find novel genes and then a detailed expression analysis was carried out by Northern blot hybridization and in situ hybridization. Results: Of the 120 cDNA clones, 35 clones (29%) were novel and 18 clones (15%) were expressed only in the testis. The expression patterns of seven novel testis-specific clones were examined on the testis sections. Three clones were expressed in spermatocytes and other germ cells, and two clones were exclusively expressed in spermatocytes. Amino acid sequences of seven novel testis-specific clones were deduced from their nucleotide sequences, suggesting that two of them contain known functional repeat structures. Conclusions: This method provides a powerful strategy to isolate novel testis-specific genes efficiently. (Reprod Med Biol 2005; 4: 231-237).