Cloning of human and mouse testis and spermatogenesis related gene 4 (TSARG4), a SPAG4 like gene

Abstract

Objective: Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme. To clone such a gene like Spag4, may do help to understand molecular mechanism of spermatogenesis, especially sperm tail development.Design: Using rat and human Spag4 amino acid sequences to hook testis ESTs in mouse and human EST database, which coded amino acid sequences were highly homology with rat and human Spag4. Cloned human and mouse testis and spermatogensis related gene 4 full-length cDNA sequences. RT-PCR and Northern blot were performed to clarify the patters of these new genes. Changes of mtsarg4 expression in mouse cryptorchidism models were observed by semi-quatitive RT-PCR.Materials and Methods: Using the new program of the National Center for Biotechnology Information (NCBI) and ExPASy Molecular Biology Server to search for proper ESTs. We hooked two ESTs BG101130 and BG100990 which expressed in mouse spermatocytes, and other testis ESTs. By information analysis, we got a hypothetical protein (GenBank accession number AK006225). Meanwhile, we found two EST BG720564 and AI700454 in human testis and the gap between these two ESTs was filled by polymerase chain reaction, and a fragment 1,252 bp which encoded a new gene was obtained. The cDNA sequences were proved in human and mouse testis by RT-PCR. Multi-tissues RT-PCR and Northern blot were performed to clarify the patterns of these new genes. Changes of mtsarg4 expression in mouse cryptorchidism models were observed by semi-quatitive RT-PCR.Results: A new fragment 1,252 bp which encoded a new gene was obtained in human testis and was named TSARG4 (testis and spermatogenesis related gene 4 (GenBank accession number AF401350). Its opening reading frame was 94–1,233 bp. The hypothetical protein AK006225 was named mtsarg4, which full-length 1,136 bp and its opening reading frame was 68–1,114 bp. The homologies of amino acid sequences were 74% between human TSARG4 and mouse mtsarg4 gene and 45% between mtsarg4 and rat Spag4 gene, respectively. Northern blot results showed human TSARG4 gene only expressed in testis, not in spleen, thymus, prostate, overy, small intestine, colon, peripheral blood leukocyte. Multi-tissues RT-PCR results showed mtsarg4 gene expressed specifically in the testis. In mouse cryptorchidism models, with the days increased after operation, the expression of mtsarg4 decreased.Conclusion: Two new genes, human and mouse TSARG4 were identified, which may do help to understand molecular mechanism of spermatogenesis, especially sperm tail development. Objective: Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme. To clone such a gene like Spag4, may do help to understand molecular mechanism of spermatogenesis, especially sperm tail development. Design: Using rat and human Spag4 amino acid sequences to hook testis ESTs in mouse and human EST database, which coded amino acid sequences were highly homology with rat and human Spag4. Cloned human and mouse testis and spermatogensis related gene 4 full-length cDNA sequences. RT-PCR and Northern blot were performed to clarify the patters of these new genes. Changes of mtsarg4 expression in mouse cryptorchidism models were observed by semi-quatitive RT-PCR. Materials and Methods: Using the new program of the National Center for Biotechnology Information (NCBI) and ExPASy Molecular Biology Server to search for proper ESTs. We hooked two ESTs BG101130 and BG100990 which expressed in mouse spermatocytes, and other testis ESTs. By information analysis, we got a hypothetical protein (GenBank accession number AK006225). Meanwhile, we found two EST BG720564 and AI700454 in human testis and the gap between these two ESTs was filled by polymerase chain reaction, and a fragment 1,252 bp which encoded a new gene was obtained. The cDNA sequences were proved in human and mouse testis by RT-PCR. Multi-tissues RT-PCR and Northern blot were performed to clarify the patterns of these new genes. Changes of mtsarg4 expression in mouse cryptorchidism models were observed by semi-quatitive RT-PCR. Results: A new fragment 1,252 bp which encoded a new gene was obtained in human testis and was named TSARG4 (testis and spermatogenesis related gene 4 (GenBank accession number AF401350). Its opening reading frame was 94–1,233 bp. The hypothetical protein AK006225 was named mtsarg4, which full-length 1,136 bp and its opening reading frame was 68–1,114 bp. The homologies of amino acid sequences were 74% between human TSARG4 and mouse mtsarg4 gene and 45% between mtsarg4 and rat Spag4 gene, respectively. Northern blot results showed human TSARG4 gene only expressed in testis, not in spleen, thymus, prostate, overy, small intestine, colon, peripheral blood leukocyte. Multi-tissues RT-PCR results showed mtsarg4 gene expressed specifically in the testis. In mouse cryptorchidism models, with the days increased after operation, the expression of mtsarg4 decreased. Conclusion: Two new genes, human and mouse TSARG4 were identified, which may do help to understand molecular mechanism of spermatogenesis, especially sperm tail development.