Functional Expression Of Three Novel Genes In Human And Mouse Testis

Objective Spermatogenesis is a highly ordered and complicated process of cellular proliferation and differentiation in the testis. Many genes specifically or highly expressed in the testis are involved in the process of spermatogenesis. According to the previous data in the lab, the present study choose three genes, TSG23, T279 and TSC21, to further investigate the expression characteristics in human and mouse testis, which may provide a new method for the diagnosis and treatment of male infertility and a target for male contraception.Methods (1) Identified and screened the genes related to spermatogenesis by microarray. (2) Analyzed the construction of the genes and its protein by bioinformatics methods. (3) Detected the expression of TSG23 and T279 in human and mouse multi-tissues, in the mouse testis in different stages and in different germ cell lines by PCR; and compared the difference of these genes in normal and azoospermic testis. (4) Detected the localization of TSG23, T279 and TSC21 mRNAs in testis by in situ hybridization. (5) Detected the subcellular localization in mouse cells by the transfection of pEGFP-N1-T279 recombinant plasmid. (6) Generated the anti-T279 monoclonal antibody, and anti-TSC21 and anti-TSG23 polyclonal antibodies. (7) Detected the specific of TSG23 and T279 proteins by Western Blot analysis. (8) Detected the expression of T279 protein in human testis by immunofluorescence. (9) Detected the expression and localization of TSG23, T279 and TSC21 proteins in testis and the difference of expression in testis from fertile men and the patients with azoospermia by immunohistochemistry.Results (1) Affymetrix Genechip analysis showed that TSG23 and T279 were highly expressed in adult human and mouse testis. (2) Human TSG23 gene was mapped to chromosome 20q13.12. The full cDNA length of TSG23 was 752bp, which encoded a protein with 227 amino acids. Mouse TSG23 gene was mapped to chromosome 2H3. The full cDNA length was 755bp, which encoded a protein with 226 amino acids. Human T279 gene was mapped to 13q34. The full cDNA length of was 773bp, which encoded a protein with 195 amino acids. Mouse T279 gene was mapped to 8A2. The full cDNA length was 761bp, which encoded a protein with 227 amino acids. Both TSG23 and T279 are human-mouse homologous genes. (3) RT-PCR analysis from multiple human and mouse tissues indicated TSG23 and T279 mRNA was only expressed in the testis. Mouse TSG23 and T279 was detected after day 15 and gradually increased with the age. There was no expression or less expression of TSG23 and T279 in azoospermic testis compared with normal testis. (4) In situ hybridization revealed that TSG23 mRNA was located in spermatocytes and round spermatids of the seminiferous tubules in human and mouse testis, and the expression could be stage specific. Mouse T279 mRNA was mainly located in spermatids and sperms of the seminiferous tubules in mouse testis. Mouse TSC21 mRNA was located in spermatocytes and spermatids in mouse testis. (5) Immunofluorescent assay revealed T279 fusion protein located in endocytoplasmic reticulum of spermatocytes. (6) Generated successfully anti TSG23, T279 and TSC21 antibody. (7) Western blot analysis demonstrated that TSG23 was mainly expressed in human testis with molecular weight of about 23 KD. And T279 was mainly expressed in human testis with molecular weight of about 23 KD. (8) Immunofluorescence showed that T279 was predominantly located in spermatocytes and round spermatids. (9) Immunohistochemistry showed that TSG23, T279 and TSC21 were predominantly located in spermatocytes and round spermatids. And intense expression of TSG23 was observed in stages III and V, whereas it was lower at stages I and II of spermatogenesis. In the testis from patients with azoospermia, the expression of the three proteins were significantly decreased or disappeared in spermatocytes and round spermatids.Conclusions (1) TSG23, T279 and TSC21 were testis-specific/enriched expressed and human-mouse homologous genes. Moreover, expression of TSG23 mRNA in mouse testis was developmental dependent. (2) In the testis from patients with azoospermia, the expression of TSG23, T279 and TSC21 were significantly decreased or disappeared at mRNA and protein levels. (3) TSG23, T279 and TSC21 may participate in testicular development and spermatogenesis, and their abnormal expression may be one of the important factors in male infertility.