Human Testis-specific Expressed Genes Hsd-3.8 Encoded Protein Function Studies

Fertilization is a complex process which involves the male(spermatozoon) and the female(oocyte) gametes. Successful fertilization is dependent on a well-choreographed series of steps being executed with precise timing and accuracy. Undoubtedly, spermatozoon is one of the most important components. After spermatogenesis, mature sperm must overcome several obstacles and undergo many changes to achieve its destiny. In these processes, many sperm genes are expressed under precise temporal and spatial regulation. Recently, more and more biologists focus on the molecular mechanisms involved in spermatogenesis and fertilization and many key molecules have been found. HSD-2.4 is one of such sperm genes found in our labs.HSD-2.4 was initially isolated by screening human testis λ gt11 library with serum from an infertile woman. In the present study, the full length of HSD-2.4, designated as HSD-3.8 cDNA was isolated and sequenced (GenBank accession number: AF311312). Compared with the genomic sequence, the HSD-3.8 gene can be delineated into 19 exons and 18 introns. By using the method of gene SOEing (gene splicing by overlap extension), the full fragment of the reading frame of HSD-3.8 was obtained. By performing the hydrophilicity analysis, the deduced polypeptide of HSD-3.8 cDNA was found to be a soluble protein without transmembrane regions. The Prosite and SMART search results showed that it has several Casein kinase II phosphorylation sites , Protein kinase C phosphorylation sites, amidation sites , a ATP/GTP-binding site motif A (P-loop) and 9 TPR motifs. The deduced polypeptide has 75% similarity to the mouse tpis and the C-terminal residues has 66% similarity to the human translocase of outer mitochondrial membrane 34(hTOM34).To identify the HSD-3.8 encoding protein in human and rat testis, western blot was performed by using anti-HSD-0.7 polyclonal antibodies raised before as the primary Ab. It was shown that the molecular weight of the protein which could react with the antibodies specifically in the testis extracts was about 55-60 kDa. It is assumed that translational or posttranslational modification is needed for the appropriate function of HSD-3.8 encoding protein.To determine the function of HSD-3.8, yeast two-hybrid systems were used to screen a human ovary and a human testis cDNA libraries to search for the potential ligands that can...