A New Spermatogenic Specific Gene Protein Separation, Identification And Functional Study

1-, 3- and 8-week old male SD rats were used in previous work of our group, Morphological observations and mRNA differential display (DDRT-PCR) were combined to compare specific gene expression in the spermatogenesis between different age-groups. A novel gene fragment named CG14 was found to be specific in 8-week old male SD rat. The in situ hybridization with the probe of CG14 demonstrated that the positive nucleic acid signals were distributed specifically in the primary spermatocytes.In the present study, northern blot analysis revealed the presence of a 1.15-kb transcript in testis and an additional 1.350-kb transcript in epididymis. No transcript was observed in cerebrum, heart, liver and kidney.CG14, as well as Factor Xa proteolytic cleavage site were ligated into the bacterial expression vector pGEX-3X and used to transform Escherichia coli strain BL21. Transformed bacteria, induced by IPTG, expressed a 36 kDa fusion protein comprising glutathione S-transferase (GST) and the target protein. The fusion protein, detected in a soluble fraction on SDS-PAGE analysis, was purified by a batch method using Glutathione Sepharose 4B, yielding 6 mg fusion protein / L culture, and used for preparation of antisera in rabbits. The GST-CG14 fusion protein was treated with factor Xa to isolate the target protein from the fused form. Western blotting indicated that the specific antibodies against the target protein, of which the molecular weight is approximately 8kD, were raised.In an effort to understand its function, we sought to localize this target protein in seminiferous epithelium by immunofluorescence. Negative control with normal rabbit serum was established. The result indicated that specific fluorescence was present through out the cytoplasm of primary spermatocytes and spermatid in a punctate pattern. Further more, proteins from adult SD rat cerebrum, heart, liver, kidney and epididymis, as well as one week old rat testis were performed western blot with the antiserum respectively, the results indicate that the target protein exsits only in adult SD rat testis.In a separate set of experiments, based on sequence of CG14...