Expression Of Stra8 In Spermatogensis

Retinoic acid, a metabolite produced from vitamin A (retinol), plays a key role in spermatogenesis. Stra8 (stimulated by retinoic acid gene) can be specifically induced by RA. It is specifically and periodically expressed in the gonads. Stra8 knockout male mice were infertile. However, its function in spermatogensis has not been explained. In this study, some experiments about the expression of Stra8 in spermatogensis have been carried out from the following three aspects: (1)Prokaryotic expression of Stra8 and preparation of its polyclonal antibody; (2) Stably expression of Stra8 in mouse spermatogonia-dervived cells by Lentivirus system; (3) Expression of Stra8 in testicular tissues of different model mice. This study established the basis for further research on mechanism of Stra8 in spermatogensis.Firstly, recombinant plasmid of Stra8 was prepared. And then the polyclonal antibody of Stra8 was acquired from immunized mice by using purified Stra8 prokaryotic protein.Stra8 cDNA sequence was amplified by PCR and was inserted into pET-28a+ vector to construct the recombinant prokaryotic expression vector Stra8-pET28a. The recombinant was transformed into BL21 and induced by IPTG. Stra8 prokaryotic expression protein was obtained and purified by His-Nickel affinity chromatography. After renaturation, Stra8 protein was infected into peritoneal cavity of the BalB/C mice. Two months later, the polyclonal antibody against Stra8 was collected and identified by Western blot, immunofluorescence, ELISA. The results showed that the prepared polyclonal antibody has high titer and specificity.Secondly, Lentiviral vector carrying Stra8 gene has been successfully constructed and maintains high expression in cells.The whole open reading frame of Stra8 gene was inserted in the lentivirus-GV341(a lentiviral expression vector). Then GV341-Stra8 was transfected into 293T cells together with the other two helper vectors. Two days after, the lentiviral particles were collected and used to transfect mouse spermatogonia GC1 cells. The stable over-expression of Stra8 protein was identified by Western Blot and immunofluorescence. The results confirm that the cell line expressing enhanced Stra8 protein and expressed in cytoplasm and nuclei simultaneously, especially in cytoplasm.Thirdly, Vitamin A deficiency (VAD) male mice and VAD recovery male mice model have been successful prepared. The expression of Stra8 in VAD male mice and VAD recovery male model was studied by Western Blot and immunofluorescence.Preperation of VAD model mice:After feeded with Vitamine A deficient diet for two weeks, male and femal mice were mated. The Newborn male mice were feeded 13-14 weeks with VAD diet. Preparation of VAD recovery model mice: Feeded the VAD mice with normal diet for thirty-five days. Paraffin sections of different stages testis were prepared and stained by H&E. And then Western blot and Immunohistochemical methods were applied to observe the expression of Stra8 in these testis. The results showed that the process of spermatogenesis was damaged after the mice kept in VAD diet for 81 to 100 days. The histological analysis showed that there were only Sertoli cells and a small amount of Spermatogonium cells in the seminiferous tubules. In VAD recovery male mice, primary spermatocyte began to generated in seminiferous tubule after feeded with normal diet 9 days. The number of spermatocyte was gradually increased at the following days. After 35 days, structure of the testis were completely rescued to normal. From 72nd to 100th day, the expression of Stra8 protein in VAD mice tapered off; while with the improvement of spermatogenesis, the expression of stra8 protein gradually increased in VAD recovery male mice model.In conclusion, we have prepared polyclonal antibody against Stra8 and established a Stra8-overexpressed spermatogonia cell line. We also successfully prepared two kind of mice models (VAD and VAD recovery mice) and analyzed the expression of Stra8 in the testis of these mice. The results here provided an experiment foundation for further studies of Stra8 function.