Mouse Mesenchymal Stem Cell Differentiate Into Spermatogonial Stem Cell After Induced By RA And The Study Of Stra8 Promoter Region

Infertility affects 13-18% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The studies accumulated in recent years shows that it is hopeful to cure male infertility. Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra8 (Stimulated by Retinoic Acid) was the one of the gene in this group. In mouse, Stra8 is restrictively expressed in male germ line cells, and its function is related to the development of sperm. In order to investigate the feature of Stra8 gene expression, the 1.4 kb (-1407-+7) promoter region of Stra8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4 kb promoter region and the reporter gene of EGFP that was regulated by 1.4 kb Stra8 promoter. To test the specificity of Stra8 promoter, the vector pStra8-EGFP was transfected into undifferentiated mouse stem cells such as ES-129, bone marrow mesenchymal stem cell (mMSC) and spermatogonial stem cell (mSSC). The results shown that the expression of GFP was only revealed in the mSSC cell, which indicated that Stra8 gene was specially regulated in testis tissue. As the gene marker, vector pStra8-EGFP was then transfected to undifferentiated mMSC cells. After drug selection by G418 for 2 weeks, the mMSC cells were induced by retinoic acid. After 12 hours induction, some induced cells started to express GFP protein which was observed under the fluorescence microscope. At the same time, several stem cell specificity biomarker such as Oct4 and spermatogonial stem cell biomarker such as CyclinA2 and Stra8 were detected in the induced cells by RT-PCR method. This results shown that our construct pStra8-EGFP can be used as a tool to select spermatogonial stem cells though cell sorting equipment such as flow cytometry. Two constructs were built, which contains 405bp Stra8 promoter and 65bp Stra8 promoter. After transfecting these two constructs into mMSCs, the constructs which has 405bp Sta8 promoter including harboring two RXR heterodimers and some key factors of promoter can promote the expression of EGFP, but the constructs which contains 65bp Stra8 promoter can't promoter the expression of EGFP.