Study On The Regulatory Mechanism Of Gap Junction Protein Connexin43 Mediated By Androgen In Mouse Testis Environment

Spermatogonial stem cells(SSCs)determine the productivity of livestock husbandry.Self-renewal and differentiation of SSCs are regulated by many factors.The microenvironment of SSCs is a special structure containing two supporting cells.There are gap junctions between two supporting cells,and Cx43 is a key component of gap junction,and is involved in the regulation of spermatogonial stem cells.It has been reported that gap junction protein Cx43 plays an important role in the development of germ cells and spermatogenesis,and Cx43 has been proved to be the key to male fertility,but the regulatory mechanism is not defined.In this study we investigated expression of Cx43 in mouse testis by immunohistochemistry staining.Cx43 signals were mainly detected in Sertoli cell at the basal membrane,and may also in spermatogonia.In 42-day mouse testis,Cx43 was also detected in the differentiation of spermatogonial cells.Then,the expression of Cx43 was analyzed by semi quantitative analysis.The results demonstrated that the expression of Cx43 in testes of 5-day,10-day and 20-day mice exhibited an increasing trend,but slightly decreased in 42-day testis.Then we investigated the regulatory role of androgen on Cx43 expression by supplement or deprivation of androgen in Sertoli cells,and observed that androgen up-regulated Cx43 expression.Our previous research suggested that AR indirectly regulates WT-1 in Sertoli cells,thus we did ChIP-seq again and the result predicted Cx43 is a direct target gene of WT-1.Then we verified this result by WT-1 overexpression or WT-1 interference in Sertoli cells,and observed that WT-1 negatively regulates Cx43 expression.To further confirm this conclusion,we intraperitoneally injected six week old male mice with busulphan to eliminate germ cells in seminiferous tubules,and observed an intact niche only composed of Sertoli cells only in the tube.Immunohistochemical staining exhibited normal Cx43 expression,indicating a model for microenvironment investigation has been established.Using this in vivo model,we injected bicaluatamide again to analyze the impact of androgen deprivation on Cx43 in Sertoli cells,and observed androgen up-regulated Cx43 in vivo.This study investigates the regulatory role of androgen on Cx43 expression in Sertoli cells,and confirms WT-1 directly inhibits Cx43 expression by binding on Cx43 gene.These results preliminarily reveal the regulatory mechanism of androgen on Sertoli cells-SSCs interaction,and may provide implications for basic research and clinic study.