BMAL1基因介导的山羊睾丸间质细胞生物钟调控睾酮合成的分子机制研究

睾丸间质细胞睾酮的合成分泌与雄性生殖力密切相关，其调控功能紊乱可导致雄性生殖力低下，甚至不育。睾酮合成的调控机制是当前生殖生物学研究的一项重要课题。生物钟基因BMAL1-/-小鼠研究证实生物钟影响睾酮的合成，但其具体分子机制尚不清楚。本研究拟以“BMAL1介导的睾丸间质细胞生物钟调控睾酮合成”为理论假说，从睾丸间质细胞生物钟调控下游生物钟控制基因转录为切入点，采用分子生物学和细胞生物学技术，在山羊睾丸间质细胞上开展研究。本研究将在首先明确生物钟基因在山羊睾丸、睾丸间质细胞时空分布表达规律的基础上，运用RNA测序、BMAL1过表达及沉默、启动子活性分析等技术探寻确证睾丸间质细胞中睾酮合成通路生物钟控制基因，从而阐明BMAL1调控睾酮合成的分子通路。本研究将揭示生物钟调控睾酮合成的新机制，为研究雄性不育以及通过靶向调控睾丸间质细胞生物钟提高雄性生殖力提供理论依据。

Testosterone synthesis and secretion of leydig cells are closely related to male fecundity. The abnormality of testosterone synthesis could cause low male fecundity, and even male stertility. The regulation mechanism of testosterone synthesis is an important research topic of reproductive biology at present. It is proved that circadian clock system has an effect on testosterone synthesis in BMAL1-/- mice. However, the molecular mechanism is still unclear. In order to test the hypothesis that leydig cells circadian clock regulates testosterone synthesis mediated by BMAL1, this study takes transcriptional control of downstream clock controlled genes by leydig cells circadian clock as point of penetration, and the goat leydig cells line is selected as a cell model. The molecular biological techniques and cellular biological techniques will be adopted in this study. Firstly, the temporal and spatial expression pattern of circadian clock genes in goat testis and leydig cells will be investigated. And then, the clock controlled genes of testosterone biosynthetic pathway in goat leydig cells will be explored and confirmed using RNA sequencing, BMAL1 gene over-expression and silencing, and promoter assay. Thus the molecular pathways of BMAL1 on regulating testosterone synthesis will be elucidated. In conclusion, this study will uncover a new mechanism of circadian clock on regulating testosterone synthesis, which will provide us theoretical basis for male stertility research, and improving male fecundity through the targeted regulation of leydig cells circadian clock.