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

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.

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。

生物钟基因Bmal1作为生物钟系统的核心转录因子，与雄性生殖密切相关。有研究证据表明，Bmal1-/-小鼠血清睾酮含量显著低于野生型小鼠，但Bmal1调控睾酮合成的具体分子机制尚不清楚。本项目围绕这一科学问题，主要以山羊睾丸间质细胞为研究模型，开展了一系列研究工作。我们研究发现山羊血清睾酮含量存在明显的昼夜节律性变化，大约在ZT11时间点达到峰值。免疫组化分析结果显示，BMAL1高表达于山羊睾丸间质细胞。Cosinor分析结果表明，地塞米松同步化后的山羊睾丸间质细胞中存在生物钟基因（Bmal1，Per1，Per2，Dbp，Nr1d1）和睾酮合成通路关键基因（StAR，Hsd3b2，Cyp17a1，Cyp11a1，Hsd17b3）mRNA表达的24 h节律性变化。进一步研究发现，Bmal1干扰显著抑制了部分生物钟基因（Per2，Nr1d1，Dbp）和睾酮合成通路关键基因（StAR，Hsd3b2）在山羊睾丸间质细胞的表达，造成细胞上清睾酮含量下降。而Bmal1过表达显著提高了生物钟基因Nr1d1和部分睾酮合成通路关键基因（StAR，Hsd17b3）在山羊睾丸间质细胞的表达，细胞上清睾酮含量显著上升。我们的研究结果阐明了山羊睾丸间质细胞生物钟通过影响睾酮合成通路关键基因的转录来调控睾酮合成的分子机制，为后期利用生物钟理论开发雄性生殖调控新技术提供理论依据。本项目计划发表论文3-4篇，截止目前项目负责人以共同通讯作者兼并列第一作者身份发表基金标注论文3篇。本项目共培养硕士生3名，协助培养博士后1名、博士生2名、硕士生1名。