Kisspeptin调控绵羊GnRH基因表达的作用机制解析

The gonadotropin-releasing hormone (GnRH) operates as the first and most important output signal for the hypothalamic regulation of the downstream elements of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining normal reproductive cycle. Recent development in reproductive regulation has revealed that kisspeptins, a family of neuropeptides encoded by the kiss1 gene and their receptor GPR54, are pivotal upstream regulators of GnRH neurons. Though regulatory process of GnRH expression via kisspeptin has been explored using experimental animals such as mouse, the detailed mechanism is unclear until now. In the proposed research project, firstly we will clone the promoter region of sheep GnRH gene, and look into the potential transcript factors binding loci in the region, and possible transcript factors which interact with the loci; then we will determine the key cis-acting GnRH promoter elements by sequential deletions of the sheep GnRH (mGnRH) gene promoter fused to the luciferase (LUC) reporter gene; thirdly, we will localize the kisspeptin-response element (KsRE) by using formaldehyde assisted isolation of regulatory elements (FAIRE) technique; finally we will identify factors that interact with KsRE of GnRH promoter to regulate sheep GnRH expression by using chromatin immunoprecipitation (ChIP), and confirm their functions by RNAi assay. Take all the information together, we will be able to expound molecular mechanism of GnRH expression regulated by kisspeptin in sheep.

促性腺激素释放激素（GnRH)对调控HPG轴下游其他元件的活动和维持动物正常的繁殖周期具有重要的作用。最近研究表明，几乎所有脊椎动物GnRH的合成和分泌都受kisspeptins及其受体GPR54调控。但有关绵羊GnRH的合成和分泌调控的分子机理依然不清。本项目以当地小尾寒羊为研究对象，首先，克隆绵羊下丘脑GnRH基因的启动子区，利用生物信息学分析启动子特征及相关的转录因子；其次，构建系列缺失的GnRH启动子与荧光素酶融合载体，转染下丘脑原代细胞并kisspeptins处理，利用荧光报告系统筛选绵羊GnRH启动子受kisspeptins控制的关键元件；接着，利用FAIRE技术鉴定kisspeptin处理时被激活的GnRH启动子元件；最后，利用ChIP技术确定与关键元件相互作用的转录调控因子；RNAi验证确定的转录调控因子。综上结果，阐明绵羊kisspeptin调控GnRH基因表达分子机制。

促性腺激素释放激素（Gonadotropin-releasing hormone, GnRH）对调控HPG轴下游其他元件的活动和维持动物正常的繁殖周期具有重要的作用。但绵羊GnRH的合成和分泌调控的分子机理依然不清楚。因此，为了揭示GnRH基因在绵羊繁殖过程中的转录调控机制，构建了8个系列缺失片段荧光素酶报告基因重组载体，与pGL3-Basic以及pRL-SV40共转染到绵羊下丘脑原代神经元细胞，通过双荧光素酶报告系统鉴定出绵羊GnRH基因核心启动子位于-1912bp~-1461bp的区域。利用生物信息学分析发现，该核心启动子区存在一个转录因子Otx-2（-1786~-1770bp）的结合位点，并在不同的物种中高度保守。利用定量PCR分析Kiss-1, Otx-2与GnRH基因表达模式，结果表明Kiss-1, Otx-2与GnRH均在绵羊垂体和下丘脑中表达，且Kiss-1, Otx-2与GnRH在繁殖期和繁殖间期、日间和夜间下丘脑中的表达模式一致。另外，通过Kisspeptin处理体外培养的下丘脑神经元细胞，结果显示Kisspeptin会诱导影响GnRH和Otx-2的表达。这些研究结果表明，kisspeptin介导转录因子Otx-2 来调控 GnRH 基因的表达，从而实现对绵羊的繁殖调控。.对绵羊SPATA7、SPATA16、SPEF2、FSHR、LHCGR、MBL2、CD4和ID2的48个SNPs进行遗传多态性分析。SPATA7基因的rs418082626A>G位点与精液量密切相关；SPEF2基因的rs400991588、rs407363126、rs419749948G位点均与精子活率密切相关。SPATA7基因的A-G-G、SPATA16基因的A-C-C、SPEF2基因的G-G-T均与精子顶体完整率密切相关。这3个候选基因的若干SNPs与多项血液生理生化指标密切相关。rs398233545与种公羊精液品质性状无关；而rs398733991、rs401909751、rs403107881、rs420477086与种公羊部分精液品质性状有密切关联，这些SNP有望作为分子标记应用于肉羊育种。