BAD基因调控绵羊季节性发情启动的机制研究

The breeding season of the most domestic ewes normally begins at some point following the summer solstice, and the period of anestrus normally begins in late winter or early spring. Due to a rather long anestrous period it is usually only possible for an ewe to give birth once per year. It is important to know the mechanisms of the onset of estrus in order to increase the fertility of ewes and to obtain lambs twice in one year or three times in two years. The preliminary RNA-Seq of our group revealed that BAD gene was differentially expressed in the different reproductive status between two breeds of sheep and enriched in insulin signaling pathway. It is relatively explicit that BAD gene plays an important role in mediating communication from different upstream signal (insulin) transduction pathways to the Bcl-2 regulated apoptotic decision step in the field of medicine and model animals, but the mechanism of how does it regulate the onset of seasonal estrus in Sheep remains unclear, so we put forward a scientific hypothesis that BAD gene enriched in insulin signaling pathway regulate the onset of seasonal estrus in sheep. In this project, the concentration of hormones in peripheral blood will be tested，the expression pattern of the BAD gene and the other important genes in this pathway and the BAD phosphorylation in ovary will be analyzed in the anestrus, proestrus, estrus and diestrus of Small Tail Han sheep (year-round estrus) and Tan sheep (seasonal estrus). The association between hormone level of insulin and BAD gene expression will also be determined in cultured granulosa cells to test the function of BAD in ovarian. The role and molecular mechanism of BAD gene in insulin signaling pathway regulating the onset of seasonal estrus will be elucidated according to the results in in vivo and in vitro, thus validating and elucidating of our scientific hypothesis. The genetic basis onset of sheep seasonal estrus will be preliminary revealed, this will provide theoretical basis for improvement of lambs production and cultivating of new sheep breeds in China.

多数绵羊为季节性发情品种，在夏末秋初开始发情，一年只能繁殖一胎。因此，要增加胎次，启动非繁殖季节发情尤为重要。本课题组前期卵巢转录组测序结果显示，胰岛素通路中BAD基因只在发情前期高表达，其主要功能在医学和模式动物领域已较为明确，但与绵羊发情启动的关系尚不清楚，因此提出胰岛素调控卵巢中BAD基因影响发情启动的科学假设。本项目将以不同发情类型（常年发情和季节性发情）绵羊为研究对象，研究不同繁殖状态下（休情期、发情前期、发情期和发情间期）外周血中胰岛素及性激素浓度变化规律；分析卵巢组织和胰岛素诱导的颗粒细胞中BAD基因及胰岛素通路关键基因表达量；探索BAD基因表达水平变化对颗粒细胞激素分泌和凋亡的影响。在群体和细胞水平阐明不同发情类型的绵羊中胰岛素调控BAD基因发情启动的作用机制，验证并阐释本课题组提出的科学假设，初步揭示季节性发情启动的遗传基础，为增加绵羊胎次及新品种培育提供理论基础。

中国大多数绵羊是短日照发情的季节性发情品种，因此羊肉的四季均衡供应受到了限制。本实验室前期针对不同发情期绵羊卵巢RNA-Seq测序结果表明，BCL2相关的细胞凋亡拮抗因子(BCL2-associated agonist of cell death, BAD)基因随着绵羊由发情前期到发情期的转换，表达量也随之发生显著下降。绵羊发情受到颗粒细胞凋亡的影响，属于Bcl-2家族的凋亡相关基因BAD参与了卵巢中卵泡发育和闭锁。本研究以常年发情的小尾寒羊和季节性发情的滩羊为研究素材，分析BAD所在凋亡通路中相关基因Bax、Bcl-2和Bcl-xL在不同繁殖状态下参与繁殖活动中表达规律，并利用RNA干扰和过表达的卵巢颗粒细胞中BAD基因，初步揭示了BAD基因可能通过促进卵巢颗粒细胞凋亡而降低孕酮水平，进而调控绵羊发情启动。主要结果如下：.成功获得绵羊BAD基因的全长cDNA序列，并进行蛋白序列预测。检测了该基因在小尾寒羊中的表达情况。发现BAD基因在绵羊下丘脑、肺和垂体中的表达水平最高，而在肝脏中表达水平最低。卵泡期和黄体期的小尾寒羊中，凋亡相关基因BAD、Bcl-2和Bcl-xL在黄体期卵巢的表达量均高于卵泡期，这3个基因在黄体期输卵管的表达量均低于卵泡期表达量。卵巢中，Bax基因表达规律与上述三个基因相反，在输卵管中，黄体期Bax基因的表达量极显著高于卵泡期（P<0.01）。在小尾寒羊黄体期和卵泡期卵巢中，BAD和Bcl-2基因表达量存在显著差异，表明它们可能主要参与调控小尾寒羊发情周期中卵泡发育和闭锁，并参与小尾寒羊黄体期到卵泡期的转换从而启动发情。.在绵羊卵巢颗粒细胞中进行BAD功能验证，发现BAD经RNAi干扰后，孕酮浓度显著升高，而BAD过表达后孕酮浓度则出现和干扰相反的变化规律。初步推测BAD基因可能通过促进卵巢颗粒细胞凋亡而降低孕酮水平，进而调控绵羊发情启动。.