小鼠卵泡局部MLT/MT1信号在FSH依赖期卵泡发育过程中的作用研究

FSH stimulates follicle development by binding with FSHR. FSHR can couple with Gs protein to promote cAMP synthesis and stimulate downstream signaling. As an important reproductive hormone, Melatonin can stimulate downstream signaling pathway by binding with MT1. In contrast to FSHR, MT1 couples with Gi protein and promote signal transduction by inhibiting cAMP synthesis. In the previous study, we have found that: (1) The appearance of LH surge activated MLT/MT1 signaling in the granulosa cells before ovulation. Besides, MLT/MT1 signaling was involved in the regulation of luteinization as a local signal. (2) MLT/MT1 signaling in granulosa cells could be activated by FSH/FSHR signaling during follicle development. Moreover, exogenous melatonin treatment could suppress the expression of genes related with proliferation and differentiation, including PCNA、p450scc、p450aro、LHcgr ect. The hypothesis of the present study is that MLT/MT1 can worked as a local signal to regulate follicle development by negatively regulating FSH/FSHR signaling. To further identify the hypothesis, firstly, we are planning to study the mice follicle development and fecundity of MT1 knockout or/and conditional overexpression. Then, explore the interaction between MLT/MT1 and FSH/FSHR signaling by using the cell culture model. Finally, we are going to clarify the complex network to explain how MLT/MT1 involves in the regulation of follicle development by transcriptome sequencing. Hopefully, this project will provide the theoretical basis for utilizing of melatonin in animal reproduction.

FSH通过受体FSHR调控卵泡发育。FSHR 偶联Gs蛋白，通过上调cAMP水平传递信号。褪黑素(MLT)可通过MT1受体传导信号，与FSHR相反，MT1通过Gi蛋白抑制cAMP合成传递信号。申请人前期研究发现:(1)排卵前LH峰激活颗粒细胞MLT/MT1信号，MLT/MT1作为卵泡局部信号参与黄体形成调控；(2)超排时，PMSG(FSH)激活颗粒细胞MLT/MT1信号，褪黑素处理抑制颗粒细胞增殖、分化相关基因的表达。据此推测“卵泡局部MLT/MT1作为FSH负调节信号调控卵泡发育”。拟以小鼠为研究对象，通过基因敲除与条件过表达技术，研究MLT/MT1信号缺失和过度激活对卵泡发育的影响；并在细胞水平阐明卵泡局部MLT/MT1信号与FSH/FSHR信号的相互调控关系；最后通过转录组测序进一步揭示MLT/MT1调控卵泡发育的信号网络。本项目将为围绕褪黑素开发提高动物繁殖力的新技术提供理论依据。

FSH依赖期卵泡发育作为雌性动物发情周期更替的内在动力与第二性征维持的生理基础，历来是生殖领域的研究热点。在本项目的资助下，我们系统地研究了FSH依赖期卵泡发育规律，并揭示了褪黑素对卵泡发育的调节作用。所取得的理论成果如下：（1）绘制了FSH依赖期卵泡发育图谱，提出了FSH依赖期卵泡发育“三阶段”模型，即FSH依赖期卵泡需经历准备期、快速生长期与减速生长期三个发育阶段。各阶段中卵泡所经历的发育事件与调控信号也得到了充分的验证；（2）发现在FSH依赖期卵泡发育过程中，褪黑素可促进卵母细胞生长与母源物质储备。同时通过提高卵源因子GDF9的分泌，间接促进FSH依赖期卵泡的发育；（3）在早期卵泡发育阶段，褪黑素通过PI3K-Akt信号抑制原始卵泡的激活与早期卵泡生长，从而延缓卵泡储备的损耗与卵巢衰老。上述成果不仅揭示了卵泡发育的一些新规律，也发现了内源性褪黑素调节卵泡发育的直接证据，并为利用褪黑素改善雌性动物繁殖性能提供了理论可行性。我们始终围绕申报书中的任务目标开展研究，顺利完成了预先制定的考核指标。发表SCI论文2篇（一篇为第1标注，另一篇为第2标注），申请国家发明专利1项。此外培养硕士研究生3名，其中1人已经获得硕士学位，另外2人预计2021毕业。除了已经取得的成果外，我们在项目执行过程中还发现了一些新的科学现象。比如，传统观点认为卵母细胞在FSH依赖期卵泡发育过程中不再生长，但我们却发现了褪黑素可以刺激该阶段卵母细胞进一步生长，并提高线粒体功能与母源物质的储备量。因此，我们期待在未来的工作中对这一问题进行深入研究，从而阐明褪黑素刺激卵母细胞生长的分子机制及其运用前景。