雌激素对斑马鱼两种kiss基因反馈调节的分子机制研究

Kisspeptin has recently drawn much attention as a candidate for the "missing link" in the estrogen feedback regulation. However, the roles of Kisspeptin in conveying the estrogen feedback in vetebrate remain superficially characterised. In mammal,only one kisspeptin gene has been found. However, two genes encoding kisspeptin were identified in teleost fish, indicating a distinct picture of kisspeptin signals in mediating the estrogen feedback with theoretical significance in telosts. In the present project, zebrafish (Danio rerio) is employed to elucidate the molecular mechanism of kisspeptin in mediating estrogen feedback regulation in teleost fish. Firstly, the expression profiles of two kiss genes will be examined after the treatments of exogenous estrogen and aromatase inhibitor, as well as the ovariectomy and hormone compensation experiment, in immature and sexually mature female fish.Secondly, co-localization among kiss genes and estrogen receptor (ERs) will be carried out by in situ hybridizationthe, and the expression of ERs in kisspeptin neurons will also analyzed with real-time PCR combined with the laser capture microdissection (LCM) techique.Thirdly, the promoters of two kiss genes will be cloned and characterized, and the transcription factor binding site of estrogen receptors will be identified. Finally, the TALEN techniques will be applied to inactivate the estrogen receptors binding sites in the kiss gene promoter so as to eluciate the molecular mechanism of estrogen feedback regulation on kisspeptin genes. Taken together, our study in this project will expand our understanding on molecular mechanism of neuroendocrine regulation of reproduction in fish.

近年的研究表明，Kisspeptin信号是介导雌激素对生殖轴反馈调节的关键衔接点，成为脊椎动物生殖生理学研究的新热点。与哺乳动物不同的是，鱼类存在两种kiss基因，因此，比较鱼类与哺乳类kisspeptin信号在介导雌激素对生殖轴反馈作用的分子机制具有重要的理论意义。本项目以斑马鱼为材料，通过外源雌激素处理和阻断内源雌激素生成的方法，从生理水平探讨雌激素对鱼类kiss基因表达的反馈作用；通过原位杂交和激光显微切割等技术，研究脑kiss与雌激素受体的共定位以及雌激素受体在kiss神经元上的表达情况，从细胞水平为雌激素调控kiss基因表达提供直接的证据；通过克隆kiss基因启动子，分析并确定雌激素调控的作用位点；运用TALEN技术，在体条件下突变kiss基因启动子区域雌激素的作用位点，揭示雌激素对kiss基因表达的转录调控机制,从而阐明雌激素对kiss基因表达的反馈调控作用的信号通路。

哺乳动物的生殖受“下丘-垂体-性腺”轴所调控，然而在鱼类中，该理论还不是非常清晰。本项目，利用TALENs 靶向基因敲除技术，敲除了生殖轴上相关的基因。结果显示，鱼类与哺乳动物在进化过程中形成了不同的生殖调控策略。这个结论主要基于以下结果：（1）kiss/kissr 信号系统并不是斑马鱼生殖调控所必需的；（2）雌激素与性腺型芳香化酶在斑马鱼性别分化过程中扮演重要角色；（3）雌激素并不是雄性斑马鱼生殖调控所必需的；（4）雄激素受体基因敲除斑马鱼中发现成熟精子；（5）在雌性青春期启动斑马鱼中，雌激素可以在垂体水平直接刺激LH 的表达；（6）核孕激素受体在LH 诱导的斑马鱼排卵过程中发挥关键功能；（7）LH 诱导ptgs2a 的表达是斑马鱼排卵所必需的。总之，本项目的结果不仅提供了直接证据阐明了生殖轴相关基因的功能，同时也为硬骨鱼类和哺乳动物的生殖内分泌调控策略上的差异提供了新观点。