硬骨鱼类（黄鳝）垂体促性腺激素Lh与Fsh差异表达调控机制研究

The pituitary gonadotropins, luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh), play key roles in the reproduction of vertebrates. As in other vertebrates, Lh and Fsh exhibit differential expression profiles during reproductive cycles of teleosts, but the underlying mechanisms remain to be elucidated. The ricefield eel Monopterus albus is emerging as an important species for aquaculture in China, but the large-scale artificial breeding of this species is still under intensive investigation. The present project, taking advantages of cellular localization by double immunofluorescence histochemistry or in situ hybridization, analysis of promoter activities, and pituitary tissue and cell culture techniques, is aimed to analyze the extracellular and intracellular signals involved in the regulation of Lh and Fsh in the pituitary of ricefield eels. The major goals includes: 1) the differential regulatory roles of Gnrhs (pjGnrh, sGnrh and cGnrh-II) on Lh and Fsh; 2) the potential differential signaling through Gnrhr-I and Gnrhr-II in Lh and Fsh cells; 3) the feedback regulation on Lh and Fsh by Activins and sex steroidal hormones; 4) the paracrine regulation of Fsh by Lh; 5) the possible differential regulation by transcription factors via lhb and fshb promoters; and 6) the comparison of transcriptomes of Lh and Fsh cells in ricefield eels. The results of this project will contribute to understand the regulatory mechanisms of differential Lh and Fsh synthesis and secretion in teleosts during reproductive cycles, and to tackle the problems in the large-scale artificial breeding of ricefield eels.

促性腺激素Lh和Fsh是脊椎动物生殖活动的关键调控因子。与其它脊椎动物一样，鱼类Lh和Fsh在生殖活动中具有不同的变化模式，但其调控机制并不清楚。黄鳝是重要的养殖鱼类，其规模化人工繁育问题仍未解决。本项目拟采用双色荧光免疫组化或原位杂交定位、启动子活性分析、组织和细胞培养等手段，从促性腺激素细胞外和细胞内信号变化等角度，探讨Lh和Fsh差异表达调控，包括脑部促性腺激素释放激素Gnrh（pjGnrh, sGnrh和cGnrh-II）的调控作用；Gnrh受体（Gnrhr-I和Gnrhr-II）介导Gnrh信号的调控作用；外周信号激活素（Activin）和性类固醇激素的反馈作用；垂体内Lh对Fsh的旁分泌作用；lhb和fshb启动子介导转录因子的作用；Lh和Fsh细胞转录组差异分析等。研究成果对阐明鱼类Lh和Fsh差异表达机制有重要理论价值，对规模化黄鳝人工繁育也有重要指导意义。

与其它脊椎动物一样，鱼类垂体促性腺激素Lh 和Fsh是生殖活动的关键调控因子。鱼类垂体Lh 和Fsh在生殖活动中具有不同的表达模式，但其调控机制并不清楚。本项目探讨了黄鳝和斜带石斑鱼垂体中lhb 和fshb差异表达的调控机制，取得如下研究结果和结论：1）GnRH对体外孵育黄鳝垂体碎片中lhb和fshb表达都有刺激作用。2）黄鳝垂体Lh细胞上表达促性腺激素释放激素受体1（Gnrhr1），但不表达促性腺激素释放激素受体2（Gnrhr2），黄鳝垂体Fsh细胞上两种受体都不表达，提示GnRH可通过Lh细胞上GnRHR1直接调控lhb表达，而对fshb表达可能是间接作用。3）黄鳝Activin刺激垂体fshb表达，而对lhb表达没影响，而且Activin下游信号分子Smad仅激活fshb启动子，这些结果提示Activin-Smad信号参与lhb和fshb差异表达调控；4）性类固醇激素包括雌二醇和睾酮上调黄鳝垂体lhb基因表达和lhb启动子活性，而对fshb基因表达没影响，提示性类固醇激素参与黄鳝垂体lhb和fshb差异表达调控。5）黄鳝Nr5a转录因子对lhb和fshb启动子没有作用。然而，斜带石斑鱼Nr5a转录因子包括Nr5a2和Nr5a4-a与Lhb共定位，但与Fshb没有共定位，而且Nr5a2和Nr5a4-a结合在lhb启动子上，而没有结合到fshb启动子上，因此Nr5a2和Nr5a4-a参与调控斜带石斑鱼lhb表达，而对fshb表达没有作用；更有趣的是Nr5a2抑制lhb启动子基础活性以及Forskolin的刺激作用，而Nr5a4-a上调lhb启动子活性。6）在雌性黄鳝垂体中，Foxl2与Lh细胞和Fsh细胞都有部分共定位现象，但在间性和雄性黄鳝垂体中没有共定位现象；Foxl2都能上调lhb和fshb启动子活性。这些结果提示Foxl2参与雌性黄鳝垂体中lhb和fshb基因表达调控。7）GnRH下调黄鳝精巢中DNA甲基转移酶基因表达和DNA甲基化水平。上述研究结果揭示了鱼类lhb和fshb差异表达调控的新机制以及GnRH在垂体外组织（精巢）的新作用，为进一步阐明脊椎动物下丘脑-垂体-性腺生殖内分泌轴的调控和功能具有重要意义。