硬骨鱼类黄鳝腺垂体中雌激素膜受体和核受体信号通路及其功能分析

The adenohypophysis of vertebrates synthesizes and releases several polypeptide hormones, which play important roles in the regulation of growth, development, and reproduction. In mammals, estrogens have been shown to regulate the function of the adenohypophysis via the membrane receptor pathway in addition to the classic nuclear receptor pathway. In teleosts, however, the expression and function of the estrogen membrane receptor, Gper, in the pituitary has not been established yet. We have previously found that identified gper transcripts in the pituitary via transcriptomic analysis, and esr1 and esr2a transcripts were localized to different regions of the adenohyophysis. To follow up, the present project is aimed to characterize functionally the membrane receptor and nuclear receptor pathways of estrogens in the adenohypophysis of ricefield eels. The full-length cDNA encoding ricefield eel esr2a, esr2b and gper will be cloned via the conventional PCR methods, and specific antisera against Gper, Esr1, Esr2a, and Esr2b will be generated. The expression of Gper, Esr1, Esr2a, and Esr2b is planned to be analyzed at the mRNA level via realtime PCR and at the protein level via immunohistochemistry in the adenohypophysis of ricefield eels at different gonadal stages. The possible localization of Gper, Esr1, Esr2a, and Esr2b in the various hormone-secreting cells of the adenohyophysis of ricefield eels will be examined with the double fluorescent immunohistochemical method. The intracellular signaling pathways mediating the actions of ricefield eel Gper and membrane-associated Esr(s) will also be examined. More importantly, the effects of estrogens on the expression of growth- and reproduction-related hormonal genes in the adenohypophysis of ricefield eels via the classic genomic and/or non-genomic pathways will be investigated. The results of the present project are sure to provide new information on the regulation of the adenohypophyseal functions in teleosts as well as other vertebrates.

脊椎动物腺垂体分泌多种蛋白激素，在生长发育和生殖活动中发挥重要调控作用。哺乳动物中研究表明，雌激素不仅可通过核受体信号通路，还可通过膜受体信号通路调控腺垂体的功能，但目前有关鱼类垂体中雌激素膜受体信号通路及功能还未见报道。我们前期研究发现黄鳝垂体转录组中存在雌激素膜受体样转录本，而且腺垂体中两种雌激素核受体esr1和esr2a mRNA分布在不同区域。因此，本项目拟研究黄鳝垂体中雌激素膜受体和核受体信号通路，及其对生殖和生长相关垂体蛋白激素基因表达调控和机制，内容包括垂体中雌激素膜受体（Gper）和核受体（Esr1，Esr2a和Esr2b）cDNA克隆和表达分析，腺垂体生殖和生长相关激素分泌细胞中雌激素受体表达，雌激素膜受体或与细胞膜相联的核受体所介导的胞内信号转导途径分析，雌激素调节腺垂体生殖和生长相关激素分泌细胞功能的机制。结果将为阐明鱼类及脊椎动物垂体功能的调节机制提供新颖的资料。

脊椎动物的垂体合成和分泌多种蛋白激素，在生长发育和生殖活动中发挥重要调控作用。雌激素不仅可通过核受体信号通路，还可通过膜受体信号通路调控垂体的功能。本项目主要探讨了黄鳝雌激素核受体和膜受体信号通路及其对垂体激素细胞的调控，取得如下进展：1）克隆获得黄鳝雌激素核受体基因（erβ1和erβ2）和膜受体基因（gper1a和gper1b）cDNA序列。2）制备了抗黄鳝Erα，Erβ1，Erβ2和Gper1a特异性抗血清。3）黄鳝erα，erβ1，erβ2，gper1a和gper1b基因表达具有明显的组织特异性。4）黄鳝脑中Erβ1免疫阳性信号分布较为广泛，而Erβ2免疫阳性信号分布较为集中，主要在视前区和松果体附近。5）黄鳝垂体中Erβ1和Erβ2免疫信号分布具有明显的差异，其中Erβ1免疫信号分布在腺垂体，而Erβ2免疫信号分布在神经垂体，提示这两种Erβ在垂体行使不同的调节功能。6）腺垂体中，雌激素核受体Erα和Erβ1以及雌激素膜受体Gper1a表达的细胞定位差异明显，其中Lh细胞高丰度表达Erα和Gper1a，Gh细胞高丰度表达Erα和Erβ1，Prl细胞高丰度表达Erβ1，但Fsh细胞中这些受体不表达或低丰度表达，这些结果提示雌激素信号差异调节腺垂体的各种激素细胞。7）在黄鳝卵巢卵泡中，gper1a和gper1b基因均表达于卵母细胞上，而不在滤泡细胞层表达，而且gper1a表达水平在卵黄发生晚期剧烈升高，而到成熟期又剧烈下降，提示gper1a可能在卵泡成熟中发挥重要作用。8）黄鳝Gper1a和Gper1b都可能通过cAMP信号通路起作用。9）E2可能主要通过核受体途径调控垂体中雌激素核受体erα表达，而通过膜受体途径调控gper1a和gper1b表达。10）尽管Gh细胞高表达Erα和Erβ1，但E2刺激并不影响gh基因表达；进一步分析发现Gh细胞上也表达GnRH受体，GnRH可能通过结合II型GnRH受体（GnRHR2），激活PLC/IP3/PKC和Ca2+等胞内信号通路，诱导Gh分泌。这些研究结果对于理解垂体蛋白激素细胞功能调控具有重要的理论价值。