雄激素对雌雄同体斜带石斑鱼促性腺激素反馈调节作用机制研究

The feedback regulation mechanism of sex steroids in reproductive activities has been elucidated in mammalian. However, the feedback regulation of steroids in fish, as the most abundant species in vertebrates, remains unclear. Recent studies using gene knockout reveal that gonadotropins play a prominent role in fish reproductive activities and may serve as a breakthrough in the regulation of sex steroids feedback. The preliminary study of the sex steroid regulation on gonadotropins has only focused on hermaphroditic fishes, but the study on hermaphrodite fishes is still blank. Based on this, we have conducted preliminary studies in the hermaphrodite grouper. Our results showed that the androgen had different regulation on the two kinds of gonadotropins, but with the molecular mechanism of this difference needs further study. This project, using orange-spotted grouper as the experimental model, intends to investigate the molecular mechanisms effects of sex steroid on the regulation of gonadal hormones in hermaphrodite fish by using two-color in situ hybridization, gonadectomy and hormone replacement, promoter point mutation and CHIP method. The research of this project will fill in the blank of the research on the sex steroid feedback regulation and will also deepen our understanding of vertebrate reproductive regulation and provide a reference for revealing the mechanism of hermaphroditism.

性类固醇激素的反馈调节在哺乳动物生殖活动中的作用和机制已被阐明。但是，作为脊椎动物中种类最多的鱼类，其类固醇激素的反馈调节仍不清楚。最近基因敲除的证据揭示，促性腺激素在鱼类生殖活动中的地位突出，可能作为性类固醇激素反馈调节的一个突破口。目前性类固醇激素对促性腺激素的调节，仅集中在雌雄异体鱼类中的初步研究，而雌雄同体鱼类中的这方面研究还是空白。基于此，我们在在雌雄同体斜带石斑鱼中初步研究显示：雄激素对垂体中两种促性腺激素的调节作用不同。但具这种差异调节的分子机制尚需进一步研究。本项目拟以斜带石斑鱼为研究对象，采用双色原位杂交共定位、性腺切除与激素补偿、启动子点突变及CHIP方法，旨在解答性类固醇激素调控雌雄同体鱼类促性腺激素的作用及分子机制。本项目的研究将填补鱼类性类固醇激素反馈调节研究中的空白，也将加深我们对脊椎动物生殖调控的认识，同时也将为揭示雌雄同体鱼类性逆转的机制提供参考。

鱼类中性类固醇激素的反馈调节作用是如何发生的？石斑鱼为典型的雌雄同体雌性先成熟鱼类，雄激素如何诱导石斑鱼性逆转以及如何调节促性腺激素的分泌，一直困扰着大家。本项目通过性腺摘除和摘除后补偿雄激素，结合垂体组织原位杂交共定位技术和启动子删减试验，解析了雄激素调控石斑鱼两种促性腺激素的作用及分子机制。本项目较好地完成了各项研究内容，并取得较好的研究进展和结果：1）揭示睾酮能够在垂体水平显著差异调控斜带石斑鱼两种GtH，即睾酮显著地抑制fshβ的表达，而显著性地促进lhβ；2）垂体组织中分泌fshβ和lhβ的细胞上均有雄激素受体arβ亚型的表达，结果展示出在雌雄同体鱼中同样存在着雄激素在垂体水平的反馈调节的直接证据；3）在293T细胞系中，分别对fshβ和lhβ启动子进行克隆并且插入启动子缺失的pGL4.10质粒中，与arβ亚型表达载体pcDNA3.1质粒共转染后通过启动子删减试验得出：雄激素在arβ的介导下，是通过fshβ启动子5’端-431位置的ARE实现对fshβ的转录调控；对lhβ的转录调控主要发生在lhβ启动子序列-226至转录起始位点间的雄激素应答元件实现的。以上结果对于鱼类生殖调控研究提供了重要参考，同时也为揭示雌雄同体鱼类性逆转的机制提供参考。