DHP反馈调控雌性大弹涂鱼促性腺激素表达的神经内分泌机理研究

In teleosts, the feedback regulation of gonadal steroids on reproductive neuroendocrine system is an important process of reproductive physiology. Much progress has been made in understanding the molecular mechanisms of feedback effects of androgens and estrogens, but potential effects of progestins on reproductive neuroendocrine system have received little attention. Our recent studies in zebrafish showed that 17α,20β-dihydroxy-4-pregnen-3-one (DHP), a most potent and biological relevant progestin produced in teleost, exhibited clear stimulatory effects on gonadotropins (Fshb and Lhb) expression. During reproductive peak period, the female mudskipper (Boleophthalmus pectinirostris) exhibits typical semilunar spawning rhythm (the fish stock spawns twice around the first and the last lunar quarters within a month). Our previous studies have demonstrated that melatonin and its receptors were involved in the synchronization of the semilunar spawning rhythm by acting directly on ovarian tissues to stimulate the production of DHP. However, the melatonin can not induce the oocyte maturation, which may suggest a potential feedback effects of DHP on the expression of gonadotropins which will enhance the maturation competence of oocyte. Based on these primary results, the present project aims to investigate the neuroendocrine pathway of the feedback effects of DHP on gonadotropins expression in the female mudskipper, by using intraperitoneal injection, tissue culture, primary cell culture, RNA interference, cloning and functional characterization of progesterone receptors and transcriptome analysis. The results from this study can not only further our understanding in reproductive endocrinology in teleost, but also provide useful scientific knowledge for improving the biotechnology of fish aquaculture in China.

性类固醇激素反馈调控神经内分泌系统是鱼类生殖过程的一个重要环节。然而，目前的研究多关注雄激素和雌激素的调控机理，而涉及孕酮激素的研究较少。我们近期研究明确发现，在鱼类生殖过程中发挥重要作用的孕酮类激素（17α，20β-双羟孕酮，DHP），能反馈调控斑马鱼垂体促性腺激素的表达。雌性大弹涂鱼在生殖高峰期呈现独特的半月周期产卵习性；我们的前期研究证实了褪黑素通过其位于卵泡上的受体参与了这一产卵习性的调控。然而，褪黑素虽能提高DHP的产量，但无法诱导卵子成熟，提示卵巢产生的DHP可能反馈调控促性腺激素表达，进而促进卵母细胞的成熟过程。基于上述研究基础，本项目拟以雌性大弹涂鱼为研究对象，应用活体注射、组织器官离体培养、原代细胞培养、RNA干扰、受体功能验证和转录组等技术方法，从细胞和分子水平，系统地研究孕酮类激素反馈调控促性腺激素表达的神经内分泌机理，为鱼类人工繁殖技术的完善提供科学依据。

性类固醇激素反馈调控神经内分泌系统是鱼类生殖过程的一个重要环节，为阐明孕酮类激素反馈调控促性腺激素表达的神经内分泌机理，本项目探究了鱼类主要的孕酮激素DHP通过内分泌和外分泌途径，调控促性腺激素以及其他神经内分泌因子表达的机理，获得以下结果：.1.与其他鱼类相比，大弹涂鱼的端脑发达；脑组织中细胞结构的差异主要出现在端脑和间脑；其端脑中缺失已在其他鱼类中报道的NT、Vi和Vp细胞群，另外间脑中的细胞群数量总体偏少。.2.对不同月相收集的大弹涂鱼大脑样品进行转录组分析，筛选到95个基因的表达与月相密切相关，包括了促卵泡激素β亚基（Fshb）。qPCR结果表明，生殖季节垂体中Fshb mRNA水平呈现半月周期的表达趋势，且在产卵前（农历月份的七号和廿二号）显著降低。活体注射DHP，也可引起垂体中Fshb mRNA水平降低。.3.对DHP离体处理大弹涂鱼大脑样品进行转录组分析，得到205个显著上调基因。经qPCR验证发现，95个与月运节律相关的基因中，有58个是受DHP调控，其中包括了已知的生物钟基因和多个与前列腺素合成及其信号通路相关的基因。.4.克隆了大弹涂鱼四种黑视蛋白基因亚型（opn4m1、opn4m3、opn4x1、opn4x2）。首次在鱼类中观察到端脑表达opn4m1和opn4x1，且opn4x1的表达呈现明显的半月周期表达规律。.5.通过构建转基因斑马鱼Tg(pgr:eGFP)家系，利用绿色荧光蛋白示踪Pgr.在垂体中的表达分布情况，结果表明绿色荧光蛋白主要表达于PPD（proximal pars distalis）区域，说明在Pgr在垂体中可能起到直接与DHP发生作用从而影响下游基因的调控。.6. 以中华乌塘鳢为对象，观察到DHP可通过激活其表达在嗅上皮细胞的孕酮膜受体mPRβ，进而将神经信号通过嗅神经传递到脑组织，并最终上调垂体中Lhβ基因的表达水平。.综上，DHP可通过内分泌或外分泌的形式，促进性成熟鱼类促性腺激素的表达与释放。此外，DHP参与了生物钟基因的调控，同时增强了脑组织中与生殖行为相关的前列腺素功能途径，从而调控生殖节律。