Activin/follistatin信号系统对中华鲟卵巢发育的调控及作用机制

The Chinese sturgeon (Acipenser sinensis) is currently endangered due to the deteriorated natural habitat caused by anthropogenic interferences. The artificial propagation of the Chinese sturgeon has already been successful, but with limited population of females for propagation, which hampers the speed of species recover. It mainly because it is rather difficult for the breeding females to reach final sexual maturation with a long period of sex maturity. The activin/follistatin system has been reported to play significant roles in the regulation of fish ovary development, by interactions with the gonadotropins. This study aims to identify the key genes in the activin/follistatin systems and demonstrate the expression patterns of the acquired genes by real-time PCR, RNA in situ hybridization, Western blot and fluorescence immunochemistry. Besides, in vivo injection, in vitro oocyte incubation and recombinant protein addition methods will be adopted to investigate their regulation of the ovary development during stageⅡand Ⅲ and the specific pathway involved. The results in this study will be helpful to understand the molecular endocrine regulation of the ovary development, to explore the ovary development promotion of the propagated Chinese sturgeon through recombinant protein addition, and to further offer the theoretic basis and technological support for both the ovary maturation regulation and the species conservation of the endangered Chinese sturgeon.

由于人类活动的影响，中华鲟生存环境持续恶化，物种处于极度濒危状态。中华鲟全人工繁殖虽已突破，但养殖雌性亲本的卵巢发育周期过长且成熟困难，导致可用于人工繁殖的雌性亲本数量非常有限，严重限制了其物种恢复速度。Activin/follistatin信号系统已被证明可通过与促性腺激素相互作用调节鱼类卵母细胞的发育。本项目拟鉴定中华鲟activin/follistatin信号系统关键基因，运用荧光定量PCR、RNA原位杂交、Western blot和免疫组化等方法揭示所得基因的表达特征。并采用体内注射、离体细胞孵育及重组蛋白投喂等技术研究其在中华鲟Ⅱ期及Ⅲ期卵巢发育中的调控功能及作用机制。本研究将有助于深入了解中华鲟卵巢发育的内分泌调控机制，探索通过重组蛋白投喂促进养殖中华鲟的卵巢发育成熟技术，最终为养殖中华鲟的卵巢成熟调控及物种保护提供理论基础和技术支撑。

极度濒危鱼类中华鲟的全人工繁殖虽已突破，但其养殖雌性亲本的卵巢发育周期过长且成熟困难，导致可用于人工繁殖的雌性亲本数量非常有限，严重限制了其物种恢复速度。Activin/follistatin信号系统被证明可通过与促性腺激素相互作用调节鱼类卵母细胞的发育。本研究克隆得到Activin/follistatin信号系统中activinβA、follistatin、activinRA、activinRB、smad2、smad3、smad4七个关键基因的全长cDNA序列，分别为1663 bp、1392 bp、2890 bp、2279 bp、1882 bp、2017 bp、2499 bp。荧光定量PCR检测表明，activinβA 只在雌性中华鲟的卵巢和脑中转录，其余6个基因为泛在表达模式。制备得到Activin、Follistatin特异的多克隆抗体，并采用Western blot技术检测发现其在卵巢和脑中蛋白表达量较高。免疫荧光组织化学技术检测结果显示Activin、Follistatin在卵巢组织体细胞及卵母细胞中均有分布。为了研究Activin蛋白在中华鲟性腺发育中的功能，我们首先进行了插管灌喂实验。Activin重组蛋白灌喂可显著上调雌性中华鲟血液中促滤泡激素FSH的分泌。卵巢细胞离体孵育实验结果显示，Activin重组蛋白孵育可显著上调Activin/Follistatin信号通路上述七个基因以及卵巢发育相关基因cyp19a1a、erα、erβ的转录。cAMP信号通路的阻断剂IBMX与Activin重组蛋白共同孵育处理体外培养的卵巢细胞6 h后，可显著下调上述10个目标基因的转录水平。另外，人绒毛膜促性腺激素hCG处理体外培养中华鲟卵巢细胞，使得activin、follistatin、activinRA、smad2以及cyp19a1a、erα、erβ基因的转录得到显著提高。最后，通过雌二醇腹腔注射实验证明雌二醇可通过下调芳香化酶以及雌激素受体β发挥对长江鲟smad2、smad3的正反馈调节作用。以上结果表明，Activin/follistatin信号系统可在hCG的介导下发挥对中华鲟卵巢发育的促进效果。该研究结果将可为养殖中华鲟的卵巢成熟调控及物种保护提供理论基础和技术支撑。