Gsdf在罗非鱼卵子发生和育性中的功能研究

The study of oogenesis is always an important research area, since egg formation is a prerequisite for the fertility. Oogenesis is controlled by the hormones, cytokines, transcriptional factors etc in the hypothalamic-pituitary-gonad axis. TGF-β superfamily members are important cytokines for oogenesis. Gsdf is a fish specific TGF-β superfamily member and is dominantly expressed in the somatic cell surrounding the oogonia in the ovary. Recently, researchers found that most gsdf-/- female medaka were infertile, while some female are sub-fertile indicating Gsdf is important for oogenesis. In our previous study, we established the gsdf knockout line in Nile tilapia and found the fertility of the gsdf mutated female was also influenced. In this project, in order to elucidate the function of the Gsdf on oogenesis and fertility in detail, we propose to (1) investigate the phenotype of the gsdf knockout female and identify the target genes of Gsdf, (2) elucidate the molecular mechanisms of Gsdf in regulating its target gene and identify the type I and II receptors of Gsdf, (3) rescue the fertility of the gsdf knockout fish. Our study will not only help to reveal the causes of the infertility in the female fish with gsdf null mutation, but also extend the comprehension of the function and mechanism of TGF-β signal pathway in the process of oogenesis and fertility. Moreover, our study will provide valuable information on the reproduction control of tilapia.

卵子发生是雌性生物育性的细胞学基础。卵子发生受下丘脑-垂体-性腺轴的激素、细胞因子和转录因子等调控。细胞因子TGF-β超家族成员在卵子发生中具有重要作用。 Gsdf是鱼类特有的TGF-β超家族成员，在卵巢主要表达于卵原细胞周围的体细胞。最近人们发现gsdf敲除雌性青鳉大多不育，少数亚不育，表明Gsdf在卵子发生中有重要作用。项目申请人前期建立了尼罗罗非鱼gsdf敲除系，发现gsdf突变也会影响雌鱼育性。本研究将从以下三个方面深入探讨Gsdf调控罗非鱼卵子发生和育性的机制：（1）分析gsdf敲除雌鱼的表型，筛选Gsdf可能作用的靶基因；（2）分析Gsdf调控靶基因的分子机制，鉴定Gsdf的I型与II型受体；（3）回救gsdf敲除雌的鱼育性。本研究有助于解析gsdf-/-雌鱼不育的机制，帮助我们认识TGF-β 信号通路在卵子发生和育性中的功能和机制，还可为罗非鱼生殖控制提供理论基础。

卵子发生是雌性生物育性的细胞学基础。卵子发生受下丘脑-垂体-性腺轴的激素、细胞因子和转录因子等调控。细胞因子TGF-β超家族成员在卵子发生中具有重要作用。Gsdf是鱼类特有的TGF-β超家族成员，在卵巢主要表达于卵原细胞周围的体细胞。近年来人们发现gsdf敲除雌性青鳉大多不育，少数亚不育，表明Gsdf在卵子发生中有重要作用。项目负责人前期建立了尼罗罗非鱼gsdf敲除系，且发现gsdf突变也会影响雌鱼育性。在此基础上，从以下三个方面深入探讨Gsdf调控罗非鱼卵子发生和育性的机制：（1）分析gsdf敲除雌鱼的表型，筛选Gsdf可能作用的靶基因；（2）分析Gsdf调控靶基因的分子机制，筛选Gsdf的I型受体；（3）回救gsdf敲除雌鱼育性。取得的主要结果如下：（1）通过繁殖对照试验，证实XX gsdf -/-雌性尼罗罗非鱼完全不育，卵巢组织学观察表明2龄敲除鱼大部分卵子不能发育成熟，血清雌二醇和卵黄蛋白原水平与对照雌鱼差异不显著。（2）6月龄XX gsdf-/-雌鱼性腺GSI显著高于野生型雌鱼，XX gsdf-/-个体卵子发育明显延迟；XX gsdf-/-个体血清雌二醇和卵黄蛋白原水平与对照雌鱼差异不显著；通过qPCR和转录组测序分析发现XX gsdf-/-雌鱼卵巢中amh、amhr表达显著上调，inhibin、acvr2aa表达显著下调，而cyp19a1a、pcna和igf2bp3表达不受影响，免疫荧光组织化学分析进一步证明XX gsdf-/-雌鱼卵巢Amh表达上调；qPCR分析显示XX gsdf-/-雌鱼垂体中fshb显著下调；酵母双杂交实验发现Acvr1和Bmpr1aa是Gsdf可能的Ⅰ型受体。(3)通过腹腔注射含有人绒毛膜促性腺激素HCG的催产剂，回救了1尾XY gsdf -/-雌鱼的育性，回救个体正常繁殖。本研究主要发现XX gsdf-/-雌鱼雌激素水平正常，因而gsdf-/-不育并不是由于雌激素缺乏或不足导致，TGF-β信号通路基因在敲除鱼中表达明显变化可能是导致gsdf-/-鱼不育的原因。本研究有助于我们认识TGF-β信号通路在卵子发生和育性中的功能和机制，还可为罗非鱼生殖控制提供理论基础。