黄鳝具有雌雄同体的天然性逆转特性，鳗鱼的性腺发育具有幼龄雌雄同体的特点。黄鳝和鳗鱼是水产养殖业的重要品种，但是黄鳝和鳗鱼都无法进行规模化人工繁殖，养殖业面临着苗种极为缺乏的瓶颈。申请人及合作者将分别以黄鳝和鳗鱼为模式研究雌雄同体鱼性腺分化的机制。鱼类性腺分化是脑、垂体和性腺间复杂相互作用的结果，GnRH是HPG轴的关键信号因子，GnRH相关肽（GAP）是GnRH前体结构变异最大的部分，我们推测GnRH/GAP可能在鱼类性腺分化中扮演重要角色。本项目将分析GnRH/GAP在黄鳝卵巢分化、性逆转过程的间性性腺和性逆转完成后的精巢中的表达特征；研究GnRH/GAP对性腺组织分化的影响，对垂体和性腺中GtH、DMRT1和Cyp19a等信号分子的调控，以及性类固醇激素水平的变化；研制转GAP基因和GnRH （GAP）敲除的鱼；以期揭示GnRH/GAP在雌雄同体黄鳝两性性腺分化的功能及其作用机制。

The ricefield eel (Monopterus albus) is a protogynous hermaphroditic fish that changes sex from a functional female to a male. The Japanese eel (Anguilla japonica) is a juvenile hermaphroditic fish. Both the ricefield eel and the Japanese eel are the important aquaculture fish in China. However, it is very difficult to carry out large-scale artificial breeding for the ricefield eel and Japanese eel aquaculture.The collaborators of the Mainland and Taiwan will study the mechanisms of gonadal differentiation in hermaphroditic fish with the ricefield eel and the Japanese eel, respectively.The endocrine control of gonadal differentiation in fish requires a complex interplay between the brain, pituitary and gonads.Gonadotropin-releasing hormone (GnRH) is a crucial regulatory factor in the hypothalamic-pituitary-gonadal axis (HPG axis). GnRH-associated peptide (GAP) is the most variable construct in the precursor molecular for GnRH. We infer that both GnRH and GAP would play an important role in the fish gonadal differentiation. The developmental expression traits of GnRHs and GAPs will be analyzed in the ricefield eel’s gonads duringthe onset of ovarian differentiation, in the process of sex reversal and in the testes. The effects of GnRH and GAP on the regulation of GtH, DMRT1, Cyp19a, sex steroids in pituitary and gonad will be studied in both in vivo and in vitro models. Moreover, we will generate the GAP transgenic fish. We will also employ Talens to knockout the GnRH (GAP)to develop the GnRH (GAP) mutant fish. The function and regulatory mechanism for gonadal differentiation in hermaphroditic fish of GnRH/GAP will beelucidated with the models.

黄鳝是第一个被发现具有雌雄同体、天然性反转特性的脊椎动物，黄鳝性反转的分子遗传机理研究一直是极富挑战性的科学问题，而且黄鳝性反转的特性在生产上又导致严重的苗种短缺和养殖效率低下，亟需建立黄鳝分子遗传育种技术。但是，由于缺乏有效的基因功能研究和分子遗传育种技术，严重制约了黄鳝性反转机制的基础研究和遗传育种工作的深入进行。本项目首次建立了黄鳝分子设计的生殖操作育种技术平台：包括黄鳝全人工繁殖、黄鳝人工雌核发育和高效特异的黄鳝靶向基因编辑等核心技术，利用该技术平台对黄鳝性别分化相关基因Dmrt1、Cyp19a1a和Foxl2成功进行了基因编辑，研究表明Foxl2可能在黄鳝早期卵巢分化发育以及性逆转中发挥着重要的作用。在黄鳝性腺中扩增出一种包含第二内含子的GnRH2新转录本----GnRH2-SV，该转录本编码形成一种新的缩短了的GnRH2相关肽（GnRH associated peptide, GAP）----New GAP2，通过组织表达分析，体外孵育和体内注射实验，发现GnRH2、GAP2和New GAP2在黄鳝性反转的启动和精巢发育中发挥着重要作用。采用CRISPR/Cas9技术获得GnRH3基因突变的试验鱼，首次发现生殖轴的信号分子GnRH3在鱼的性别分化调控中具有重要作用，而且，GnRH3对鱼的性别分化调控作用是通过影响早期性别分化相关基因的表达，影响鱼类早期胚胎原始生殖细胞（Primordial germ cells, PGCs）的增殖，导致迁移至生殖嵴的PGCs数量减少而实现。本项目发表SCI论文4篇，撰写国际学术专著章节1篇，申请中国发明专利2项。