中华绒螯蟹原始生殖细胞的形成与性分化机制的研究

In most prawns and crabs，the eggs are centrolecithal and opacity. it is difficult to reveal the process of the primordial germ cell (PGC) specification based entirely on cytological morphological observation. The mechanism of sex differentiation remains mysterious. Our previous studies demonstrated that VASA, a germ cell-specific gene, is a reliable molecular marker to examine the differentiation of PGCs in decapod embryos as seen in many other animals. Meanwhile, another two novel germ cell/gonad-specific genes, EsSox21b-like and EsDmrt-like, were also isolated and characterized in the Chinese mitten crab Eriocheir sinense. To further characterize the role of those germ cell/gonad-specific genes and insight into the early events of sex differentiation in the crab, firstly we will trace the origin, migration, and formation of PGCs during embryogenesis and examine germ cell differentiation in juvenile by taking advantage of those genes as markers. Then the expression profiles of vasa, Sox21b-like and Dmrt-like in the early sex differentiation will be comparatively analysed on both mRNA and protein levels to provide information on the possible function of those genes during germ cell differentiation and development. Finally, the exact role of those genes will be characterized by RNAi approach. The present studies will further our understanding of regulatory mechanism of germ cell differentiation in the decapod species and will be of benefit to the sex control in the crab aquaculture.

虾蟹类卵母细胞多为多黄卵，应用传统组织细胞形态学方法难以观察早期原始生殖细胞分化过程，早期性分化机制一直是个未解之谜。本课题组之前证明了以只在生殖细胞中特异表达的VASA基因为标记研究虾蟹类原始生殖细胞分化的可行性，同时分离鉴定了另外两个只在中华绒螯蟹性腺中特异表达的新基因EsSox21b-like和EsDmrt-like， 本研究拟进一步以这些生殖细胞和性腺特异表达基因作为标记，在组织器官发生形态学上追踪观察原始生殖细胞产生、在早期胚胎发育时期的迁移路线以及幼体发育时期生殖细胞的分化过程；在mRNA和蛋白水平上描绘这些基因在早期性别分化过程中的时空表达谱，探究基因的表达与早期性别分化的关系；同时利用RNAi方法干扰这些性别分化相关基因的表达，解析它们在性别分化和生殖细胞发育过程中的功能。蟹类性别分化机制的揭示在学术理论上具有重要科学价值，同时将为进一步开展蟹类性别控制研究打下坚实基础。

虾蟹类卵母细胞多为多黄卵，应用传统组织细胞形态学方法难以观察早期原始生殖细胞分化过程，性分化机制一直是个未解之谜。本项目首先克隆鉴定了只在性腺中特异表达的新基因EsVasa, EsSoxb2-1和EsDmrt-like，同时制备相应的多抗，利用这些基因和抗体作为标记，在组织器官发生形态学上追踪观察了原始生殖细胞产生、在早期胚胎发育时期的迁移路线以及生殖细胞的分化过程；在mRNA和蛋白水平上检测、定位了这些基因在生殖细胞发育与分化过程中的时空表达谱，探究基因的表达与生殖细胞成熟与性别分化的关系；同时利用RNAi方法干扰这些生殖细胞特异表达基因的表达，解析它们在生殖细胞发育过程中的功能。结果表明，（i）卵裂早期只有其中一个卵裂球继承了卵母细胞中的VASA mRNA和蛋白质，只有这个卵裂球才能进一步分裂，最终分化形成原始生殖细胞。在原肠期，含有VASA mRNA和蛋白质的细胞向原口集中，内卷进入原肠内，在溞状幼体期，原始生殖细胞沿后肠分布，随着胚胎发育推进，原始生殖细胞顺着后肠往头胸部生殖嵴方向迁移，该结果首次证明了虾蟹原始生殖细胞遵循先成模式；（ii）EsSoxb2-1 mRNA在精原细胞开始表达,但其蛋白只在精细胞和精子核中表达，RNAi干扰EsSoxb2-1基因表达导致精子变态异常，辐射臂降解，说明EsSoxb2-1基因在精核成熟过程特别是核辐射臂的形成具有必不可少的作用；(iii) EsDmrt-like mRNA 和蛋白在精巢都特异表达，RNAi方法干扰EsDmrt-like 基因表达能有效抑制精巢发育，性腺指数下降75%，精子发生受阻，证明EsDmrt-like 基因对于精巢及其生殖细胞正常发育具有重要作用。本项目研究解析了原始生殖细胞发生模式，鉴定了EsVasa, EsSoxb2-1和EsDmrt-like在生殖细胞发育过程中的功能，对于虾蟹人工繁殖与性别控制研究具有重要科学价值。