中华绒螯蟹GnRH信号系统的鉴定及其在卵母细胞成熟中的调控作用

Vertebrate gonadotropin-releasing hormone (GnRH) plays a central role in the regulation of reproduction. However, so far the correspondent GnRH signaling system has not been characterized in invertebrates including shrimps and crabs. In our previous studies, we isolated a GnRH homolog from Chinese mitten crab Eriocheir sinensis. In vivo injection experiments demonstrated that the crab GnRH can induce germinal vesicle breakdown (GVBD) and meiotic maturation of oocytes. To further explore the potential GnRH signal pathway and insight into its role on regulation of the oocyte maturation, firstly we will perform comparative analysis on the transcriptom of the brain, thoracic ganglion,and ovary to isolate the genes in response to the crab GnRH treatment and deduce the GnRH signal pathway. By means of real-time PCR, in situ hybridization and immunofluoresence, then we will examine the spatio-temporal expression profiles of GnRH-responsed genes including GnRH receptor on both mRNA and protein levels to provide information on the possible function of those genes during oocyte maturation. Finally, the exact GnRH signal pathway will be characterized by proteins interaction and RNAi approach. The present studies will further our understanding of regulatory mechanism of oocyte maturation in the decapod species and the evolution of GnRH signal system in invertebrates.

促性腺激素释放激素（GnRH）在脊椎动物生殖调控中起核心作用，但在虾蟹类等无脊椎动物中相应的激素信号系统尚未建立，本课题组之前分离鉴定了中华绒螯蟹GnRH，同时证明该激素能够刺激卵母细胞生发泡破裂、促进卵母细胞减数分裂成熟，本研究拟进一步利用转录组学、基因芯片分析方法筛选鉴定脑、胸神经团、卵巢组织GnRH效应基因，推导潜在的GnRH信号通路；利用荧光定量PCR、原位杂交、免疫荧光定位等方法分析GnRH受体等关键通路基因的时空表达谱，探究基因的表达与卵母细胞成熟的关系；利用蛋白相互作用、RNAi实验进一步验证GnRH信号通路，解析GnRH信号系统在调控卵母细胞成熟中的作用，蟹类GnRH信号系统的揭示对于解明卵母细胞成熟机制和无脊椎动物GnRH信号系统的进化具有重要学术价值。

促性腺激素释放激素（GnRH）在脊椎动物生殖调控中起核心作用，但在虾蟹类等无脊椎动物中相应的激素信号系统尚未建立，之前我们研究组分离鉴定了中华绒螯蟹（Eriocheir sinensis）GnRH，同时证明了该激素能够刺激卵母细胞生发泡破裂（GVBD）、促进卵母细胞减数分裂成熟，本研究进一步利用转录组学分析方法筛选鉴定脑、胸神经团、卵巢组织GnRH效应基因，推导潜在的GnRH信号通路；减数分裂成熟GV与GVBD两个时期差异表达的基因共13517个，7140个基因在GV时期的卵巢中显著高表达，而6375个基因在GVBD时期高表达，其中31个基因注释到了GnRH通路上，随机挑选23个基因进行荧光定量PCR，21个基因的表达量在GVBD时期显著上升；利用荧光定量PCR、原位杂交等方法分析通路关键基因GnRHR和LHR的时空表达谱，RNAi敲降实验表明GnRHR 表达抑制能导致GnRH通路中其它下游基因表达的变化，活体注射GnRH类似物能显著增加LHR基因表达量和Cdc2激酶活性（P<0.05），表明GnRH通过诱导LHR表达、调节成熟促进因子（MPF）活性促进卵母细胞的减数分裂成熟，从而证明中华绒螯蟹与脊椎动物类似存在GnRH信号通路，GnRH信号系统在调控卵母细胞成熟中起关键作用，蟹类 GnRH信号系统的发现对于解明卵母细胞成熟机制和无脊椎动物GnRH信号系统的进化具有重要学术价值。