拟穴青蟹促性腺激素释放激素（GnRH）生殖旁分泌作用及信号通路分析

This project plans to investigate the reproductive paracrine of gonadtropin releasing hormone (GnRH) and its signaling pathways in the mud crab, Scylla paramamosain, including following topics: ⑴ The reproductive paracrine function of GnRH, by observing GnRH's effect on germinal vesicle breakdown (GVBD), the activation of maturation promoting factor (MPF), the change of Ca2+ current, and the induction of the GnRH-receptor expression; ⑵ The analysis of GnRH signaling pathways, by studying the relationship between cAMP, MAPK and phosphatidylinositol signaling pathways and the activation of MPF, and confirm whether they accept the regulation of GnRH. This project takes a lead in studying the paracrine function of GnRH in crustacean, thus develops a new domain in crustacean endocrinology. It is for the first time that the relationship between oocytes signaling pathways and activation of MPF is analyzed, and signaling pathways which accept the regulation of GnRH is identified. This project will break through the limits of lacking cell lines in crustacean in that oocytes are taken as experimental materials, and patch clamp techniques were used to study the maturation mechnism of oocyte. The results would provide new scientific proofs for spawning induction in crustacean. In brief, this project has great theoretical importance and potent application value.

本项目开展拟穴青蟹GnRH 生殖旁分泌作用和信号通路分析，主要内容：（1） GnRH 的生殖旁分泌作用，主要观察GnRH 对卵母细胞生发泡破裂（GVBD）、成熟促进因子（MPF） 激活、钙离子电流变化、GnRH 受体表达的影响。（2）GnRH 信号通路分析，研究卵母细胞cAMP 信号通路，MAPK 信号通路和磷脂酰肌醇信号通路三者与MPF 激活的关系，并确认接受GnRH 调控的信号通路。本项目率先开展了甲壳动物GnRH 旁分泌作用的研究，开辟了甲壳动物内分泌学研究的一个新领域；首次分析了甲壳动物卵母细胞信号通路与MPF 激活的关系，并对接受GnRH 调控的信号通路进行了鉴定。本项目以卵母细胞为研究材料，利用膜片钳技术等手段分析卵母细胞成熟的机制，突破了甲壳动物没有细胞系的平台限制，相关研究结果可为甲壳动物诱导产卵的技术创新提供科学依据。

甲壳动物GnRH生殖旁分泌作用和信号通路迄今尚不清楚。本项目以拟穴青蟹为对象开展相关研究，主要实验结果如下：经免疫组化检测，在青蟹卵巢组织中发现GnRH受体存在于一种未被鉴定的细胞。由于未能分离获得青蟹的GnRH，我们应用章鱼GnRH（oct-GnRH）开展研究，在离体条件下发现oct-GnRH直接促进卵黄发生期卵巢中雌二醇和卵黄蛋白的合成，促进卵巢成熟期卵母细胞生发泡破裂、成熟促进因子激活和钙离子浓度上升。信号通路分析揭示了oct-GnRH诱导的卵母细胞成熟主要与cAMP信号通路和磷脂酰肌醇信号通路相关。cAMP浓度升高抑制了oct-GnRH诱导的卵母细胞的成熟过程，表明cAMP信号通路起着负调控的作用。卵母细胞成熟需要磷脂酰肌醇信号通路的激活：磷脂酶C浓度的降低能够抑制oct-GnRH诱导的卵母细胞成熟；蛋白激酶C浓度的降低则会促进卵母细胞的成熟。本项目以卵母细胞为研究材料，突破了甲壳动物没有细胞系的平台限制，相关研究结果可为甲壳动物诱导产卵的技术创新提供科学依据。