雌激素及其受体对三疣梭子蟹卵巢发育的生理调控

Estrogen is one of the important sex steroid hormones during the ovarian development of crustacean. However, up to date, it is not clear for the regulatory mechanism of estrogen during the crustacean ovarian development. In our previous researches, we have found that the exoestrogen (17β-estradiol, E2) can enhance the oocyte development, and there are two type of estrogen receptor protein (named ERα and ERβ) and the transcripts of estrogen receptor candidate gene could be detected in both ovary and hepatopancrea during the ovarian development of swimming crab, Portunus trituberculatus. The current project will foucus on the physiological regulation of estrogen and its receptors on the ovarian development of female P. trituberculatus. Firstly, by vitro experiment (tissue culture with different E2 concentration) and vivo injection with different E2 dose, we try to exhaustedly evaluate the physiological regulation effect of E2) on ovarian development and vitellogensis for female P. trituberculatus. Then, we will squence and analyse the transcriptom of some samples from our previous vivo and vitro experiment using transcriptomics and bioinformatics, then establish the potentionally regulatory pathway and its network.. Finally, we will identificate of gene functions of estrogen receptor candidate gene during the ovarian development of female P. trituberculatus via three comprehensive methods, including estrogen receptor protein binding assay, gene transfection study and gene RNAi knockdown study using vivo experiment. Therefore, the expected outcomes of this project could be not only improve our understanding of crustacean reproductive endocrinology and reproductive toxicology of environmental endocrine disruptors (EEDS), but also provide the basic information for the fattening female P. trituberculatus. Furthermore. the methodology and criteria developed in this project will be applied in other crustacean species, which may facilitate the improvements of Chinese academic status of crustacean reproduction.

雌激素是甲壳动物卵巢发育过程中的重要性类固醇激素, 目前有关雌激素对甲壳动物卵巢发育的作用机制研究甚少。本项目在确认外源雌二醇可以促进三疣梭子蟹卵黄合成、发现三疣梭子蟹雌激素受体蛋白及其候选基因和完成三疣梭子蟹卵巢发育转录组测序的基础上, 首次系统研究"雌激素及其受体对三疣梭子蟹卵巢发育的生理调控"。项目首先通过外源雌二醇对三疣梭子蟹活体注射和组织离体培养, 全面评估雌二醇对三疣梭子蟹卵巢发育过程中的生理调控作用; 进一步通过转录组学和生物信息学研究, 构建雌激素对三疣梭子蟹卵巢发育的调控路径及网络; 最后, 通过同位素标记雌二醇与受体基因重组蛋白结合实验、受体基因细胞转染和活体RNAi这三个层次的深入研究, 确认三疣梭子蟹雌激素受体基因在卵巢发育中的生理功能。预期研究结果对深入理解甲壳动物的生殖内分泌机制、环境内分泌干扰物的生殖毒理、三疣梭子蟹的人工育肥均具有重要的理论意义和现实意义。

雌激素是甲壳动物卵巢发育过程中的重要性类固醇激素，本项目较系统研究“雌激素及其受体对三疣梭子蟹卵巢发育的生理调控作用及其可能机制”。项目首先系统研究了外源雌二醇对不同卵黄发生阶段卵巢发育和卵黄发生的影响，接着通过免疫组化技术研究了卵巢发育过程中雌二醇及其受体类似物的免疫定位和变化，筛选出合适的拮抗剂种类、注射剂量和注射频率，最后通过特异性结合实验、细胞转染和RNAi等技术研究雌激素相关受体基因的功能, 结果表明, 1. 组织离体培养结果显示一定浓度的外源雌二醇(10-9mol/L和10-8mol/L组)可以促进小生长期卵巢中Vn含量、Vg-mRNA和ERR-mRNA表达水平的提高，但对肝胰腺中Vg-mRNA表达无显著影响；外源雌二醇可以促进卵巢大生长期肝胰腺中Vg和ERR基因表达。活体注射条件下，雌二醇可以显著促进卵巢II期和III期早期雌体的卵巢发育，可以显著提高肝胰腺中的Vg和ERR基因的表达。2. 他莫昔芬(TAM)和氟维司群(ICI)都可以抑制III早期雌体的卵巢发育，均可以抑制Vg基因的表达，ERR和EcR可能参与了雌激素或雌激素拮抗剂对三疣梭子蟹卵巢发育的调控过程。卵巢中E2的免疫阳性主要分布于滤泡细胞和中后期的卵母细胞胞质(II-V期)，肝胰腺中的E2免疫阳性主要分布于F细胞及R细胞胞核中，R细胞胞核中的E2免疫阳性从III期开始显著下降，E2免疫阳性在神经组织和大颚器也有分布；ER-like阳性在三疣梭子蟹卵巢和肝胰腺中也广泛存在，且与卵巢发育周期相关，ERα和ERβ的分子量分别为53kDa和120kDa。3. 三疣梭子蟹雌激素相关受体(Pt-ERR)基因表达与三疣梭子蟹卵巢发育关系密切, 重组蛋白分子量约49KD, 受到雌激素的诱导表达, 敲降Pt-ERR会严重影响三疣梭子蟹的卵巢发育,该蛋白不能直接和雌激素结合,这暗示雌激素调控甲壳动物卵巢发育还存在其他的非核受体调控路径, 传递雌激素信号调控卵黄发生和卵巢发育。本研究结果对深入理解雌激素对甲壳动物卵巢发育的调控机制具有重要理论价值，同时对三疣梭子蟹的人工繁殖和池塘养殖也具有一定的现实意义。