GPR30介导的雌激素非基因组效应对硬骨鱼类精子发生过程中生殖细胞凋亡的调控

项目名称： GPR30介导的雌激素非基因组效应对硬骨鱼类精子发生过程中生殖细胞凋亡的调控

项目编号： No.30871906

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 刘晓春

作者单位： 中山大学

项目金额： 30万元

中文摘要： 本项目在斑马鱼中克隆了雌激素膜受体GPR30（亦称GPER）。RT-PCR 结果显示，斑马鱼GPR30 (zfGPR30) 在脑和性腺丰度最高。原位杂交结果发现，zfGPR30 主要分布在雄性早期生殖细胞如精原细胞和精母细胞以及Sertoli 细胞，而在精子细胞中检测不到信号。提示GPR30 蛋白可能在斑马鱼早期精子发生过程中发挥作用。配体受体结合率检测发现，雌二醇与zfGPR30的亲和常数为4.6nM。zfGPR30 在各种浓度的雌二醇处理下都不能显著刺激c-fos 基因启动子偶联的萤光素酶的表达；而在10-8 M 雌二醇浓度下即可显著性地启动带有腺苷酸环化酶应答元件的启动子偶联的萤光素酶基因的表达。外源性雌二醇浸泡处理成年雄性斑马鱼，能诱导精子细胞或精子的明显凋亡，而核受体拮抗剂ICI182, 780 和芳香化酶抑制剂Fadrozole 则对早期和晚期生殖细胞的凋亡都起促进作用。通过检测斑马鱼凋亡相关基因，提示17β2、ICI182, 780 和Fadrozole 通过Fas 途径引发精子细胞的凋亡，而后两者同时通过p73 途径引起精子发生早期精母细胞的凋亡。

中文关键词： 斑马鱼；雌二醇；雌激素膜受体（GPR30）；精子发生；生殖细胞凋亡

英文摘要： A seven-transmembrane G protein coupled receptor was isolated in zebrafish using bioinformatics and RACE (rapid amplification of cDNA ends) techniques. Phylogenetic analysis and multiple alignments of amino acid sequences indicated that the cloned receptor protein was of huge homology with the novel human estrogen membrane estrogen receptor GPR30. As the results of RT-PCR shown, zfGPR30 expressed most abundantly in brain and gonads. In situ hybridization results displayed that zfGPR30 mRNA mainly expressed in the early germ cells including spermatogonia and spermatocytes as well as somatic cells like Sertoli cells, while no staining was detected in spermatids, implying that zfGPR30 protein may take effects in early spermatogenesis of zebrafish. Binding test of zfGPR30 with estradiol-17βhowed the presence of a single, high-affinity and saturable estradiol-binding sites with a dissociation constant (Kd) of 4.6nM. Functional study on zfGPR30 transactivating c-fos and adenylyl cyclase showed that zfGPR30 could activate the pCRE promoter at E2 concentration from 10-8M but failed to stimulate the c-fos promoter at a wide range of E2 concentrations. Exogenous estradiol could induce a significant apoptosis of spermatids or sperms in adult male zebrafish, while ICI182, 780 and Fadrozole facilitated apoptosis effects on both spermatocytes and late germ cells. As to the checked apoptosis related genes, Fas and FasL expressed with huge increase in estradiol-17βreated testis. Bax, Fas and p73 significantly elevated their abundance after ICI182, 780 treatment, and Fas and p73 greatly increased in Fadrozole treated testis as well. It indicated that estradiol-17βICI182, 780 and Fadrozole induced apoptosis of spermatids by activating transcription of Fas. Meanwhile, ICI182, 780 and Fadrozole also facilitated apoptosis of early spermatocytes through activating expression of p73.

英文关键词： zebrafish (Danio rerio); estradiol-17βGper (GPR30); spermatogenesis; germ cell apoptosis