G protein-coupled receptor 30 mediates meiosis resumption and gap junction communications downregulation in goat cumulus-oocyte complexes by 17 beta-estradiol
G protein-coupled receptor 30 mediates meiosis resumption and gap junction communications downregulation in goat cumulus-oocyte complexes by 17 beta-estradiol
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G 蛋白偶联受体 30 通过 17 β-雌二醇介导山羊卵丘-卵母细胞复合物减数分裂恢复和间隙连接通讯下调
DOI：
10.1016/j.jsbmb.2018.11.001
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发表时间：
2019
影响因子：
4.1
通讯作者：
Ma Baohua
中科院分区：
文献类型：
--
作者：
Zhang Hui;Wei Qiang;Gao Zhen;Ma Chiyuan;Yang Zhenshan;Zhao Hui;Liu Chen;Liu Jie;Zhao Xiaoe;Ma Baohua
在哺乳动物卵巢中，雌激素在卵母细胞和颗粒细胞之间的缝隙连接调节中起着关键作用。G蛋白偶联受体30(GPR30)是一种膜雌激素受体，参与调节卵母细胞减数分裂恢复和缝隙连接细胞间通讯(GJICs)的快速非基因组信号传递。本研究旨在探讨GPR30在山羊卵丘卵母细胞复合体(COCs)中的表达和定位及其在卵母细胞减数分裂进程和GJICs中的作用。免疫荧光实验表明，GPR30主要定位于山羊卵丘细胞和卵母细胞的质膜。17-β-雌二醇可促进卵母细胞的减数分裂进程，但在体外培养早期可被选择性GPR30拮抗剂G15阻断，但不能被核雌激素受体抑制剂ICI182780阻断。用荧光黄(LY)微量注射和钙黄素-AM荧光染料扩散法检测17β-雌二醇对GJICs的影响。17β-雌二醇处理山羊COCs后，GJICs的表达迅速下调。钙黄素从卵丘细胞到卵母细胞的转运可被已知的缝隙连接阻断剂甘草酮、17β-雌二醇或GPR30激动剂G1显著抑制，这种抑制作用可被G15逆转，但不能被ICI182780逆转，表明GPR30介导了17β-雌二醇对GJICs的快速下调作用。体外培养4h、6h和8h时，17β-雌二醇均能显著促进细胞外信号调节蛋白43(ERK1/2)和Cx43的丝氨酸368磷酸化，而G15、表皮生长因子受体(EGFR)酪氨酸激酶抑制剂AG1478和ERK1/2抑制剂PD98059均能抑制ERK1/2和Cx43的磷酸化，提示EGFR-ERK1/2信号转导通路参与了这一过程。这些结果支持GPR30介导17β-雌二醇刺激的山羊COCs减数分裂恢复和GJIC减少的假说。因此，本研究为阐明类固醇激素在卵母细胞成熟调控中的作用机制提供了新的见解。
Estrogen plays a critical role in the regulation of gap junctions between oocytes and granulosa cells in mammalian ovaries. G protein-coupled receptor 30 (GPR30) was identified as a membrane estrogen receptor, mediating rapid, nongenomic signaling events that might be responsible for the regulation of oocyte meiosis resumption and gap junction intercellular communications (GJICs). The present study aimed to determine the expression and localization of GPR30 and its role in oocyte meiotic progression and GJICs in goat cumulusoocyte complexes (COCs). Immunofluorescence experiments revealed that GPR30 was primarily located in the plasma membrane of cumulus cells and oocytes in goats. 17β-estradiol could promote oocyte meiotic progression, which was blocked by G15 (a selective GPR30 antagonist) but not ICI182780 (a nuclear estrogen receptor inhibitor) in the early stage of in vitro culture. The effect of 17β-estradiol on the GJICs was quantified by lucifer yellow (LY) microinjection and calcein-AM fluorescent dye diffusion. 17β-estradiol treatment of goat COCs resulted in rapid downregulation of GJICs. The transfer of calcein from cumulus cells to oocytes could be significantly inhibited by carbenoxolone (a known gap junction blocker), 17β-estradiol or G1 (a GPR30 agonist), and this inhibition could be reversed by G15 but not ICI182780, indicating that GPR30 mediates the effect of 17β-estradiol on the rapid downregulation of GJICs. 17β-estradiol also stimulated the serine 368 phosphorylation of connexin 43 (Cx43) when COCs were in vitro cultured for 4 h, 6 h, and 8 h. More importantly, 17βestradiol or G1 could separately promote the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and Cx43 significantly when COCs were cultured for 4 h. Furthermore, both ERK1/2 and Cx43 phosphorylation could be inhibited by G15 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or by the ERK1/2 inhibitor PD98059, indicating that EGFR-ERK1/2 signaling was involved in these events. These results supported the hypothesis that GPR30 mediated 17β-estradiol-stimulated meiotic resumption and GJIC reduction in goat COCs. Thus, the present study provides novel insights into elucidating the mechanisms for steroid hormone action in the regulation of oocyte maturation.