College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, People's Republic of China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, People's Republic of China.
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, People's Republic of China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, People's Republic of China.
J Steroid Biochem Mol Biol. 2019 Mar;187:58-67. doi: 10.1016/j.jsbmb.2018.11.001. Epub 2018 Nov 8.
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 cumulus-oocyte 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, 17β-estradiol 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.
雌激素在哺乳动物卵巢中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）被鉴定为一种膜雌激素受体，介导快速的非基因组信号事件，可能负责调节卵母细胞减数分裂恢复和缝隙连接细胞间通讯（GJIC）。本研究旨在确定 GPR30 在山羊卵丘-卵母细胞复合物（COC）中的表达和定位及其在卵母细胞减数分裂进展和 GJIC 中的作用。免疫荧光实验显示，GPR30 主要位于山羊卵丘细胞和卵母细胞的质膜中。在体外培养的早期，17β-雌二醇可促进卵母细胞减数分裂的进展，这一过程可被 G15（一种选择性 GPR30 拮抗剂）阻断，但不能被 ICI182780（核雌激素受体抑制剂）阻断。通过 Lucifer Yellow（LY）微注射和 calcein-AM 荧光染料扩散定量 17β-雌二醇对 GJIC 的影响。17β-雌二醇处理山羊 COC 可导致 GJIC 迅速下调。用 carbenoxolone（一种已知的缝隙连接阻断剂）、17β-雌二醇或 G1（一种 GPR30 激动剂）可显著抑制 calcein 从卵丘细胞向卵母细胞的转移，而 G15 可逆转这种抑制，但 ICI182780 则不能，表明 GPR30 介导了 17β-雌二醇对 GJIC 快速下调的作用。当 COC 在体外培养 4、6 和 8 小时时，17β-雌二醇还刺激连接蛋白 43（Cx43）的丝氨酸 368 磷酸化。更重要的是，当 COC 培养 4 小时时，17β-雌二醇或 G1 可分别显著促进细胞外信号调节激酶 1/2（ERK 1/2）和 Cx43 的磷酸化。此外，G15 和表皮生长因子受体（EGFR）酪氨酸激酶抑制剂 AG1478 或 ERK1/2 抑制剂 PD98059 均可抑制 ERK1/2 和 Cx43 的磷酸化，表明 EGFR-ERK1/2 信号通路参与了这些事件。这些结果支持了 GPR30 介导 17β-雌二醇刺激的减数分裂恢复和山羊 COC 中 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。
