College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, 010018, PR China; Key Laboratory of Animal Embryo and Development Engineering of Autonomous Region Universities, Inner Mongolia Agricultural University, Hohhot, 010018, PR China.
Inner Mongolia People's Hospital, Hohhot, 010020, PR China.
Theriogenology. 2024 Jan 15;214:81-88. doi: 10.1016/j.theriogenology.2023.10.009. Epub 2023 Oct 14.
Although 17β-estradiol (E2) and its receptors (ERs) are reported to play important roles in regulating oocyte maturation, the specific mechanism remains unclear. First, we performed immunohistochemistry analyses to determine the expression of the ERα and ERβ proteins in ovine ovarian tissue. Second, E2 (0.5 ng/mL and 1 μg/mL) were added to pre-IVM medium for 0 h, 1 h and 2 h. The effects of E2 (0.5 ng/mL and 1 μg/mL) on cyclic adenosine monophosphate (cAMP) level in cumulus-oocyte complexes (COCs) and on oocyte meiotic progression were evaluated by ELISA and DAPI staining respectively. Third, the effects of E2 on the gene and protein expression of ERα and ERβ in COCs were investigated by Western blotting and real-time PCR. Afterward, ERβ and cAMP regulators were added to the 2-h pretreatment medium with or without E2 (0.5 ng/mL) to explore the possible interactions among E2, cAMP and ERβ. The results showed that both ERα and ERβ proteins were expressed in ovine cumulus layers and oocytes. E2 significantly increased intra-COC cAMP levels, maintained oocyte meiotic arrest, and promoted ERβ transcript and protein expression. E2 treatment increased the cAMP concentration, which was enhanced by ERβ agonist treatment and remarkably attenuated by ERβ inhibitor treatment. Forskolin plus IBMX treatment increased ERβ protein expression in COCs (P < 0.05), and this was attenuated by Rp-cAMP treatment. In conclusion, E2 (0.5 ng/mL) increased intra-COC cAMP levels by promoting ERβ expression, thereby maintaining oocyte meiotic arrest. cAMP in COCs has a positive feedback effect on ERβ expression, which provides a novel explanation for the positive role of E2 in regulating ovine follicle development and oocyte maturation.
虽然 17β-雌二醇（E2）及其受体（ERs）被报道在调节卵母细胞成熟中发挥重要作用，但具体机制仍不清楚。首先，我们进行了免疫组织化学分析，以确定绵羊卵巢组织中 ERα 和 ERβ 蛋白的表达。其次，将 E2（0.5ng/mL 和 1μg/mL）添加到预-IVM 培养基中 0h、1h 和 2h。通过 ELISA 和 DAPI 染色分别评估 E2（0.5ng/mL 和 1μg/mL）对卵丘-卵母细胞复合物（COCs）中环磷酸腺苷（cAMP）水平和卵母细胞减数分裂进展的影响。第三，通过 Western blot 和实时 PCR 研究 E2 对 COCs 中 ERα 和 ERβ 的基因和蛋白表达的影响。随后，在含有或不含有 E2（0.5ng/mL）的 2h 预处理培养基中添加 ERβ 和 cAMP 调节剂，以探讨 E2、cAMP 和 ERβ 之间的可能相互作用。结果表明，ERα 和 ERβ 蛋白均在绵羊卵丘层和卵母细胞中表达。E2 显著增加 COC 内的 cAMP 水平，维持卵母细胞减数分裂阻滞，并促进 ERβ 转录本和蛋白表达。E2 处理增加了 cAMP 浓度，该浓度被 ERβ 激动剂处理增强，并被 ERβ 抑制剂处理显著减弱。forskolin 加 IBMX 处理增加了 COCs 中 ERβ 蛋白的表达（P<0.05），而 Rp-cAMP 处理则减弱了这一表达。总之，E2（0.5ng/mL）通过促进 ERβ 表达增加 COC 内的 cAMP 水平，从而维持卵母细胞减数分裂阻滞。COCs 中的 cAMP 对 ERβ 表达具有正反馈作用，这为 E2 在调节绵羊卵泡发育和卵母细胞成熟中的积极作用提供了新的解释。
