College of Animal Science and Technology, Key Laboratory of Animal Genetic, Breeding and Reproduction in Shaanxi Province, Northwestern A&F University, Yangling, Shaanxi, China.
Reprod Domest Anim. 2022 Sep;57(9):989-998. doi: 10.1111/rda.14165. Epub 2022 Jun 1.
The aim of our study was to determine whether silencing or overexpression of estrogen receptor β (ERβ) regulates cell proliferation, steroidogenesis, autophagy and signalling pathways in bovine ovarian granulosa cells in vitro. In this study, bovine ovarian granulosa cells (BGCs) were cultured and transfected with ERβ siRNA (si-ERβ) or a plasmid overexpressing ERβ (oe-ERβ), and CCK-8 kit was used to assess cell proliferation. Real-time PCR was used to measure gene transcription. Western blotting was used to measure protein expression, and a specific kit was used to measure the production of steroid hormones. The results showed the expression level of ERβ affects BGC proliferation according to the gene transcription levels of FSHR, CYP19A1, HSD3β1 and STAR and the production of E2 and P4. ERβ was identified as an important nuclear receptor that induced BGC autophagy based on the mRNA and protein expression of autophagy-related genes. Furthermore, the role of ERβ in BGC autophagy was confirmed through treatment with rapamycin (RAPA) or 3-methyladenine (3-MA) in BGCs by cotransfection with si-ERβ or oe-ERβ in BGCs. The results related to AKT/mTOR signalling and phosphorylation suggested that ERβ induces BGC autophagy through attenuating AKT/mTOR signalling. In summary, this study demonstrates that silencing or overexpression of ERβ regulates BGC proliferation and function and induces BGC autophagy by targeting AKT/mTOR signalling. These data reveal a novel regulatory mechanism of autophagy via ERβ and provide insights into the role of autophagy in BGCs.
本研究旨在确定雌激素受体β（ERβ）的沉默或过表达是否调节体外牛卵巢颗粒细胞的细胞增殖、类固醇生成、自噬和信号通路。在这项研究中，培养牛卵巢颗粒细胞（BGCs）并转染 ERβ siRNA（si-ERβ）或过表达 ERβ 的质粒（oe-ERβ），使用 CCK-8 试剂盒评估细胞增殖。实时 PCR 用于测量基因转录。Western blot 用于测量蛋白质表达，并用特定试剂盒测量类固醇激素的产生。结果表明，根据 FSHR、CYP19A1、HSD3β1 和 STAR 的基因转录水平以及 E2 和 P4 的产生，ERβ 的表达水平影响 BGC 的增殖。根据自噬相关基因的 mRNA 和蛋白质表达，将 ERβ 鉴定为诱导 BGC 自噬的重要核受体。此外，通过在 BGC 中转染 si-ERβ 或 oe-ERβ，用雷帕霉素（RAPA）或 3-甲基腺嘌呤（3-MA）处理 BGC，证实了 ERβ 在 BGC 自噬中的作用。与 AKT/mTOR 信号和磷酸化相关的结果表明，ERβ 通过减弱 AKT/mTOR 信号诱导 BGC 自噬。总之，本研究表明，沉默或过表达 ERβ 调节 BGC 的增殖和功能，并通过靶向 AKT/mTOR 信号诱导 BGC 自噬。这些数据揭示了 ERβ 通过自噬的新调节机制，并为自噬在 BGCs 中的作用提供了新的见解。
