Chongqing Key Laboratory of Forage &Herbivore, College of Veterinary Medicine, Southwest University, Beibei, Chongqing, 400715, China; Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China.
Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China.
Theriogenology. 2023 Dec;212:91-103. doi: 10.1016/j.theriogenology.2023.09.005. Epub 2023 Sep 9.
Follicular fluid (FF) is rich in extracellular vesicles (EVs), which have regulatory effects on follicular growth and oocyte development. EVs can be divided into two subtypes, i.e. HD-sEVs and LD-sEVs. In this study, HD-sEVs were successfully isolated from bovine follicular fluid (BFF) by density gradient ultracentrifugation. By western blot, quantitative polymerase chain reaction (qPCR), flow cytometry, transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA), this study found HD-sEVs promoted autophagy in bGCs by increasing the protein and mRNA expression of LC3II/LC3I ratio and Beclin1, and inhibiting the protein and mRNA expression of p62. HD-sEVs promoted mitophagy in bGCs by increasing the protein and mRNA expression of VDAC1, CTSD, and HSP60. Flow cytometry showed that HD-sEVs inhibited bGCs apoptosis rate. HD-sEVs promoted estradiol secretion by increasing steroidogenesis-associated proteins and mRNA, such as CYP19A, HSD3B in bGCs. HD-sEVs promoted autophagosome formation and mitochondrial structure swelling in bGCs, and decreased p-mTOR/mTOR ratio. The above phenomenon was reversed when wortmannin was added. Collectively, BFF HD-sEVs promote bGCs autophagy and mitophagy, inhibit bGCs apoptosis and promote estradiol secretion through the autophagy pathway-mTOR signaling pathway.
卵泡液（FF）富含细胞外囊泡（EVs），对卵泡生长和卵母细胞发育具有调节作用。EVs 可分为两种亚型，即高密度-sEVs 和低密度-sEVs。本研究通过密度梯度超速离心成功从牛卵泡液（BFF）中分离出高密度-sEVs。通过 Western blot、定量聚合酶链反应（qPCR）、流式细胞术、透射电子显微镜（TEM）和酶联免疫吸附试验（ELISA），本研究发现高密度-sEVs 通过增加 LC3II/LC3I 比值和 Beclin1 的蛋白和 mRNA 表达，抑制 p62 的蛋白和 mRNA 表达，促进 bGC 中的自噬。高密度-sEVs 通过增加 VDAC1、CTSD 和 HSP60 的蛋白和 mRNA 表达促进 bGC 中的细胞自噬。流式细胞术显示高密度-sEVs 抑制 bGCs 的凋亡率。高密度-sEVs 通过增加 bGC 中的类固醇生成相关蛋白和 mRNA，如 CYP19A、HSD3B，促进雌二醇的分泌。高密度-sEVs 促进 bGC 中自噬体的形成和线粒体结构肿胀，并降低 p-mTOR/mTOR 比值。当添加渥曼青霉素时，上述现象被逆转。总之，BFF 高密度-sEVs 通过自噬途径-mTOR 信号通路促进 bGC 自噬和细胞自噬，抑制 bGC 凋亡并促进雌二醇的分泌。
