Abstract
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.