Extracellular vesicles of bovine small follicular fluid promote ovarian cortical stromal cell proliferation and steroidogenesis.

The aim of this study was to investigate the effects of extracellular vesicles (EVs) on the proliferation and steroid hormone synthesis of bovine ovarian cortical stromal cells in vitro. The release and uptake of EVs are the new mechanisms of cell-to-cell communication. Using reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, TUNEL and other experiments, we found that EVs in bovine follicular fluid can promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. Moreover, 100 µg/ml EVs caused the most significant effect. We conclude that EVs at 100 µg/ml can significantly promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. This research is of great significance for further elucidating the regulatory role of follicular fluid EVs in follicular development and atresia and for research on the interaction of ovarian stromal cells, granulosa cells and oocytes.

Follicular fluid HD-sevs-mir-128-3p is a key molecule in regulating bovine granulosa cells autophagy.

Follicular fluid (FF) is rich in extracellular vesicles (EVs). EVs carries a variety of miRNA involved in regulating follicular development, the function of cells in follicles, primordial follicular formation, follicular recruitment and selection, follicular atresia, oocyte communication, granulosa cells (GCs) function and luteinization and other biological processes of follicular development. Previous studies in our laboratory have shown that bovine follicular fluid (bFF) high density-small extracellular vesicles (HD-sEVs)-miRNA was enriched in autophagy-related pathways. However, the mechanism of bFF EVs carrying miRNA regulating GCs autophagy is not clear. Thus, this study carried out a series of studies on the previous HD-sEVs sequencing data and miR-128-3p contained in bFF HD-sEVs. A total of 38 differentially expressed genes were detected by RNA-Seq after overexpression of miR-128-3p in bovine GCs (bGCs). Through cell transfection, Western blot (WB) and Immunofluorescence (IF), it was proved that overexpression of miR-128-3p could promote the expression of LC3 (microtubule-associated protein I light chain 3), inhibit p62, promote the number of autophagosome, promote the formation of autophagy lysosome and autophagy flow, and activate bGCs autophagy. MiR-128-3p inhibitor significantly inhibited the expression of LC3 and monodansylcadaverine (MDC) in bGCs, and promoted the expression of autophagy substrate p62, indicating that HD-sEVs-miR-128-3p could activate bGCs autophagy. In addition, through double luciferase assay, bioinformatics analysis, WB and RT-qPCR, it was concluded that bFF HD-sEVs-miR-128-3p could target TFEB (transcription factor EB) and FoxO4 (Forkhead box O4) and activate GCs autophagy.

Large extracellular vesicles in bovine follicular fluid inhibit the apoptosis of granulosa cell and stimulate the production of steroid hormones.

The cargo carried by extracellular vesicles (EVs) plays an important physiological role in their corresponding target organs or target tissue cells. Extracellular vesicles are classified into large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs) according to their diameters. Since different subtypes contain different contents, their roles are also different. In this study, the morphology and size of LEVs were analyzed by transmission electron microscopy and nanoparticle size, and the marker proteins of LEVs (CD63, GP96, TSG101, ALB) were identified by western blot, and high-purity LEVs were obtained. Through the uptake of extracellular vesicles by purified ovarian granulosa cells and the determination of granulosa cell viability, cell apoptosis, and steroid hormone production, the result indicated that LEVs significantly enhanced cell viability (P < 0.05), reduced the rate of granulosa cell apoptosis (P < 0.05). Meanwhile, LEVs promoted the secretion of estradiol in granulosa cells (P < 0.05). This study provides a reference for the in-depth study of the function of follicular fluid extracellular vesicle subtypes and the research on the regulation of extracellular vesicles on follicle and oocyte development.