Characterization and Small RNA Content of Extracellular Vesicles in Follicular Fluid of Developing Bovine Antral Follicles

Exosomes and microvesicles (i.e., extracellular vesicles: EVs) have been identified within ovarian follicular fluid and recent evidence suggests that EVs are able to elicit profound effects on ovarian cell function. While existence of miRNA within EVs has been reported, whether EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth remains unknown. Extracellular vesicles isolated from follicular fluid of small, medium and large bovine follicles were similar in size, while concentration of EVs decreased progressively as follicle size increased. Electron microscopy indicated a highly purified population of the lipid bilayer enclosed vesicles that were enriched in exosome biomarkers including CD81 and Alix. Small RNA sequencing identified a large number of known and novel miRNAs that changed in the EVs of different size follicles. Ingenuity Pathway Analysis (IPA) indicated that miRNA abundant in small follicle EV preparations were associated with cell proliferation pathways, while those miRNA abundant in large follicle preparations were related to inflammatory response pathways. These studies are the first to demonstrate that EVs change in their levels and makeup during antral follicle development and point to the potential for a unique vesicle-mediated cell-to-cell communication network within the ovarian follicle.