MicroRNA and Extracellular Vesicle Mediated Cellular Communication within the Bovine Follicle.

During ovarian follicular development, a rich and complex fluid containing numerous proteins, nucleic acids, and other macromolecules accumulates within the follicular antrum to effectively isolate and simultaneously nurture the cumulus-oocyte-complex. Recent studies have shown that extracellular vesicles (EVs), which include both exosomes and microvesicles, are abundant within antral fluid and are a rich source of microRNA (miRNA). Small RNAseq analyses have identified miRNA that are differentially abundant dependent on the stage of development of the bovine antral follicle. Furthermore, our laboratory and several others have now also demonstrated that follicular fluid EVs can elicit functional effects on ovarian granulosa cells and cumulus-oocyte-complexes. Extracellular vesicles isolated from small 3-5 mm bovine follicles were shown to stimulate ovarian granulosa cell proliferation, while EVs from larger periovulatory follicles (>9 mm diameter) exhibited a diminished ability to stimulate cell proliferation. Ongoing studies are evaluating the factors (e.g. lipid, RNA, protein) that might be responsible for these effects seen in the EVs using a combination of proteomic and RNAseq strategies. We also observed that EVs from small follicles were more readily taken up by granulosa cells, which might partly explain their increased activity. We are currently evaluating whether CD81, an established EV biomarker that is differentially expressed in EV from small versus large follicles, is important in this differential uptake by granulosa cells. Interestingly, we have also observed that EVs isolated from periovulatory follicles from cows prior to the LH surge are rich in CD81, while those isolated immediately following the LH surge have very few CD81-positive EVs. This is particularly interesting as previous studies in mice have demonstrated that loss of CD81 caused a pronounced decrease in sperm/egg fusion. Although, the specific mechanism of action via which CD81 faciltiates the process of fertilization remains to be determined. In addition to stimulating granulosa cell proliferation, EVs have been shown to impact another key physiologic event within the periovulatory follicle, the expansion of cumulus granulosa cells surrounding the oocyte. We showed that bovine EVs from both small and large follicles were able to stimulate cumulus expansion, however they had divergent effects on gene expression within the cumulus. In conclusion, we believe that EVs may present a novel cell to cell signalling mechanism that allows mural granulosa cells to interact with the cumulus and oocyte and vice versa. Funded by NIH-HD082484