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.
Annotation breakdown and description of miRNA within the
EVs using a comprehensive and unbiased approach
(A) The flow chart shows the total number of loci and the classification of derivation (i.e., known, homologous to other species, and novel) that were associated with sequence loci with >100 read average within a follicle size group that met the Sanger requirements to be considered a miRNA (see detailed description in Methods). (B,C) show two representative miRNA sequences derived from EVs and compares them to the established human and bovine annotated sequences. (B) The current miR-103 sequence is 5 bases shorter than the previous annotated bovine and human miR-103 transcripts. (C) The current miR-224 sequences were of two lengths 22 and 24 bases (denoted by two overlapping arrows) this differs from the previous annotated bovine sequence which was 23 bases.
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.