BACKGROUND

Within the follicular fluid, extracellular vesicles (EVs) guide oocyte growth through their cargo microRNAs (miRNAs). Here, we investigated the role of EVs and their cargo miRNAs by linking the miRNAs found in EVs, derived from the fluid of an individual follicle, to the ability of its oocyte to become a blastocyst (competent) or not (non-competent).

METHODS

Bovine antral follicles were dissected, categorized as small (2-4 mm) or large (5-8 mm) and the corresponding oocytes were subjected to individual maturation, fertilization and embryo culture to the blastocyst stage. Follicular fluid was pooled in 4 groups (4 replicates) based on follicle size and competence of the corresponding oocyte to produce a blastocyst. Follicular fluid-derived EVs were isolated, characterized, and subjected to miRNA-sequencing (Illumina Miseq) to assess differential expression (DE) in the 4 groups. Functional validation of the effect of miR-34c on embryo development was performed by supplementation of mimics and inhibitors during in vitro maturation (IVM).

RESULTS

We identified 16 DE miRNAs linked to oocyte competence when follicular size was not considered. Within the large and small follicles, 46 DE miRNAs were driving blastocyst formation in each group. Comparison of EVs from competent small and large follicles revealed 90 DE miRNAs. Cell regulation, cell differentiation, cell cycle, and metabolic process regulation were the most enriched pathways targeted by the DE miRNAs from competent oocytes. We identified bta-miR-34c as the most abundant in follicular fluid containing competent oocytes. Supplementation of miR-34c mimic and inhibitor during IVM did not affect embryo development. However, blastocyst quality, as evidenced by higher cell numbers, was significantly improved following oocyte IVM in the presence of miR-34c mimics, while miR-34c inhibitors resulted in the opposite effect.

CONCLUSION

This study demonstrates the regulatory effect of miRNAs from follicular fluid-derived EVs on oocyte competence acquisition, providing a further basis for understanding the significance of miRNAs in oocyte maturation and embryonic development. Up-regulation of miR-34c in EVs from follicular fluid containing competent oocytes and the positive impact of miR-34c mimics added during IVM on the resulting blastocysts indicate its pivotal role in oocyte competence.