MicroRNA-34c from follicular fluid-derived extracellular vesicles is a modulator of embryo quality

Oocyte competence is progressively acquired during follicle growth, with large follicles containing more often competent oocytes than small follicles. A competent oocyte is able to mature in vitro, be fertilized, and develop to the blastocyst stage. Extracellular vesicles (EVs) and their cargo micro RNAs (miRNAs) are critical in mediating intercellular communication inside the follicle and facilitating oocyte maturation. Whether follicular EVs are also involved in the acquisition of oocyte competence in the growing follicle remains unclear. Here, we have been using an individual culture system to distinguish follicular fluid and their corresponding bovine oocytes as competent or noncompetent. We used a qEV single-column method to isolate EVs from follicular fluid. RNA sequencing revealed 16 differentially expressed (DE) miRNAs in EVs from follicular fluid derived from either competent or noncompetent oocytes . Among the up-regulated miRNAs, we selected bta-miR-34c to further validate its effect on oocyte competence during in vitro maturation using mimics and inhibitors. By supplementing miR-34c mimics to the oocyte maturation medium, we could significantly improve blastocyst quality, as evidenced by higher cell numbers, while supplementation of miR-34c inhibitors produced the opposite effect. Taken together, the up-regulation of miR-34c in EVs derived from follicular fluid from competent oocytes is indicative of its regulatory role, and, when added during in vitro maturation of unselected oocytes, is able to increase total cell number and inner cell mass of resulting embryos, thus contributing to the development of healthy offspring Ovarian follicles (2-4 mm) from slaughterhouse-derived bovine ovaries were used to collect oocytes. Bovine oocytes were distributed in five different categories according to the in vitro maturation protocol used: control, microRNA-34c mimic supplementation, microRNA-34c inhibitor, negative control mimic supplementation, and negative control inhibitor supplementation. After 22 hours of in vitro maturation, oocytes were fertilized, and the resulting zygotes were put in in vitro embryo culture. After 8 days post-insemination, 10 embryos/group were pooled for transcriptome profile analysis. We performed 5 replicates of microRNA-34c mimic, 4 replicates of Control, 3 replicates of microRNA-34c inhibitor, negative control mimic and negative control inhibitor.