Modulation of bovine oocyte developmental competence through exogenous miRNA delivered via engineered EVs

In vitro maturation (IVM) of oocytes plays a crucial role in assisted reproductive technologies in livestock production, enabling efficient large-scale embryos production and supporting genetics improvement programs. However, the efficiency of this technology remains limited as only a small proportion of cumulus-oocytes complexes (COCs) matured in vitro reach the blastocyst stage, which exhibit lower quality than those produced in vivo. Recent efforts have focused on delivering biological materials to cumulus cells during IVM to improve oocyte quality. Herein, we hypothesized that extracellular vesicles (EVs) isolated from ovarian follicular fluid (FF) can be engineered to deliver synthetic microRNAs (miRNAs) to cumulus cells during IVM. Here, we demonstrate for the first time that follicular fluid-derived EVs are enriched with synthetic miRNAs and successfully delivered to COCs, resulting in altered gene expression in cumulus cells, and transfer of miRNAs to the oocyte cytoplasm. Specifically, EVs loaded with bta-miR-20a-5p were effectively taken up by cumulus cells during IVM, enhanced PI3K/AKT signaling with significant increase in downstream genes, such EIF4b and GSK3, which are involved in cell cycle progression, DNA repair and protein synthesis. Additionally, bta-miR-20a-5p-loaded-EVs supplementation to COCs, during IVM, appeared to reduce cleavage and blastocyst development rates. Collectively, our findings demonstrated that engineered EVs can deliver a synthetic miRNA to COCs during IVM, representing highlighting their potential to modulate oocyte cytoplasm content and embryo development.