Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts

Extracellular vesicles (EVs) and their cargo microRNAs (miRNAs) are important regulators of embryo development to the blastocyst stage and beyond. Before implantation can take place, hatching of blastocysts from their zona pellucida is required. However, underlying mechanisms by which blastocyst formation and hatching are initiated remain largely unknown. Here, we provide evidence that embryonic EVs containing bta-miR-378a-3p play a crucial role in blastocyst hatching, using a bovine model. A customized procedure was used to isolate EV-miRNAs from culture droplets conditioned by individual bovine embryos that either developed to the blastocyst stage or did not (nonblastocyst). RNA sequencing identified 69 differentially expressed miRNAs between EVs derived from blastocyst conditioned medium (CM) and nonblastocyst CM. Among the miRNAs up-regulated in blastocyst CM, we selected bta-miR-378a-3p for further validation by functionality testing on developing in vitro embryos by means of mimics and inhibitors. Supplementing the embryo culture medium with miR-378a-3p mimic significantly improved blastocyst quality, with higher cell numbers and reduced apoptosis, and improved hatching, while the opposite was found after supplementation with miR-378a-3p inhibitor (P < 0.01). Transcriptomic analysis of embryos treated with miR-378 mimic/inhibitor showed differential expression (P < 0.01) of genes associated with embryo development and implantation, including RAP1GAP, ARFGEF2, SLC7A6, CENPA, SP1, LDLR, PYCR1, MYD88, TPP1, and NCOA3. In conclusion, miR-378a-3p is up-regulated in EVs secreted by embryos that develop to the blastocyst stage, and this EV-derived miR-378a-3p increases blastocyst quality and regulates embryo hatching, which is essential for embryo implantation.