193 Extracellular vesicles coupled miRNAs modulate bovine oocyte response to thermal stress

Oxidative stress induced by elevated temperatures is known to cause damage to cellular DNA, lipids, and proteins that results in growth arrest and apoptosis. We have recently reported that extracellular vesicles (EVs) released from granulosa cells (GCs) in response to thermal stress mitigate the detrimental impact of thermal stress on recipient GCs, oocytes, and developing embryos by reducing reactive oxygen species (ROS) accumulation, improving cell viability, and decreasing apoptotic damage. MicroRNA (miRNA) cargo analysis revealed the enrichment of miR-1246, miR-374a, and miR-2904 in these EVs compared with those released under thermoneutral conditions. We hypothesized that heat-stress EV-associated miRNAs (miR-1246, miR-374a, and miR-2904) provide critical protection to oocytes under thermal stress, thereby enhancing their subsequent development and functional viability. Target gene prediction indicated that these candidate miRNAs regulate genes involved in the JNK, p38 MAPK, and p53 signaling pathways, suggesting potential anti-inflammatory/anti-apoptotic functions. To test this hypothesis, we overexpressed the three candidate miRNAs individually by supplementing cumulus–oocyte complexes (COCs) with synthetic miRNA mimics (1 μM) during IVM, followed by exposure to 41°C thermal stress for 8 h. Expression of the miRNAs, their subsequent predicted target genes, and stress response-related genes were then quantified in the cumulus cells (CCs) of transfected COCs, normalized to ACTB and GAPDH reference genes, and analyzed using the ΔΔCT method. Intracellular ROS accumulation in oocytes (n = 30 per group) was assessed using the H2DCFDA Cellular ROS Assay Kit, visualized under an inverted fluorescent microscope, and analyzed using ImageJ software. Data from each miRNA mimic group were statistically compared with the non-supplemented negative control group using student’s t-test. Results demonstrated that transfection of COCs with synthetic miRNA mimics significantly (P < 0.05) increases the expression of each corresponding miRNA in the CCs. Overexpression of miR-1246, miR-374a, and miR-2904 resulted in the suppression of HSP70 to control levels, while HSP90 expression was inversely upregulated. Furthermore, the overexpression of miR-374a and miR-2904 suppressed MAP2K6 and MAPK12 expression, and the overexpression of miR-1246 suppressed the pro-apoptotic gene BID. Additionally, overexpression of the three candidate miRNAs significantly reduced (P < 0.05) the accumulated ROS in the resulting oocytes following thermal stress compared with the negative controls. In conclusion, miR-1246, miR-374a, and miR-2904 have the potential to in part modulate the stress response and improve the survival and viability of bovine oocytes subjected to thermal stress by altering stress-induced transcriptomic changes, particularly in the JNK and p38 MAPK signaling pathways, and through modulating the subsequent ROS production and accumulation.