The histone methyltransferase SETD2 regulates the PI3K/AKT signaling pathway to affect the in vitro fertilization of yak oocytes.

SET domain-containing 2 (SETD2) mediated H3K36me3 plays a pivotal role in maternal epigenetic regulation; however, its function during in vitro fertilization (IVF) remains largely unexplored. This study investigated the impact of SETD2 inhibition on yak oocyte fertilization competence. Pharmacological inhibition of SETD2 using EZM0414 significantly increased ROS levels and reduced GSH levels in yak oocytes. In parallel, it decreased the second polar body extrusion rate, increased zona pellucida thickness, and downregulated essential genes associated with sperm-oocyte interaction and oocyte activation (Zp2, Zp3, Zp4, Tle6, Wee2, Patl2). Further transcriptomic analysis of mature oocytes and fertilized zygotes using Smart-seq2 revealed that the differentially expressed genes between mature oocytes and zygotes were significantly enriched in the PI3K/AKT signaling pathway. Key survival-related genes (Pik3r1, Pik3c2b, Akt1, Akt2, Creb5, Mcl1) were markedly upregulated in zygotes, indicating activation of PI3K/AKT during fertilization. SETD2 inhibition also suppressed PI3K/AKT pathway activity at both mRNA and protein levels. Furthermore, SETD2 inhibition shifted the apoptotic balance by decreasing BCL2, increasing BAX and Caspase-3, and elevating TUNEL-positive signals in both oocytes and zygotes. Inhibition of SETD2 suppressed H3K36me3 expression in yak oocytes and zygotes. Finally, we found that inhibition of SETD2 significantly increased the rate of early embryonic arrest while reducing the blastocyst formation rate. Collectively, these findings demonstrate that SETD2 is essential for maintaining PI3K/AKT signaling, preventing apoptosis, and preserving fertilization competence in yak oocytes. This work provides new mechanistic insight into the epigenetic regulation of fertilization and offers a potential strategy for improving assisted reproductive efficiency in livestock.