IP3R1 regulates AMPK-mTOR-mediated mitochondrial function to influence the oocyte-to-embryo transition of porcine

Deciphering the biological regulatory mechanisms involved in mammalian oocyte-to-embryo transition (OET), which is initiated by postfertilization mature oocyte activation, is highly important. Oocyte maturation and activation are simultaneously regulated by inositol 1,4,5-triphosphate receptor (IP3R1)/Ca²⁺ signaling. This study investigated the potential mechanisms of IP3R1 in the OET process. The present study revealed that treatment with 2-aminoethyl diphenylborinate (2-APB group), a specific IP3R1inhibitor, markedly decreased intracellular calcium ion levels, as well as the rates of pronuclear formation, embryo cleavage and blastocyst formation. To clarify the mechanism underlying the role of IP3R1 during the OET, siRNA targeting IP3R1 was subsequently microinjected into oocytes at the pre-MII stage to interfere with IP3R1 expression. We use Smart-seq analysis to compare and analyze the differentially expressed genes and potential pathways between the control group and the treating oocytes with a noncompetitive IP3R1 inhibitor-treated group (siIP3R1 group). Through differential gene screening at three critical time points (MⅡ, Zygote, and 2-cell), combined with GO and KEGG pathway enrichment analysis, we revealed that IP3R1 regulates AMPK/mTOR balance in OET after fertilization via CAMKK2. This regulation modulates mitochondrial function and mitochondrial energy metabolism, decreases PGC1 gene expression, upregulates TOMM40 and Caspase3 gene expression, promote ATP production, upregulates ND1 and ATP6 gene expression, reduces mitochondrial oxidative stress, and decreases reactive oxygen species(ROS)levels and BAX、SOD2 and CAT gene expression Collectively, these effects enhance eukaryotic translation initiation factor 4E (eIF4E) activity and promote histone methylation levels, as demonstrated by increased SETD2 expression and H3K36me3 levels. In conclusion, this study demonstrated that IP3R1 activity in both in vitro matured oocytes and in vitro activated oocytes initiates the OET through regulation of the AMPK-mTOR-eIF4E pathway mediated by Ca²⁺/CAMKK2, thereby facilitating the OET and embryonic development. This study aims to provide theoretical guidance for optimizing the in vitro production system of porcine gametes.