Phosphorylated Parkin (Ser65) alleviates MCU-mediated mitochondrial dysfunction in vitrified porcine oocytes through coordinated mitophagy and mitochondrial fission

Porcine oocyte vitrification-warming causes severe mitochondrial defects and inefficient cryopreservation due to its low cryotolerance, accompanied by induction of phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1)/Parkin-mediated mitophagy. Here, micro-proteomics revealed that differentially expressed proteins in vitrified oocytes were enriched in mitochondrial function and mitophagy-related pathways with the mitochondrial calcium uniporter (MCU) identified as a key upregulated protein. MCU-driven mitochondrial calcium overload triggered mitochondrial permeability transition pore (mPTP) opening, oxidative stress, and ultimately oocyte death. To restore mitochondrial homeostasis, the Parkin agonist T0467 was evaluated. In fresh oocytes, T0467 concentration-dependently increased PINK1/Parkin expression and enhanced mitophagy. Importantly, 10 µM T0467 treatment markedly promoted Parkin Ser65 phosphorylation, coordinately activating mitophagy and mitochondrial fission in vitrified oocytes. This response attenuated MCU-mediated calcium overload, preserved mitochondrial homeostasis, oocyte viability and in vitro fertilization (IVF). Collectively, these findings demonstrated that phosphorylation of Parkin at Ser65 protected vitrified porcine oocytes against MCU-mediated mitochondrial dysfunction by coordinating mitophagy and mitochondrial fission, highlighting T0467 as a promising approach for improving oocyte cryopreservation efficiency.