Deficiency of ZP3 activates oocyte degeneration via mito-ROS/GPX4 pathway.

Mutations in glycoprotein, zona pellucida glycoprotein 3 (ZP3), cause human ZP loss and empty follicle syndrome (EFS), yet the underlying mechanisms remain unknown. In this study, we generated Zp3-knockout rats (rZp3-/-) using CRISPR-Cas9, which recapitulated the EFS phenotype with follicular arrest and oocyte degeneration. Transmission electron microscopy revealed complete absence of the zona pellucida and transzonal projections, alongside shortened microvilli that correlated with radixin downregulation, indicating impaired oocyte-granulosa communication. Single-oocyte transcriptomics revealed dysregulated mitochondria-associated signaling pathways, such as glutathione metabolism and oxidative phosphorylation, consistent with energy deprivation and oxidative stress resulting from impaired cell dialogs between oocyte and granulosa cells. In oocytes of rZp3-/-, mitochondrial dynamics were severely disrupted, featuring reduced mitochondrial area, cristae hyperfusion. In addition, electron transport chain activity was impaired in oocytes of rZp3-/-, driving mitochondrial reactive oxygen species (ROS) overproduction. Consequently, rZp3-/- oocytes displayed ferroptosis hallmarks, including elevated lipid peroxidation, iron dysregulation, and glutathione peroxidase 4 (GPX4) inactivation. Pharmacological inhibition of ferroptosis in vitro using deferiprone partially rescued oocyte defects by restoring GPX4, attenuating mitochondrial ROS and lipid peroxidation, therefore inhibit apoptosis in rZp3-/-.This study establishes ZP3 as a guardian of mitochondrial homeostasis and implicates ferroptosis as a key pathway in EFS pathogenesis, offering novel therapeutic targets.