Effects of Stem Cell-Derived Mitochondrial Transfer on H₂O₂-Induced Oxidative Stress in COV434 Granulosa Cells

Background and Objectives: Oxidative stress-induced mitochondrial dysfunction contributes to granulosa cell apoptosis and ovarian dysfunction. This study investigated the therapeutic potential of human Wharton's jelly-derived mesenchymal stem cell-derived mitochondria (hWJ-MSCs-mito) in an H₂O₂-induced oxidative stress model of COV434 granulosa cells. Materials and Methods: Oxidative stress was induced using increasing concentrations of H₂O₂, and cell viability was assessed by the WST-8 assay. Isolated hWJ-MSC-mito were labeled with TMRM, transferred into COV434 cells, and confirmed by confocal microscopy. The expression of oxidative stress- and mitochondrial-related genes (CYP19A1, GPX1, HO1, SOD1, and TOMM20) was analyzed by qRT-PCR, whereas proliferative and apoptotic responses were evaluated by PCNA and active caspase-3 immunofluorescence, respectively. Results: Exposure to 1.5 mM H₂O₂ reduced cell viability by approximately 50%, establishing an oxidative stress model, while 10 µg hWJ-MSCs-mito provided an optimal mitochondrial dose. Transferred mitochondria were successfully internalized by recipient cells and enhanced cell viability, increased PCNA immunoreactivity, and reduced active caspase-3 immunoreactivity. Furthermore, it attenuated H₂O₂-induced upregulation of CYP19A1 and HO1, moderately reduced SOD1 expression, preserved GPX1 expression, and showed a modest increase in TOMM20 expression. Conclusions: hWJ-MSC-mito supplementation was associated with improved cell viability, increased PCNA immunoreactivity, reduced active caspase-3 immunoreactivity, and modulation of selected transcriptional responses in H₂O₂-treated COV434 cells. These findings support further investigation of mitochondrial supplementation in oxidative stress-induced granulosa cell injury.