194 Effect of nobiletin on oocyte meiotic maturation in pigs

Nobiletin is a natural flavonoid found in citrus fruits with beneficial effects that possess anti-inflammatory, anti-cancer, anti-oxidant properties, and it has been shown to prevent aging by enhancing mitochondrial function in muscle cells. The aim of this study was to evaluate the potential of using nobiletin to improve mitochondrial function in porcine oocytes and examine the underlying mechanism. Oocytes enclosed by at least three layers of cumulus cells were cultured in TCM-199 for 44 h with 0.1% dimethyl sulfoxide (control) or supplemented with 10, 25, or 50 µM nobiletin (Nob10, Nob25, Nob50, respectively). The key mitochondrial pathways in this study were categorized as follows: oxidative stress, mitochondrial activity, mitochondria DNA (mtDNA) copy number, and cell death. The protein levels were assessed by western blot analysis and immunofluorescence staining. The mitochondrial membrane potential (MMP), ATP levels, and levels of reactive oxygen species (ROS) were measured using the JC-1 probe, firefly luciferase assay, and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, respectively. Mitochondrial quantity was measured based on mtDNA copy number using real-time PCR. Statistical analysis was performed by Student’s t-test using the GraphPad Prism 6 software. The results showed that oocyte maturation rate was significantly enhanced in Nob10 (70.88 ± 1.16%) compared with the other groups (control: 59.5 ± 0.91%; Nob25: 63.59 ± 1.08%; Nob50: 47.13 ± 1.02%). The addition of nobiletin induced lower levels of ROS and higher glutathione levels, indicating a reduction in oxidative stress. Nob10 promoted mitochondrial biogenesis by up-regulating the protein levels of sirtuin 1 and peroxisome proliferator-activated receptor-gamma coactivator 1α. Additionally, it resulted in an increase in the number of active mitochondria, mtDNA copy number, MMP, and ATP production, thereby enhancing mitochondrial functions. Mitochondria-mediated apoptosis was significantly attenuated due to decreased p53 levels and high expression of B-cell lymphoma 2 proteins in Nob10. Thus, these results revealed that nobiletin has a great function to improve IVM of porcine oocytes by suppressing oxidative stress and apoptosis and promoting mitochondrial functions.