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This study investigated the effect of the flavonoid-based compound isorhamnetin (ISO) on maturation and developmental competence in oxidative stress-exposed porcine oocytes in vitro. Treatment with 2 μM ISO (2 ISO) increases the developmental rate of oxidative stress-exposed porcine oocytes during in vitro maturation (IVM). The glutathione level and mRNA expression of antioxidant-related genes (NFE2L2 and SOD2) were increased in the 2 ISO-treated group, whereas the reactive oxygen species level was decreased. Treatment with 2 ISO increased mRNA expression of a cumulus cell expansion-related gene (SHAS2) and improved chromosomal alignment. mRNA expression of maternal genes (CCNB1, MOS, BMP15 and GDF9) and mitogen activated protein kinase (MAPK) activity were increased in the 2 ISO-treated group. The total cell number per blastocyst and percentage of apoptotic cells were increased and decreased in the 2 ISO-treated group, respectively. Treatment with 2 ISO increased mRNA expression of development-related genes (SOX2, NANOG, and POU5F1) and anti-apoptotic genes (BCL2L1 and BIRC5) and decreased that of pro-apoptotic genes (CASP3 and FAS). These results demonstrate that 2 ISO improves the quality of porcine oocytes by protecting them against oxidative stress during IVM and enhances subsequent embryo development in vitro. Therefore, we propose that ISO is a useful supplement for IVM of porcine oocytes.
Our previous studies have already revealed that β-cryptoxanthin (BCX), hesperetin (HES), and icariin (ICA) antioxidants are effective for in vitro maturation (IVM) of porcine oocytes. In this study, we investigated which of BCX, HES, or ICA was more effective for IVM of porcine oocytes. The antioxidant properties were assessed with aged porcine oocytes and embryos by comparing 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH), reducing power, and H2O2 scavenging activity assays. The chemical assay results demonstrated that BCX had a greater DPPH scavenging activity and reducing power than HES and ICA, compared with controls. However, the H2O2 scavenging activity of the antioxidants was similar when tested at the optimal concentrations of 1 μM BCX (BCX-1), 100 μM HES (HES-100), and 5 μM ICA (ICA-5). The biological assay results showed that BCX-1 treatment was more effective in inducing a significant reduction in reactive oxygen species (ROS), improving glutathione levels, and increasing the expression of antioxidant genes. In addition, BCX-1 inhibited apoptosis by increasing the expression of anti-apoptotic genes and decreasing pro-apoptotic genes in porcine parthenogenetic blastocysts. BCX-1 also significantly increased the blastocyst formation rate compared with the ageing control group, HES-100 and ICA-5. This study demonstrates that damage from ROS produced during oocyte ageing can be prevented by supplementing antioxidants into the IVM medium, and BCX may be a potential candidate to improve assisted reproductive technologies.
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