(39) Betulinic Acid Enhances Porcine Oocyte Maturation by modulating Nrf2/Keap1-mediated antioxidant defense

1. Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Republic of Korea
2. Department of Functional Genomics, University of Science and Technology, Daejeon, Republic of Korea

Abstract Text: Betulinic acid (BA) is a pentacyclic triterpenoid that naturally occurs in sources such as the bark of white birch or rosemary. BA is commonly used as an anti-cancer agent by inhibiting topoisomerase, but recent studies show that BA can possess antioxidant properties in various cells through the activation of antioxidant genes. Despite its antioxidant activity, the effect of BA on oocyte maturation is still unknown. Therefore, we investigated the effect of BA treatment during porcine oocyte maturation, expecting that BA can enhance oocyte quality through its antioxidant activity and the underlying mechanism. During in vitro maturation (IVM), various concentrations of BA (0, 0.01, 0.1, and 1 µM) were added to the medium. The treatment of 0.1 µM BA significantly increased the proportion of MII oocytes compared with controls. After parthenogenetic activation, BA-treated groups showed significantly enhanced embryo developmental parameters, such as blastocyst formation rate, trophectoderm and total cell number, and cell survival. Additionally, BA treatment significantly reduced ROS levels and increased GSH levels. To evaluate the antioxidant effect of BA, oocytes were exposed to H₂O₂, a potent ROS activator. The H₂O₂ supplementation significantly increased ROS levels, decreased GSH levels, and decreased expression of antioxidant genes, resulting in a significant decrease in nuclear maturation rate and embryo developmental parameters, while these were significantly restored by BA treatment. Interestingly, these antioxidant-related genes are regulated by Nrf2/Keap1 signaling pathway, and decreased these genes by H₂O₂ was significantly mitigated by BA treatment. To further confirm whether the Nrf2/Keap1 signaling pathway was involved in the increase in antioxidant gene expression, we used brusatol, an NRF2 inhibitor, with or without BA. The BA treatment alleviated the negative effects of brusatol on meiotic maturation, ROS levels, GSH levels, and embryonic development, and regulated the expression of Nrf2/Keap1 signaling pathway related antioxidant genes. In conclusion, BA appears to play an effective role as an antioxidant in porcine oocyte maturation through the modulation of Nrf2/Keap1 signaling pathway.