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During in vitro maturation, excess levels of reactive oxygen species (ROS) are a major cause of developmental defects in embryos. Betulinic acid (BA) is a naturally produced antioxidant in white birch bark. Recent studies have shown that BA exhibits antioxidant properties in various cells through the activation of antioxidant genes. Therefore, we investigated the effect of BA treatment on porcine oocytes and its underlying mechanism during oocyte maturation. Treatment with 0.1 μM BA significantly increased the proportion of MII oocytes compared with controls, and BA-treated oocytes had significantly higher development rates, trophectoderm cell numbers, and cell survival rates than controls. These results demonstrate that BA treatment improved the developmental competence of oocytes. Following BA treatment, oocytes exhibited reduced ROS levels and elevated glutathione (GSH) levels, accompanied by the enhanced expression of antioxidant genes, compared with control oocytes. To evaluate the antioxidant effects of BA, oocytes were exposed to H2O2, a potent ROS activator. Impaired nuclear maturation, ROS levels, and GSH levels induced in oocytes by H2O2 exposure was restored by BA treatment. As these antioxidant genes are regulated by the Nrf2/Keap1 signaling pathway, which is involved in antioxidant responses, we applied the Nrf2 inhibitor brusatol to investigate the effects of BA on this pathway. The negative effects of brusatol on meiotic maturation and oocyte quality, including levels of ROS, GSH, and antioxidant-related gene expression, were mitigated by BA treatment. Our results suggested that BA plays an effective role as an antioxidant in porcine oocyte maturation through adjusting the Nrf2/Keap1 signaling pathway. This finding provides valuable insights into the mechanisms governing oocyte maturation and embryonic development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress., Competing Interests: The authors have declared that no competing interests exist.

Successful production of transgenic pigs requires oocytes with a high developmental competence. However, cumulus-oocyte complexes (COCs) obtained from antral follicles have a heterogeneous morphology. COCs can be classified into one of two classes: class I, with five or more layers of cumulus cells; and class II, with one or two layers of cumulus cells. Activator [e.g., epidermal growth factor (EGF)] or inhibitors (e.g., wortmannin and U0126) are added to modulate kinases in oocytes during meiosis. In the present study, we investigated the effects of kinase modulation on nuclear and cytoplasmic maturation in COCs. Class I COCs showed a significantly higher developmental competence than class II COCs. Moreover, the expression of two kinases, AKT and ERK, differed between class I and class II COCs during in vitro maturation (IVM). Initially, inhibition of the PI3K/AKT signaling pathway in class I COCs during early IVM (0-22 h) decreased developmental parameters, such as blastocyst formation rate, blastomere number, and cell survival. Conversely, EGF-mediated AKT activation in class II COCs enhanced developmental capacity. Regarding the MAPK signaling pathway, inhibition of ERK by U0126 in class II COCs during early IVM impaired developmental competence. However, transient treatment with U0126 in class II COCs increased oocyte maturation and AKT activity, improving embryonic development. Additionally, western blotting showed that inhibition of ERK activity negatively regulated the AKT signaling pathway, indicative of a relationship between AKT and MAPK signaling in the process underlying meiotic progression in pigs. These findings may help increase the developmental competence and utilization rate of pig COCs with regard to the production of transgenic pigs and improve our understanding of kinase-associated meiosis events., Competing Interests: The authors have declared that no competing interests exist.

Despite the application of numerous supplements to improve in vitro culture (IVC) conditions of mammalian cells, studies regarding the effect of fetal bovine serum (FBS) on mammalian early embryogenesis, particularly in relation to redox homeostasis, are lacking. Herein, we demonstrated that early development of in vitro-produced (IVP) porcine embryos highly depends on the combination of FBS supplementation timing and embryonic reactive oxygen species (ROS) requirements. Interestingly, FBS significantly reduced intracellular ROS levels in parthenogenetically activated (PA) embryos regardless of the developmental stage. However, the beneficial effect of FBS on early embryogenesis was found only during the late phase (IVC 4-6 days) treatment group. In particular, developmental competence parameters, such as blastocyst formation rate, cellular survival, total cell number and trophectoderm proportion, were markedly increased by FBS supplementation during the late IVC phase. In addition, treatment with FBS elevated antioxidant transcript levels during the late IVC phase. In contrast, supplementation with FBS during the entire period (1-6 days) or during the early IVC phase (1-2 days) greatly impaired the developmental parameters. Consistent with the results from PA embryos, the developmental competence of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) embryos were markedly improved by treatment with FBS during the late IVC phase. Moreover, the embryonic stage-specific effects of FBS were reversed by the addition of an oxidant and were mimicked by treatment with an antioxidant. These findings may increase our understanding of redox-dependent early embryogenesis and contribute to the large-scale production of high-quality IVP embryos.