(303) Antioxidant Strategy Mitigates Heat Stress-Induced Oxidative Damage in Bovine Oocytes

Heat stress (HS) disrupts the redox homeostasis in the ovary, a key factor in producing viable gametes capable of fertilization for the establishment of a successful pregnancy. At the cellular level, HS triggers excessive levels of reactive oxygen species (ROS) accumulation, known as oxidative stress (OS), leading to mitochondrial dysfunction and apoptosis. In response to HS-induced OS, oocytes activate various cytoprotective mechanisms, including the induction of heat shock proteins (HSPs) and glucose-regulated proteins (GRPs). The NRF2/KEAP1/ARE pathway, a master regulator of the antioxidant response, plays a crucial role in mitigating OS. This project explores the potential of plant-derived compounds such as quercetin (Que) and sulforaphane (SFN), known for activating the NRF2 pathway, to enhance antioxidant defenses and mitigate the cellular damage inflicted by HS during oocyte maturation. We hypothesize that Que and SFN mitigate the detrimental effects of HS through the NRF2/KEAP1/ARE pathway. Initially, bovine cumulus-oocyte complexes (COCs) were collected by aspirating small follicles (3mm–8mm) from abattoir-derived ovaries. Groups of 50 COCs were placed into 4-well culture dishes containing distinct equilibrated mediums: 5μM Que, 1μM SFN, and a vehicle control group (DMSO) that will be cultured under normothermic (NT; 38.5°C) and HS (41°C) conditions. IVM lasted 23±1h and began with four groups incubated under NT conditions for the first 8h. Subsequently, the Que, SFN, and HS control groups were exposed to HS conditions for the remaining 15h, while the NT control group remained at NT conditions.  Concluding IVM, selected stress-associated makers were investigated among the resected cumulus cells (CCs) and the denuded matured oocytes. Fluorescence microscopy was used to measure ROS accumulation in matured denuded oocytes from four biological replicates, while confocal microscopy was performed in duplicates to assess mitochondrial activity. Fluorescence intensity was measured in individual oocytes, with a total of 20 observations per treatment. qRT-PCR was performed to quantify the expression of selected stress-related genes in oocytes and CCs and cumulus expansion genes in CCs only. Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. The results showed the transcript levels of HSPs and GRPs remained unaltered in oocytes, but in the CCs HSP90 expression was reduced in the Que-treated group compared to HS control (p=0.04), while GRP94 expression was also lower in the Que-treated group (p=0.008). On the other hand, NRF2 expression was higher in both antioxidant-supplemented groups than in NT control group and was significant for Que (p=0.04). Similarly, SOD1 expression was upregulated in CCs from Que group (p=0.003) and the SFN group (p=0.0001) compared to HS control group. Additionally, CAT expression was significantly higher in the SFN group (p=0.02) relative to HS control group. Additionally, a significant increase in PTX3 expression was noted relative to the NT control for the Que-treated group (p=0.01) and SFN-treated group (p=0.03). ROS accumulation assay revealed both antioxidant treatments successfully reduced ROS accumulation, with a significant reduction observed in the SFN-treated group versus the HS control (p=0.0007). Similarly, the mitochondrial membrane potential (MMP) assay using JC-1 staining showed MMP increased in both Que (p=0.0004) and SFN-treated (p< 0.0001) groups compared to the HS control group. The results indicate that antioxidant supplementation effectively mitigates the detrimental effects of HS during oocyte maturation by reducing ROS accumulation and improving mitochondrial function of the oocyte via modulating the physiology of the surrounding CCs. Funded by CVMBS College Research Council CSU.