TARGETING THE NRF2 SIGNALING PATHWAY TO IMPROVE OOCYTE QUALITY AND EMBRYO DEVELOPMENT UNDER STRESS CONDITIONS

Abstract

The increasing frequency and severity of environmental stressors associated with global climate change pose significant challenges to livestock production systems worldwide. Among these, heat stress has emerged as a leading cause of reproductive inefficiency in cattle, compromising ovarian function, oocyte quality, and preimplantation embryonic development. In addition to environmental thermal stress, embryos produced through assisted reproductive technologies are frequently exposed to suboptimal in vitro culture conditions that further exacerbate cellular stress. Although these insults arise from different sources, a common consequence is excessive accumulation of reactive oxygen species (ROS), leading to oxidative stress, mitochondrial dysfunction, metabolic disturbances, and reduced developmental competence. Despite the recognized importance of oxidative stress in reproductive failure, the molecular mechanisms that enable reproductive cells and embryos to adapt to these challenges remain incompletely understood. The work presented in this dissertation investigated the role of oxidative stress as a common mediator of reproductive dysfunction across multiple stages of female reproduction, from the ovarian follicle to the preimplantation embryo. Particular emphasis was placed on the Nuclear factor erythroid 2-related factor 2 (NRF2) pathway, a master regulator of cellular redox homeostasis that coordinates antioxidant, cytoprotective, and metabolic responses to oxidative challenges. Through a combination of literature synthesis and experimental studies utilizing bovine granulosa cells, oocytes, and embryos, this dissertation examined how activation of NRF2-mediated antioxidant defenses influences cellular resilience under conditions of heat stress and oxidative stress induced by in vitro culture. Collectively, the findings demonstrate that oxidative stress represents a central mechanism through which heat stress and suboptimal culture environments compromise reproductive performance. Exposure to thermal and oxidative challenges disrupted redox homeostasis, increased ROS accumulation, impaired mitochondrial function, altered metabolic activity, and reduced developmental competence in granulosa cells, oocytes, and embryos. Conversely, activation of NRF2 signaling through naturally occurring antioxidant compounds enhanced cellular antioxidant capacity, preserved mitochondrial integrity, improved metabolic efficiency, and restored developmental potential. Importantly, the beneficial effects of NRF2 activation extended beyond immediate protection against oxidative damage, contributing to improvements in embryo quality and in molecular indicators of embryonic competence. This dissertation further establishes the NRF2–KEAP1 signaling axis as a critical regulator of cellular adaptation to environmental and culture-induced stress during early reproductive development. By demonstrating that targeted activation of endogenous antioxidant pathways can mitigate the detrimental consequences of both heat stress and oxidative stress, these studies provide mechanistic insight into how reproductive cells maintain functionality under adverse conditions. Moreover, the findings identify NRF2-mediated antioxidant signaling as a promising target for improving ovarian function, oocyte quality, embryo competence, and the overall efficiency of assisted reproductive technologies.