Master of Science
Physiology and Pharmacology
Watson, Andrew J.
Betts, Dean H.
Obese women experience frequent infertility; obesity-conditioned reproductive tracts expose embryos to stressors including free fatty acids (FFA). Palmitic acid (PA) exposure significantly impairs mouse preimplantation development and increases endoplasmic reticulum stress; oleic acid (OA) rescues these effects. To further understand FFA in preimplantation development, this thesis investigates the NRF2/KEAP1 pathway, which coordinates antioxidant stress response pathways. We hypothesize that PA induces the NRF2/KEAP1 pathway, while OA alleviates it. Female mice were super-ovulated, mated, and embryos were placed in culture treatments: bovine serum albumin (control), 100 µM PA, 100 µM OA, or 100 µM PA+OA. Protein localization and mRNA abundances were assessed via immunofluorescence and RT-qPCR, respectively. NRF2 displayed nuclear localization throughout preimplantation development. PA significantly decreased NRF2-positive nuclei, while OA and PA+OA did not. FFAs showed no significant effects on relative expression of Nrf2/Keap1. Current results suggest that PA suppresses NRF2/KEAP1 pathway activation, which warrants further investigation as a mechanism for PA-induced developmental impairments.
Summary for Lay Audience
Infertility impacts nearly 1 in 6 couples globally. The high prevalence of infertility has driven increased demand for assisted reproductive technologies, which are techniques—such as in vitro fertilization or embryo transfer—that assist couples to successfully conceive and carry healthy pregnancies to term. Many techniques focus on preimplantation embryo development, which is the period starting at fertilization and ending when the embryo implants in the uterus. Importantly, patients experience varying ART success rates due to factors like age and health. Obese women experience higher incidences of infertility than women with healthy BMIs, and obesity has been increasing for decades, prompting studies into why obese patients face these challenges. Obesity changes the reproductive tract environment, exposing embryos to external stressors during development, including macromolecules called fatty acids. Previous research indicates that exposing preimplantation mouse embryos to palmitic acid, an abundant fatty acid in the body, impairs embryo development and induces stress responses. In contrast, exposing preimplantation embryos to oleic acid, another abundant free fatty acid, reverses the negative effects of palmitic acid. Given that palmitic acid impacts stress responses, this thesis investigated the Nrf2/Keap1 signalling pathway, which coordinates cellular defences against stress. Keap1 normally binds to Nrf2 to promote its degradation. Under cellular stress conditions, Nrf2 enters the nucleus to promote antioxidant gene expression, which helps the cell deal with stressors. We hypothesized that treating mouse preimplantation embryos with palmitic acid would induce the Nrf2/Keap1 pathway, while treating embryos with oleic acid would alleviate it. Mouse embryos were cultured in vitro in the following treatments: fatty acid carrier, palmitic acid, oleic acid, palmitic & oleic acid, or plain culture media. Embryos were imaged to visualize localization of Nrf2/Keap1 proteins in response to fatty acids treatments. Palmitic acid significantly decreased the percentage of embryonic cells with nuclear Nrf2 localization, which indicated less activation, since Nrf2 functions inside the nucleus. Oleic acid and the combination treatment did not produce this effect. My findings suggest that palmitic acid suppresses Nrf2/Keap1 activity, which could prevent cells from properly responding to stressful conditions such as obesity, presenting a mechanism for palmitic acid impairing preimplantation embryo development.
Dionne, Grace, "Fatty Acid Regulation of the Nrf2/Keap1 Pathway During Mouse Preimplantation Embryo Development" (2021). Electronic Thesis and Dissertation Repository. 8159.
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