P66 shc regulates oxidative stress-induced developmental failure in the early bovine embryo

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Bain, Nathan Theodore

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University of Guelph

Abstract

The 'in vitro production' (IVP) of mammalian embryos suffers from low efficiency; 50-70% of fertilized oocytes fail to develop to the blastocyst stage. This high rate of developmental failure is in part due to the effects of reactive oxygen species (ROS). Increased embryo ROS content, well associated with an increased likelihood of developmental failure, has recently been linked to p66'Shc', an oxidative stress-response protein broadly implicated in the regulation of ROS, apoptosis, and longevity. It was the aim of this thesis to explore the role of p66'Shc' in a ROS-signalling pathway linking oxidative stress with early embryo developmental failure. 'In vitro' culture O2 tension significantly influenced the timing of the onset of developmental failure. H2O 2 treatments, applied separately to different stage embryos, resulted in significant, dose-dependent decreases in blastocyst development in conjunction with increases in either permanent embryo arrest or embryo apoptosis in a stage-dependent manner. Catalase supplementation reduced H2O 2-induced developmental failure resulting in a significant increase in blastocyst frequencies under high O2 culture conditions. 'In vitro' culture conditions and treatments associated with increased developmental failure (20% O2 culture, late cleaving embryos, H2O2-treated embryos) invariably correlated with increased p66'Shc' and Ser36-phosphorylated p66'Shc' content while conditions associated with increased developmental potential (early cleaving embryos, Catalase supplementation) correlated with decreased p66'Shc' and Ser36-phosphorylated p66'Shc' content. SiRNA-mediated p66' Shc' RNAi, confirmed at the mRNA and protein levels with multiple siRNA molecules, demonstrated an improved rate of blastocyst development, decreased incidence of both embryo arrest and embryo apoptosis, and diminished embryo ROS and superoxide content. The developmentally beneficial effects of p66'Shc' RNAi persisted even under direct H2O2 treatment, confirming a protective effect of p66' Shc' RNAi against oxidative stress. Lastly, a p66'Shc' molecular signalling pathway, operating through Ser36-p66'Shc' activation and FOXO3a nuclear exclusion, was found to regulate oxidative stress in the early embryo by influencing the abundance MnSOD, but not Catalase. Taken together, the results of this thesis indicate that the ability of the early bovine embryo to both generate, and cope with the effects of oxidative stress are significantly influenced by p66'Shc'.

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P66 Shc, Reactive oxygen species, Mammalian embryos, Blastocyst development, Bovine

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