VIEWS: 3 PAGES: 1 POSTED ON: 2/9/2012
Reactive oxygen species and the early bovine embryo: arrest, apoptosis and the role of p66Shc Nathan T. Bain1, Pavneesh Madan1, Dean H. Betts,2 1 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON Canada N1G 2W1 2 Department of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada N6A 5C1 The in vitro production (IVP) of embryos suffers from a high degree of early developmental failure. This inefficiency has been linked to the effects of reactive oxygen species (ROS) brought on by the unavoidable exposure of embryos to atmospheric levels of O2 during the IVP process. P66Shc, a stress-response protein involved in oxidative stress signalling, was examined as a potential mediator between oxidative stress and developmental failure. The expression of p66Shc was quantified through real-time PCR and semi-quantitative immunofluorescence. Significant differences (p <0.05) in p66Shc expression were found between groups of embryos with respect to time of first cleavage, in vitro O2 concentration, catalase supplementation and H2O2 treatment. Furthermore, all instances of increased p66Shc expression correlated with increased H2A-X staining (ROS-associated DNA damage) and DCF staining (H2O2 quantification). siRNA molecules designed to selectively knockdown p66Shc were microinjected into zygote-stage embryos. P66Shc knockdown was confirmed by mRNA and protein quantification. Two, non-overlapping anti-p66Shc siRNA molecules produced a significant decrease in developmental failure at both the 2-4 cell and 9-16 cell stage of development. P66Shc knockdown was also found to correlate with significantly decreased levels of H2A-X and DCF staining. Lastly, p66Shc knockdown also induced a resistance to H2O2-induced developmental failure. The strong correlative relationship between ROS, developmental failure and p66Shc expression, as well as the protective effects of p66Shc knockdown support the view of p66Shc as a mediator of oxidative stress-induced developmental failure.
"Bain_ Madan_ Betts"