Role of inflammatory pathways on embryonic competence and cryotolerance

Abstract

One of the main challanges to further expansion of in vitro produced of embryos is its reduced cryosurvival. When exposed to stressful situations, cells develop self-defense mechanisms that, under exacerbated conditions, can lead to cell death. NF-KB (nuclear factor kappa B) is an inducible transcription factor known to be a regulator of inflammatory cellular response and the development of defense mechanisms against a wide variety of stimuli, controlling the expression of numerous target genes. In order to elucidate the participation of NF-kB in embryonic survival mechanisms, bovine embryos will be produced in vitro with the supplementation of NF-kB modulating agents, TNF (tumor necrosis factor) inducer, IL-13 (Interleukin 13) inhibitor, the association of both (IL-13 + TNF), and subsequently compared to a control group. Embryonic development rates, relative abundance of inflammatory genes transcripts and oxidative stress will be evaluated. Blastocysts will be evaluated for cell viability (Tunel) and protein expression of NF-Kb, IKB±, CASP3 and 8-OxoG by imonofluorescence.The culture medium will be quantified for the presence of inflammatory interleukins. Secondly, the same variables will be evaluated in embryos treated with IL-13 and control subjected to stress conditions (cold and presence of H2O2) during culture. Finally, the cryotolerance of vitrified control and IL-13 treated embryos will be evaluated in two different situations: in the embryonic development medium and in the reculture medium after warming. For statistical analysis, univariate data models will be used, assuming a significance level of 5%. (AU)