The in vitro production (IVP) of bovine embryos has been applied commercially with success, however, a major obstacle for further use of this technique is the sensitivity of such embryos to cryopreservation. The lower cryotolerance is often associated with the accumulation of lipidis in the cytoplasm of IVP embryos. In adipocytes intracellular levels of cAMP and cGMP are important for regulating the rate of lipolysis. PDE5 is a cGMP degrading enzyme and when activated, lipolysis is negatively affected. cGMP levels are regulated by synthesis (guanylate cyclase - GC) and degradation enzymes (PDE5), is present in COCs. Thus this work aims to investigate the relationship between PDE5 and lipid metabolism in bovine oocytes matured in vitro. For this purpose, the activity of PDE5 will be evaluated by measuring cGMP levels in oocytes with 0, 12 and 24h of maturation with different concentrations of PDE5 inhibitor (0, 500nM, 1 µM and 10µM Sildenafil) and the lipid contents of different treatments will be determined using the fluorescent probe Nile Red. Oocytes with lower and higher lipid content will be fertilized (D0) and cultured to D7, when embryos will be split in two groups: the first will be cryopreserved (slow freezing) and the second will be fixed for actin filaments staining (indicative of lipid content in embryos). The group for cryopreservation will be thawed to assess re-expansion rate and also for actin filaments staining in order to compare the characteristics after freezing/thawing. Finally, whether the cryopreservation of IVP embryos is hampered by the presence of FCS in the culture medium by interfering in the cGMP pathway will be investigated. COCs will be cultured in maturation medium supplemented with different concentrations of FCS (2% and 10%). A control group will be matured in medium with 0.4% BSA. The effect of FCS will be evaluated on the activity of PDE5 by measuring cGMP levels in immature oocytes and after 12 and 24h of maturation, and also on the relative abundance of transcripts of components of the cGMP pathway (GUCY1B3, PKG1 and PDE5A). Greater knowledge on the control of lipid metabolism in oocytes may cause changes in cell composition, improving its ability to originate more cryotolerant embryos.
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