Melatonin (MLT) is an indoleamine that can act on reproductive competence, has antioxidant and antiapoptotic activities and influences different cell signaling pathways. In vitro studies have shown beneficial effects of MLT use on in vitro maturation (IVM) and culture (IVC) of bovine oocytes and embryos, respectively. In addition, MLT is able to induce the resumption of meiosis, but the mechanism by which this stimulus occurs is still unknown. In this study, we propose to investigate the involvement of maturation promoting factor (MPF) and cyclic adenosine monophosphate (cAMP) in meiosis resumption stimulated by MLT, as well as its effect on the development, quality and cryotolerance of bovine embryos produced in vitro. In experiment 1, cumulus-oocyte complexes (COCs) will be cultured for 9 h for IVM with MLT (10-11M, 10-9M and 10-7M) or FSH (control) and the rate of meiosis resumption will be assessed. The best concentration (highest % of meiosis resumption) will be selected for the following experiments. In the second experiment, COCs submitted to 0, 3, 6, 9 and 12 h IVM in the presence of MLT will be evaluated for the presence of the phosphorylated CDK1 protein (MPF catalytic unit) by western blotting. In experiment 3, cAMP levels will be measured in oocytes at 0, 1, 2, 3, 6 and 9 h IVM in the presence or absence of MLT. In experiment 4, MLT will be supplemented to the IVM medium for 24 h in association with gonadotropins: 1) FSH; 2) FSH+LH; 3) MLT; 4) FSH+MLT and 5) FSH+LH+MLT. Oocytes will be evaluated for meiosis progression. The best combination of MLT and gonadotropins (highest % MII) will be used in experiment 5 in which matured COCs will be submitted to in vitro fertilization (IVF) followed by IVC with MLT (0, 10-11, 10-9 and 10-7M). The embryos will be evaluated for cleavage and blastocysts rates, and quality analysis by TUNEL. In experiment 6, the embryos with the best development rates from experiment 5 will be vitrified, thawed and submitted to IVC for up to 72 h to determine the re-expansion and embryonic hatching rates.
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