Abstract
BackgroundMelatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP). The aim of our study was to investigate the presence of melatonin membrane receptors (MT1and MT2) and MT3, which is the melatonin binding site of NQO2 enzyme, in both oocytes and hatched blastocysts to consider an additional subcellular mechanism responsible for the effects of melatonin on IVP.MethodsThe presence of the high affinity melatonin receptors was investigated through an autoradiographic binding assay, using the non-permeable ligand [125I]-iodomelatonin (17 pM) in embryos. The kind of melatonin site was investigated in oocytes and embryos by immunocytochemistry. In vitro fertilized bovine embryos produced from in vitro maturated oocytes supplemented with melatonin (0.0001 to 1000 nM) were analysed to determine their cleavage and blastocyst formation rates.ResultsThe [125I]-iodomelatonin (17 pM) binding in blastocysts was blocked by pre-incubation with melatonin (30000 nM), showing the presence of the high affinity melatonin receptors. MT1, MT2 and NQO2 immunoreactivity was observed in oocytes. MT1 immunoreactivity was observed in hatched blastocysts, however MT2 and NQO2 were not observed in this embryonic stage. Melatonin (pM) triggered significant difference in both cleavage and blastocysts formation rates.ConclusionsThe high affinity MT1 melatonin receptor must be taking part in IVM events; furthermore it is the first melatonin receptor to appear during bovine embryo development in vitro.
Highlights
Melatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP)
Due to the lipophilic nature of melatonin, it is able to function by binding to receptor in the nucleus [5], a cytosolic binding site the calmodulin protein [6] and by modulating the cytosolic detoxifying enzyme NRH: Quinone Oxireductase 2 (NQO2)
The aim of the study was to evaluate the effects of melatonin on bovine oocyte IVM by measuring the cleavage rates and blastocysts formation rates, as well as investigating the presence of the NQO2 enzyme, and MT1 and MT2 melatonin membrane receptors in bovine oocytes and blastocysts
Summary
Melatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP). Due to the lipophilic nature of melatonin, it is able to function by binding to receptor in the nucleus [5], a cytosolic binding site the calmodulin protein [6] and by modulating the cytosolic detoxifying enzyme NRH: Quinone Oxireductase 2 (NQO2). The most primitive mechanism of melatonin action is the direct scavenger function on reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and the super-oxide anion (O2- ) This neurohormone has an indirect antioxidant role thought to be triggered by membrane receptors and binding sites, mainly resulting in the up regulation of the antioxidant enzymes (glutathione peroxidase, glutathione reductase, gammaglutamyl cisteine synthetase and glucose 6-phosphate deydrogenase) and the down regulation of the oxidant enzymes (NO synthase and lipoxygenases) [9,10]
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