Functional study of the melatonin receptor MT1 in bovine cumulus-oocyte complexes and its importance on oocyte maturation and embryo development in vitro under normal and heat stress culture conditions
Melatonin, a molecule derived from tryptophan and synthesized mainly by the pineal gland, mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro, including under heat stress conditions. Melatonin (MEL) may act either directly or through specific membrane receptors (MT1 and MT2), but little is known about its role in cumulus-oocyte complexes (COCs) during maturation. Recently, the melatonin receptor MT1 was detected in bovine cumulus cells (CC) and oocytes (OO). The aim of this work is to test the melatonin receptor inhibitors Luzindole (inhibits MT1 and MT2) and 4-P-PDOT (selective for MT2) to investigate the function of MT1 present in bovine COCs during in vitro maturation (IVM), and to obtain new information on the role of melatonin in promoting maturation, embryo development in vitro and protection against heat stress. Firstly, to investigate whether the effects of MEL on oocyte maturation are dependent on the MT1 receptor, bovine COCs will be submitted to IVM in the following treatments: 1) MEL (concentration defined in previous experiment); 2) Luzindole (10-6M); 3) 4-P-PDOT (10-6M); 4) MEL + Luzindole; 5) MEL + 4-P-PDOT; and 6) without drugs (control). After 22-24h IVM, OO will be evaluated for maturation rate and ROS level, CC for expression of antioxidant (SOD1, SOD2 and GPx4), pro-apoptotic (p53, Bax and caspase 3) and expansion-related genes (PTX3, HAS1 and HAS2). In a second step, COCs will be evaluated for embryo development after fertilization and in vitro culture. In a second experiment, COCs will be matured in vitro under heat stress conditions, with or without addition of melatonin, to verify the effect of MEL in protecting against heat stress. Finally, a third experiment will be carried out to investigate whether the effect of MEL on the protection against heat stress is mediated by the MT1 receptor, in which COCs will be submitted to IVM in the following treatments: 1) 38.5ºC (control); 2) 41.5°C; 3) MEL at 41.5°C; 4); 4) MEL + Luzindole at 41.5°C; 5) MEL + 4-P-PDOT at 41.5°C. After 24h IVM, the COCs will be evaluated for the same parameters of experiment 1 and also for the expression of the HSP70 gene (heat stress marker). In a second step, the COCs will be evaluated for their competence to develop into blastocysts.
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