A Preliminary Study On The Effect Of Melatonin On The In Vitro Maturation Of Buffalo Oocytes And Its Mechanism

Melatonin (N-acetyl-5-methoxytryptamine), a major multifunctional molecule of the pineal secretory product, not only influences circadian rhythms, and regulates seasonal reproductive function in photoperiodic mammals, but also plays an important role in modulating reproduction. It has been reported that melatonin plays a very important role in mammalian follicles development, oocytes maturation and early embryonic development. The purpose of this study was to investigate the effect of exogenous Melatonin on the efficiency of buffalo oocytes in vitro maturation (IVM) and its relevant regulation meschanism, in order to make further efforts to optimize the IVM culture system, and provide a theoretical basis for clarifing the mechanism of oocyte maturation in buffalo.Firstly, whether the presence and expression of melatonin receptor MT1 and MT2 genes in buffalo cumulus cells and oocytes were detected. The results showed that both of the MT1 and MT2 receptors are present in the buffalo cumulus cells and oocytes.Secondly, the effects of different concentrations of melatonin (10-9M,10-8M, 10-7M) added in the maturation medium on the efficiency of buffalo oocytes IVM and the development of the embryos after in vitro fertilization (IVF) were explored. The results showed that the first polar body exhaustrates of buffalo oocytes cultured in the maturation medium added with different concentrations of melatonin (10-9M、10-8M、10-7M) were significantly higher than the control group (50.30%vs47.56%、53.04%vs47.56%、50.63%vs47.56%, P<0.05), and the first polar body exhaust rate in the 10-8M group was significantly higher than that of 10-9M and 10-7M group (P<0.05). Subsequently, the oocytes of each group were respectively fertilized in vitro, and found that the embryonic cleavage rate in each treatment group was significantly higher than the control group (60.23%vs57.62%,65.26%vs57.62%,62.84%vs57.62%, P<0.05), moreover, more embryos developed to the blastocyst in the groups of 10-8 and 10-7M than the control group (20.48%vs15.22%、18.68vsl5.22%, P<0.05), but there was no significant difference in the blastocyst rate between the 10-9 and control group (16.44%vs 15.22%, P> 0.05).Finally, the regulation mechanism of melatonin on oocytes maturation was investigated. The results showed that:Adding equal concentration of melatonin receptor antagonist (10-8MLZU) into maturation medium, the first polar body exhaust rate of buffalo oocytes (47.44% vs 48.15%), the embryonic cleavage rate (57.18% vs58.42%) and the blastocyst rate (15.06% vs15.26%) of subsequent IVF embryos had no significant difference compared with the control group (P>0.05). However, alone added an equivalent concentration of melatonin receptor agonist (10-8M IIK7), the first polar body exhaust rate of oocytes (51.25%vs48.15%), the embryonic cleavage rate (64.15%vs58.42%) and blastocyst rate (19.04%vs15.26%) of subsequent IVF embryos were significantly higher than that of the control group (P<0.05), but there were not significantly different compared with the melatonin treatment group (10-8MMT, P> 0.05). When simultaneously adding melatonin (10-8MMT) and melatonin receptor antagonist (10-8MLZU) into the maturation medium, the first polar body exhaust rate of buffalo oocytes after IVM (48.19% vs48.15%), the embryonic cleavage rate (58.13% vs58.42%) and the blastocyst rate (15.23% vs 15.26%) of subsequent IVF embryos were no significant difference than that of the control group(P>0.05). Then, the reactive oxygen species content in buffalo oocytes was detected, and found that the oocytes were cultured in the medium added with melatonin, the intracellular active oxygen content was significantly reduced than the control group (P<0.05). Futhermore, the mitochondrial distribution and mitochondrial membrane potential changes were analysed with the confocal microscope, the results showed that the rate of mitochondrial diffusion distribution and the mitochondrial membrane potential in the oocytes of the melatonin-treated group were significantly higher than the control group(P<0.05). The concentration changes of cAMP, cGMP, Catalase and Glutathione peroxidase enzyme in the oocytes were respectively detected by Elisa, the results revealed that the concentration of cAMP in the oocyte of melatonin-treated group was significantly reduced than the control group, but the concentration of cGMP, the Glutathione peroxidase enzyme and Catalase were significantly increased than the control group (P<0.05). Moreover, the antioxidant genes (GPX4, SOD1), cumulus expansion-related genes (PTX3 and HAS1, HAS2), and the melatonin receptors genes (MT1 and MT2) were tested by QRT-PCR, and the results showed that the melatonin treatment could significantly up-regulate the expression of GPX4, SOD1, MT1 and MT2 genes in buffalo oocytes, moreover, the expression of PTX, HAS1, HAS2, MT1 and MT2 genes in buffalo cumulus cells were also significantly increased (P<0.05).In conclusion:(1) The both of MT1 and MT2 of melatonin receptors are present in buffalo cumulus cells and oocytes. (2) Adding with the appropriate concentration (10-8M) of melatonin into buffalo maturation medium is beneficial for improving the efficiency of buffalo oocytes in vitro maturation and the developmental potential of embryos after subsequent in vitro fertilization. Melatonin plays this role on buffalo oocytes may be realized by the following two aspects:One is that Melatonin binds to the MT1 and MT2 receptors in the cumulus cells and oocyte, and the synthesis of cAMP in oocyte is inhibited, but cGMP is promoted, moreover, the expression of cumulus expansion-related genes PTX, HAS1, HAS2 and melatonin receptor genes MT1, MT2 is up-regulated. The second is that adding Melatonin can promote the synthesis of antioxidant enzymes, remove reactive oxygen species, and increase the expression of antioxidant enzymes.