The Mechanisms Of Melatonin Inhibiting Oxidative Damage In Bovine Oocytes And In Vitro Fertilized Embryos

Oxidative stress induced by reactive oxygen species (ROS) is one of the main factors that decrease both the quality of in vitro matured oocytes and the efficiency of in vitro embryo production. It has been confirmed that melatonin and its metabolites, which are highly efficient free radical scavengers, can improve both the quality of matured oocytes and the developmental competence of embryos produced in vitro. In this study, the mechanisms of melatonin which inhibits paraquat (PQ)-induced damage in bovine in vitro matured oocytes and preimplantation embryos were respectively examined. The results were as follows:1. Experiment ? analyzed the protective role of melatonin against PQ-induced oxidative damage in bovine in vitro matured oocytes. The results showed that:(1) The first polar body extrusion rate was decreased to some extent in all PQ treatment groups (10,50,250, 1000?M) compared with the control group. Among them, the rates in 50,250, 1000?M PQ groups were significantly lower than that of the control group (P< 0.05). With the PQ concentration raised, the maturation competence of bovine oocytes was decreased in a dose-dependent manner (61.8%±6.8% vs.46.9%±1.8% vs. 32.4%±3.7%, P< 0.05). There was no differences between the 10 ?M,50 ?M and the control group (P> 0.05). (2) Adding 250?M PQ to the maturation medium significantly down-regulated the expression of cumulus cell expansion related HAS2, TNFAIP6, PTX3 and PTGS2 genes (P< 0.05). (3) Melatonin treatment could alleviate the oxidative damage to the oocytes caused by PQ, as the first polar body extrusion rate was significantly increased compared to the PQ group (60.4%±3.0% vs.45.9%±5.2%, P< 0.05), but it was still lower than that of the control group (81.2%±4.8%, P< 0.05). (4) Melatonin significantly reduced the intracellular ROS level in bovine oocytes exposed to PQ (P< 0.05), while the proportions of oocytes with cortical granule in peripheral and cortical distribution and mitochondria in homogeneous distribution were both increased compared with the PQ group (P< 0.05). (5) Melatonin significantly up-regulated the expressions of GDF9, DNMT1A and GPx4 genes (P< 0.05), but the CAT expression level was not affected compared with the PQ treatment group (P> 0.05).2. Experiment ? assessed the possible mechanism of melatonin involved in inhibiting PQ-induced oxidative damage in bovine preimplantation embryos. The results showed that:(1) The cleavage rate in the 1000 ?M PQ group (64.4%±12.7%) was significantly lower than that of the control group and the other treatment groups (10,50,250?M) (P< 0.05). With the PQ concentration raised, the number of 2-4 cell cleaved embryos increased, while the number in the 8-16 cell stage decreased. Moreover, PQ inhibited the morula rate and the blastocyst rate in a dose-dependent manner. In contrast to the control group, the morula rate and the blastocyst rate were both significantly decreased in 50,250, and 1000?M PQ groups (P< 0.05). No statistically significant differences in the morula rates were observed between the 10 ?M PQ group and the control group (P> 0.05), but the blastocyst rate was significantly lower in the 10 ?M PQ group than that of the control group (P< 0.05). (2) Melatonin pretreatment prior to PQ significantly promoted the development of morula (23.9%±4.3% vs. 16.7%±2.2%) and blastocyst stage embryos (20.9%±2.7% vs.14.3%±2.9%), but the proportion remained lower than that of the control group (37.0%±3.0% & 33.4%±3.3%, P< 0.05). (3) Following pre-incubation with melatonin, the apoptotic cell rate was significantly lower compared with the PQ treatment group (4.5%±0.8% vs.6.4%±1.6%, P< 0.05). Meanwhile, melatonin significantly inhibited PQ-induced apoptosis by decreasing the release of cytochrome c. (4) Melatonin application prior to PQ significantly up-regulated the expression of Trx, while down-regulating the expression of Txnip, caspase-3 and Bax in the resulted embryos. (5) Western blot analysis showed that the expression of p-p38 was clearly down-regulated by melatonin pre-incubation, while the activity of ERK and JNK were not altered.