Mechanisms Of Resveratrol And Melatonin On Oocyte Maturation And Embryo Development

In vitro oocyte maturation and embryo development are the basis researches of development biology, improve the in vitro embryo production efficiency has great significance in improve animal production and to promote the application of assisted reproductive technology. In this study, we found the mechanisms of resveratrol and melatonin in in vitro oocyte maturation and embryo development. The results were as follows:1. Experiment I analyzed the potential beneficial effects and mechanisms of action of resveratrol on the maturation of bovine oocytes that were incubated in different concentrations of resveratrol (0.1,1.0or10μM) as germinal vesicle (GV) oocytes. Resveratrol significantly increased progesterone secretion and decreased estradiol-17β secretion by cumulus cells. The elevated levels of progesterone activated the Mos/MEK/p42MAPK. cascade in the oocytes. At a concentration of1.0μM, resveratrol significantly improved cumulus expansion, polar body formation, the (hatched)blastocyst rate and the mean number of cells/blastocyst. Meanwhile, resveratrol significantly reduced the level of ROS, increased the level of GSH. For the first time, the expression of the sirtuin1gene was identified in granulosa cells, cumulus cells, oocytes and blastocysts. Further studies revealed that resveratrol promoted sirtuin1gene expression. In conclusion, resveratrol promoted bovine oocyte maturation and subsequent post-IVF embryonic development by inducing progesterone secretion and antioxidant effect, probably in a manner dependent on Sirtuin1.2. Experiment II addressed the mechanisms related to the beneficial effects of melatonin on mammalian embryonic development. To examine the potential beneficial effects of melatonin on bovine embryonic development, different concentrations of melatonin (10-11,10-9,10-7,10-5,10-3M) were incubated with fertilized embryos. Melatonin in the range of10-11to10-5M significantly promoted embryonic development both in early culture medium (CRlaa+3mg/ml BSA) and in later culture medium (CR1aa+6%FBS). The most effective concentrations applied in the current studies were10-9and10-7M. Using quantitative real-time PCR with immunofluorescence and western blot assays, the expression of melatonin receptor MT1and MT2genes were identified in bovine embryos. Further studies indicate that the beneficial effects of melatonin on bovine embryo development were mediated by the MT1receptor. This is based on the facts that luzindole, a non-selective MT1and MT2antagonist, blocked the effect on melatonin-induced embryo development while4-P-PDOT, a selective MT2antagonist, had little effect. Mechanistic explorations uncovered that melatonin application during bovine embryonic development significantly up-regulated the expression of antioxidative (Gpx4, SOD1and bcl-2) and developmentaliy important genes (SLC2A1, DNMT1A and DSC2) while down-regulating expression of pro-apoptotic genes (P53, BAX and Caspase-3). The results obtained from the current studies provide new information regarding the mechanisms by which melatonin promotes bovine embryonic development under both in vitro and in vivo conditions.3. Experiment Ⅲ evaluated the potential effects of melatonin on the kinetics of embryo development and quality of blastocyst during the process of in vitro bovine embryo culture. Bovine cumulus-oocyte complexes (COCs) were fertilized after in vitro maturation. The presumed zygotes were cultured in in vitro culture medium supplemented with or without10-7M melatonin. The cleavage rate,8-cell rate and blastocyst rate were examined to identify the kinetics of embryo development. The hatched blastocyst rate, mortality rate after thawing and the relevant transcript abundance were measured to evaluate the quality of blastocyst. The results showed that melatonin significantly promoted the cleavage rate and8-cell embryo yield of in vitro produced bovine embryo. In addition, significantly more blastocysts were observed by Day7of embryo culture at the presence of melatonin. These results indicated that melatonin accelerated the development of in vitro produced bovine embryos. Following vitrification at Day7of embryo culture, melatonin (10’7M) significantly increased the hatched blastocyst rate from24h to72h and decreased the mortality rate from48h to72h after thawing. The presence of melatonin during the embryo culture resulted in a significant increase in the gene expressions of DNMT3A, OCC, CDH1and decrease in that of AQP3after thawing. In conclusion, melatonin not only promoted blastocyst yield and accelerated in vitro bovine embryo development, but also improved the quality of blastocysts which was indexed by an elevated cryotolerance and the up-regulated expressions of developmentally important genes.4. Experiment IVverified that melatonin at its physiological concentration (10-7M) significantly promoted the in vitro development of murine pronuclear embryos. This was indicated by the increased blastocyst rate, hatching blastocyst rate and blastocyst cell number with melatonin treatment. In addition, when these blastocysts were implanted into female recipient mice, the pregnancy rates (95.0%vs. control67.8%), litter sizes (4.1pups/litter vs. control2.7pups/litter), and postnatal survival rates of offspring (96.84%vs. control81.24%) were significantly improved compared to their non-melatonin treated counterparts. Mechanistic studies revealed that melatonin treatment upregulates gene expression of the antioxidant enzyme, superoxide dismutase (SOD), and the anti-apoptotic factor bcl-2while downregulating the expression of pro-apoptotic genes p53and caspase-3. Due to these changes, melatonin treatment reduces ROS production and cellular apoptosis during in vitro embryo development and improves the quality of blastocysts. The implantation of blastocysts with higher quality leads to more healthy offspring and increased pup survival.