| In vitro fertilization and embryo transfer technology is currently the most effective methods for the treatment of infertility. To minimize the impact of external environment on the development of blastocyst, the conventional method is to transplant embryos which was in the early cleavage stage (Day 2-3) into the uterus. But now the technology is also faced with many problems, mainly due to①low rate of the pregnancy and implantation of embryo,②relatively high abortion rate, and③a high risk of multiple pregnancy. Embryo transferring at the blastocyst stage may be the alternative to solve these problems. Therefore, the study on growth arrest and development of 2-cell embryos to the blastocyst stage, as well as the influencing factors on the blastocyst development, has important reference value.Melatonin is a hormone with antioxidant effects, secreted by the pineal gland located in the posterior wall of the third ventricle. Recent studies showed that melatonin could directly affect the development of embryos in vitro, 1μg/ml of melatonin could promote the recovery capacity of freezed animal blastocysts, improve hatching rate, and protect the embryo against H2O2-induced oxidative stress damage. Studies in mice showed that high concentrations of melatonin (10-4-10-6 M) could increase the fertilization rate, while low concentrations (10-6 or 10-8M) of melatonin could improve the early development of embryos in vitro.Using 2-cell mouse embryos as a model, this study investigated the effects and physiological activities of melatonin on embryonic development in vitro, and explored the specific mechanism of melatonin on embryogenesis. We expected this study could not only help to optimize the in vitro embryo culture system environment, but also give us further aspects to clarify the physiological roles of melatonin in embryonic development.Part 1. The effects of melatonin on the development of mice 2-cell embryosObjective: To investigate the effects of melatonin on the development of mice 2-cell embryos. Methods: Eight hundred and six mice 2-cell embryos were selected and randomly assigned into seven groups. The effects of various concentrations of melatonin(10-13-10-3M)on the development of mouse embryos were detected. Cell counting was taken under the microscope, and the blastocyst rate and hatching rate of mouse embryos were recorded after treated by different concentrations of melatonin. Results: Melatonin could improve the development of mice 2-cell embryos in a bi-phasic manner. The rates of blastocyst formation and hatched blastocysts increased by melatonin at 10-13-10-5M, and the maximum effects arrived at 10-9M. However, the inhibition effects appeared at 10-3M. Conclusions: Melatonin affects the development of mice embryos depending the concentrations of melatonin. In all, the best concentration of melatonin favor the development of mice 2-cell embryos is 10-9.Part 2. The mechanisms of melatonin on the development of mice 2-cell embryosObjective: The potential mechanisms of melatonin on 2-cell embryos were explored. Methods: After treatment with different concentrations of melatonin, the level of RNS or ROS in culture medium was evaluated. Cell cycle and apoptosis-associated molecular such as Bax, Bcl-XL were identified by PI staining, and reverse-time PCR respectively. Results: Finally, after administration with different concentrations of melatonin, the detection of ROS and RNS revealed that the level of RNS presented wave properties, and the maximum scanvage effect occured between 10-9 and 10-7M. Meanwhile the level of ROS did not show any significant change after treatment of melatonin. These data proved that the RNS which released during the embryo development can be largely neutralized by melatonin at the concentration between 10-9 and 10-7 M. Cell cycle analysis showed that 2-cell embryos were arrested at G0/G1 phase after treatment with 1mM of melatonin. Results of reverse-time PCR revealed that the mRNA level of pro-apoptotic factor Bax was elevated, while the mRNA level of anti-apoptotic factor Bcl-XL was down-regulated with the increased concentrations of melatonin. Conclusions: All of these results not only ascertained the effects of melatonin on mice 2-cell embryos, but also were benefit for the explosion of the physiological role of melatonin on mammalian reproduction system. |
PDF Full Text Request