【摘要】：Melatonin (MT) is a kind of indoles hormones, secreted from the pineal gland of brains in human and mammals mainly, and other organs and cells also secrete less. The production of MT is greater affected by illumination, and presents the circadian rhythm:commonly secrete more at night, less during the day. As effective antioxidants in vivo, MT has the character of highly lipophilic and hydrophilic, and can pass through the cytomembrane and a variety of membrane structures into organelles to play the role of antioxidation. The main functions contain direct scavenging free radicals; improving the activity of antioxidant enzymes (including superoxide dismutase (SOD); glutathione reductase (GSH-Px)) and decreasing the production of free radicals.
At present there are many researches which study about the effect of MT as an antioxidant and antiapoptosis factor on the in vitro maturation of oocytes and early embryonic development in mice, pigs, sheep, chickens and other animals. However,-the further-studies of its molecular mechanism and receptor signaling pathways are reported less. In mice as:test object, this study aim to investigate the effect of oxidant hydrogen peroxide (H2O2) on mouse oocytes during IVM and the protection of MT against the oxidative damage; the influence of MT on embryo development after parthenogenetic activation and early embryonic development in vitro. It will provide the theoretical and experimental basis for optimizing the system of embryo culture in vitro. The main contents can be stated as follows:
1. The effect of melatonin on mouse oocytes threatened by H2O2during IVM and parthenogenetic embryo development.
The influence of H2O2on the meiotic resumption and progression of mouse oocytes were assessed and the dates were summarized:The proportion of oocytes meiotic resumption (GVBD to MⅡ) decreased with the addition of H2O2:the group of100μM was no significant difference in comparison with the control group; The oxidative damage was more and more obvious when more than200μM, the discharge rate of PB1was decreased significantly (P0.05) when the concentration was increased to250μM. There was no survival in300μM.Screened the maximum concentration for oxidative stress was250μM.
2. The protective effect of melatonin against the inhibitory effect of exogenous H2O2on oocyte maturation.
Different concentrations of MT (0,0.1,1,10ng/ml) were adding to the maturation medium which containing OμM,250μM of H2O2, After incubating for12h, the PB1discharged number of oocytes were analyzed. We found that when oocytes were cultured in the culture medium of the coexistence of MT and H2O2(250μM), MT dose-dependently reversed the inhibition of H2O2on oocytes maturation in vitro. It can be seen compared to the second group, the third, the four groups of maturation rate was significantly increased (42.0%vs.24.5%;47.4%vs.24.5%, P0.05), the concentration of MT reached10ng/mL, the reversal effect is the most significant (61.1%vs.24.5%, P0.05).
3. The effect of MT on parthenogenetic development of mouse oocytes.
In this experiment, different concentrations of MT (0,10-9,10-7,10-5,10-3M) were used to incubate parthenogenetic development of mouse oocytes. The results were shown that there was no significant difference in the groups of10-9-10-3M than the control group during two-cell phase (88.2%,88.8%,91.4%,91.8%,88.8%vs.88.1%,P0.05). the rate of four-eight-cell was improved when added10-9M of MT;(3O.5%vs.23.4%, p0.05):-and had emerged a significant difference. There were very significant difference among the rates of10-7-10-3M than control group (58.8%,73.8%,43.6%vs.23.4%, p0.05), Blastocyst rates were increased in the higher concentrations10-9M-10-5M (24.6%,46.6%,49.2%vs.8.0%, p0.05), but it decreased during10-3M of MT. In conclusion, the best concentration of MT for development of mouse oocytes after parthenogenetic activation was10"5M.
4. The effects of melatonin on mouse one-cell embryos development.
Through experiment, we found that MT could overcome embryo developmental block, and significantly promote the embryonic development. When treated with10-7,10-5M of MT, the blocking rates of two-cell were significantly increased (44.20%,41.60%vs.26.6%, p0.05), but further development to the blastocyst was not increased under10-5M. MT(10-9M.10-7M) increased development rates from four-cell to blastocyst (four-cell stage,25.8%;25.3%vs.15.9%,P0.05;eight-cell stage,23.4%,24.4%vs.12.1%, p0.05).but not that of two-cell stage embryos. Moreover, a promoting effect in all stages from two-cell to blastocyst was observed at10"7M (two-cell stage,44.20%vs.26.6%; four-cell stage25.3%vs.15.9%; eight-cell stage,24.4%vs.12.1%; blastocyst.22.6%vs.7.9%).
5. The Hoechst33342staining of blastocyst.
Blastocysts which were cultured at different concentrations of MT were stained by Hoechst33342, the total cell numbers of blastocyst were observed. The higher concentration of MT could increase the blastocyst rate and total cell number of blastocyst.10-7M of MT was the suitable concentration for early embryo development, and were significantly increased the total cell number of blastocyst (42.7vs.32.4, p 0.05), the other concentrations (10-9M;10-5M;10-3M) were not significantly than control group (33.1;37.4;35.9vs.32.4).
In conclusion, mouse oocytes were suffered oxidative damage by H2O2which the highest concentration was250μM. MT dose-dependently reversed the inhibition of H2O2during IVM of oocytes.10'7-10-5M of MT could improve the quality of mouse oocytes in vitro and the development rate of parthenogenetic embryo, and total number of blastocyst cells, promote the development capacity of parthenogenetic embryos in vitro.