| Resveratrol is a kind of antitoxin secreted from plant, it has extensive pharmacological and biological activity. Studies have shown that resveratrol could promote maturation of oocytes and embryo development in vitro, but its mechanism was not yet clear, especially its role on embryo development block mechanism has not been reported. The aim of this study was to understand the mechanism of 2-cell block phenomenon in KM Mouse, and the embryo development block relationship between active oxygen and resveratrol. We explored the effect of Res on early embryonic development and zygotic genome activation(ZGA) for KM Mouse, and the relationship between reactive oxygen species and the 2-cell block by adding the antioxidant resveratrol (Res) in the culture medium of M16. The study was divided into three parts:The first part was to explore the impact of resveratrol (Res) on the development of Mouse zygote developmental in vitro with different concentration. In order to obtain the appropriate concentration and adding time of resveratrol on mouse embryos, different concentrations of Res were added to mouse embryo in vitro culture medium, the effects were observed. The results showed that the 4-cell rate, blastocyst rate, blastocyst cell number of 2 μmol/L (68.75%,49.22%,51.40),4 μmol/L Res(64.17%,45%,50.53) were higher than that of non-treated group (58.29%,41.21%,44.27), compared with the blank control,2 μmol/L group was significantly higher (p<0.05),4 μmol/L had no significant difference (p>0.05). while the 4-cell rate, blastocyst rate of 5 μmol/L (61.83%,42.62%) had no significant difference (p> 0.05).10 μmol/L (36.52%,18.26%) was significantly lower than the control group (p<0.05). Compared with control group,2 μmol/L of Res could significantly increase the cell number of the blastocysts (p<0.05),4 μmol/L was no significant difference (p>0.05). To further study the effect of Res on embryo development, we also examined the number of apoptotic cells in the blastocyst, expression of apoptosis-related and development-related genes. The results found that, compared with control group,2 μmol/L Res treatment reduced the number of apoptotic cells in the blastocyst, but no significant difference (1.14 vs 1.57, p> 0.05). It significantly increased expression of anti-apoptotic gene Bcl-2 and developmental gene Oct4, cdx2, Nanog (p<0.05), significantly down-regulated pro-apoptotic gene Bax and p53 expression (p<0.05), but did not affect expression of casepase3 (p>0.05). To confirm the action time of Res, we explored the influence of Res on the zygote blastocyst rate by adding it at different times and periods, it was found that Res work time was mainly at the second day of embryo culture,2-cell to 4-cell stage.The second part explored the influence of Res on reactive oxygen species zygote. By detecting reactive oxygen species (ROS) level of 2-cell,4-cell embryos and blastocysts, we found that compared with control group, addition of 2μmol/L and 4 μmol/L Res Res did not reduce the level of reactive oxygen species in embryos of each stages (26.84 vs 26.00,25.8; 33.56 vs 32.79,35.56; 15.79 vs 17.10,17.55, p>0.05). ROS levels in 2-cell,4-cell embryos of in vivo were significantly higher than that of in vitro (39.62 vs 26.84,44.53 vs 35.56, p<0.05). To further understand the relationship between the reactive oxygen species and Res, we examined the expression of antioxidant genes GPX1, Mn-SOD, PRDX3, GLS2, and hypoxia test was explored. The results found that, the addition of Res could significantly increase the expression of these antioxidant genes, and compared with common oxygen condition (20%O2), hypoxic condition(5%O2) culture did not improve the blastocyst rate in M16 medium(41.07% vs 39.07%, p>0.05). The third part explored the mechanism of Res treatment on mouse embryo 2-cell block. qRT-PCR method was used to detect expression of zygotic genome activation (ZGA) related genes. The results found that compared with control group,2μmol/L Res could significantly increase the expression of Hsp70.1, Zscan4d, MuERV, TRC(p<0.05). To further study the mechanisms of Res, the distribution and membrane potential of early embryonic mitochondria were examined, qRT-PCR was used to detect the expression of mitochondrial DNA ND-1, ATP synthase gene ATP6, ATP8, and the maternal transcription factor Oct4, Sox2 and Nanog. The results showed that Res did not affect the distribution of 2-cell mitochondria, but could improve the membrane potential of 2-cell and 4-cell embryos (p<0.05), and the membrane potential in all 4-cell groups was higher than that of the 2-cell groups(p<0.05), expression of the ND-1, ATP6, ATP8 and Oct4 was significantly improved (p<0.05), the expression of Sox2 was decreased significantly (p<0.05).In summary,2 μmol/L of Res treatment could significantly increase the development and quality of the moues embryos in vitro, and its role was at the 2-cell to 4-cell stage. Res might not be through the antioxidant pathway, probably through other mechanism to increase the blastocyst rate. Res treatment could increase the amount of ATP synthase, promoted the mitochondria oxidative phosphorylation level, or by influencing the expression of maternal transcription factors Oct4 and Sox2 to promote the occurrence of ZGA, finally improved the quality of cultured mouse embryos in vitro. |
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