The Effects Of EGCG On Mouse Zygotic Genome Activationand The Expression Of Stem Cell Factors Sox2 And Oct4

Objective: To observe the effects of EGCG on the transition of mouse embryos from 1-cell to 2-cell stage in vitro, and investigate the effects of EGCG on mouse zygotic genome activation(ZGA) and m RNA expression of stem cell factors Sox2 and Oct4. To explore the potential function of EGCG and its mechanism in the development of mouse preimplantation embryo further.Methods: 1. KM 1-cell embryos were collected at post-h CG 21 h, and cultured in M16 medium and M16 medium supplemented with EGCG(10μg/ml and 20μg/ml) respectively for the following analysis:(1)The number of 2-cell embryos, 4-cell embryos, morula and blastocysts developed from 1-cell embryos was counted under an inverted microscope, and the developmental ratios at different stages were calculated.(2)At post-h CG 33 h, the number of 2-cell embryos from 1-cell embryos was counted under an inverted microscope, and the number of 1-cell embryos with positive or negative immunofluorescence of phosphorylated histone H3 was distinguished by the fluorescence microscope. 2. The in vivo fertilized 1-cell embryos were collected at post-h CG 21 h, and cultured in M16 medium or in M16 medium with 20μg/ml EGCG respectively for 6h in vitro, then 1-cell embryos was collected to detect as follows:(1)The levels of e IF1 A, ZSCAN4, SOX2 and OCT4 protein in the nucleus of 1-cell embryos were detected by laser scanning confocal microscopy.(2)The m RNA levels of e IF1 A, Zscan4 d, Mu ERV-L, Hsp70.1, Sox2 and Oct4 in 1-cell embryos were measured by Realtime-PCR. 3. The in vivo fertilized 1-cell embryos were collected at post-h CG 27 h, and cultured in M16 medium or in M16 medium with 20μg/ml EGCG respectively for 18 h in vitro, then 2-cell embryos were collected to detect as follows:(1)The levels of e IF1 A, ZSCAN4, SOX2 and OCT4 protein in the nucleus of 2-cell embryos were detected by Confocal laser scanning microscopy.(2)The m RNA levels of e IF1 A, Zscan4 d, Mu ERV-L, Hsp70.1, Sox2 and Oct4 in 2-cell embryos were measured by Realtime-PCR.Results: 1. The developmental ratios of 4-cell embryos, morula and blastocysts developed from 1-cell embryos was higher in EGCG group than M16 group, and the immunofluorescence positive rate of phosphorylated histone H3 was also higher in EGCG group. 2. The 1-cell embryos were cultured in vitro from post-h CG 21 h to 27 h, and then collected to detect the expression level of ZGA related genes and stem cell factors. The results showed:(1)The relative immumofluorescence intensity of ZSCAN4 and SOX2 proteins at the nucleus of 1-cell embryos in EGCG group was obvious higher than in M16 group(P<0.01).While the immumofluorescence relative intensity of e IF1 A and OCT4 protein had no significant differences between EGCG and M16 group(P>0.05).(2)The m RNA levels of Zscan4 d, Mu ERV-L, Hsp70.1 and Oct4 in EGCG group were significantly higher than M16 group(P<0.01). However, the m RNA levels of e IF1 A and Sox2 had no significant differences between EGCG and M16 group(P>0.05). 3. The 1-cell embryos were cultured in vitro from post-h CG 27 h to 45 h, and then 2-cell embryos were collected to detect the expression level of ZGA related genes and stem cell factors. The results displayed:(1)The relative immumofluorescence intensity of ZSCAN4 and OCT4 proteins at the nucleus of 2-cell embryos in EGCG group was obviously higher than in M16 group(P<0.01).While the relative immumofluorescence intensity of e IF1 A and SOX2 protein had no significant differences between EGCG and M16 group(P>0.05).(2)The m RNA levels of Zscan4 d, Mu ERV-L, Hsp70.1 and Oct4 in EGCG group were significantly higher than M16 group(P<0.01). However, the m RNA levels of Zscan4 d, e IF1 A and Sox2 had no significant differences between EGCG and M16 group(P>0.05).Conclusion: EGCG might enhance the developmental potential of mouse 1-cell embryos in vitro, and also promote the transition of mouse embryos from 1-cell to 2-cell stage. Compared with M16 group, the m RNA levels of Zscan4 d, Mu ERV-L, Hsp70.1 and Oct4 in 1-cell embryos were significantly up-regulated in EGCG group, and also the protein level of SOX2 in 1-cell embryos was significantly increased in EGCG group. Moreover, the m RNA and protein level of e IF1 A, Mu ERV-L, Hsp70.1 and Oct4 were significantly up-regulated in 2-cell embryos in EGCG group. These results suggested that EGCG might promote preimplantation embryo development in vitro, through regulating the minor and major ZGA and the expression levels of some stem cell factors.