Effect Of ER? Antagonist TPSF On Mouse Preimplantation Embryo Development In Vitro And The Expression Of Transcription Factor Such As SOX2, OCT4 And TRPS1

Objective:To elucidate the further functions of estrogen receptor alpha in the early stage of mouse preimplantation embryo development, we observed the affection of ER? antagonist(TPSF) on the development of mouse preimplantation embryo in vitro. Meanwhile, we analyzed the expression of pan ER?, p-ER?-ser118 and transcription factors such as Rpb1,Sox2,Oct4 and Trps1 in 2-cell embryos after ER? inhibition. Methods:1. KM mouse embryos were cultured in KSOM medium supplemented with 1/3000 DMSO and different concentration of TPSF respectively. The different development potential was observed under an inverted microscope.2. 2-cell embryos were obtained from KSOM and TPSF(5?M and 7.5?M) media, the different m RNA expression level of ER?were measured by Realtime-PCR, the different protein expression levels of pan ER? and p-ER?-ser118 were measured by immunofluorescence.3. 2-cell embryos were obtained from KSOM and TPSF(5?M and 7.5?M) media, the different m RNA expression levels of Sox2,Oct4 and Trps1 were measured by Realtime-PCR, the different protein expression levels of RPB1,SOX2,OCT4 and TRPS1 were measured by immunofluorescence. Results:1. The ER? specific antagonist can obviously inhibit the mouse 1-cell embryos development in vitro concentration dependently. 1- embryos treated with TPSF(5?M) continuously in vitro were blocked at 4-cell embryos, and minority of them can develop to blastosphere(P<0.001); 1- embryos treated with TPSF(7.5?M and 10?M) continuously in vitro were blocked at 2-cell embryos, even minority of them were blocked at 1-cell embryos, the very few could be developed to 4-cell embryos(P<0.001).2. In TPSF(5?M and 7.5?M) groups, the ER? m RNA levels was down –regulated slightly, while there were no significant difference compared with KSOM group(P>0.05); the p-RPB1,SOX2 and TRPS1 relative fluorescence intensity was decreased in TPSF(5?M and 7.5?M) groups(P<0. 01), the p-ER?-ser118 relative fluorescence intensity was decreased in TPSF(7.5?M) group(P<0. 01).3. In TPSF(7.5?M) group, the Sox2 m RNA levels was significantly down-regulated(P<0.05); the m RNA levels of Oct4 and Trps1 was down –regulated slightly, while there were no significant difference compared with KSOM group(P>0.05); the pan-ER? relative fluorescence intensity was increased in TPSF(5?M and 7.5?M) groups(P<0. 01); the OCT4 relative fluorescence intensity was not changed obviously in TPSF(7.5?M) group compared with KSOM group(P<0. 01).Conclusion:1. The ER? specific antagonist TPSF can inhibit the development of mouse preimplantation embryos from 2-cell to 4-cell, resulting in “2-cell block” in vitro; meanwhile, the ser118 phosphorylation level of ER? is down-regulated after ER? inhibition. The results suggest that ER? may play important role in the development of mouse preimplantation embryos, and the TPSF may function via suppressing the phosphorylation of ER? ser118 site.2. TPSF can down-regulate the expression of p-RPB1, SOX2 and TRPS1, revealing that TPSF may affect the transcriptive activity in mouse 2-cell embryos to suppress the development of mouse preimplantation embryos in vitro.