Effects Of ER? Specific Antagonist And Agonist On Mouse Trophectoderm Lineage Differentiation

Objective:To investigate whether ER? has important effects on the formation of mouse blastocyst, we observed the localization and distribution of ER? during the lineages specification of mouse embryos and the affection of ER? specific antagonist(MPP) and agonist(PPT) on the development of mouse 8-cell embryos to blastocyst in vitro. Meanwhile, we analyzed the expression of factors related to the trophectoderm and inner cell mass lineages differentiation in the mouse blastocyst development after MPP and PPT treatment.Methods:1. Embryos at 8-cell, morula and blastocyst stage were collected from hybrid(?KM×?KM). The localization and distribution of ER? protein in these embryos were observed by confocal laser scanning microscopy after immunostaining.2. Collect KM mouse 8-cell embryos:1) cultured in KSOM medium to observe time points of blastocoels formation and expansion in vitro;2) cultured in KSOM medium supplemented with different concentration of MPP and PPT respectively to record rates of embryos cavitation in above time points;3) cultured in KSOM medium supplemented with 5?M and 10?M MPP for 24 h, and then observe blastocyst formation status of each group after washing three times and transferring to MPP-free KOSM medium;3. KM mouse 8-cell embryos were collected to culture in KSOM medium and KSOM medium containing 5?M MPP and 10?M PPT for 24 h, and then take the morula embryos of each group to:1) observe the expression of ER? and protein related to lineages differentiation,including OCT4, CDX2, GATA3 and TEAD4 by confocal laser scanning microscopy;2) detect the m RNA levels of Tead4, Gata3 and Cdx2 by Realtime-PCR;4. After cultured at different concentration of MPP for 24 h, MCF-7(a ER?-positive breast cancer) cells were collected to test cell inhibition rates by CCK-8 method. Collect MCF-7 cells cultured at medium containing 10?M MPP for 24 h to detect the expression of ER? protein by Western-blot.5. KM mouse 8-cell embryos were collected to respectively culture in KSOM medium and KSOM medium containing 10?M PPT, and then took the morula embryos of each group to detect the expression of Occludin, a transmembrane tight-junction protein, and E-Cadherin, a cell-adherin related protein by confocal laser scanning microscopy after immunostaining.Results:1. Immunofluorescence results showed that ER? protein located predominantly in the nuclear of 8-cell, morula and blastocyst stage embryos. And nuclear-localized ER? was mainly observed in the outer cells of morula and cells of blastocyst trophectoderm.2. 24-30 h was the time period of large-scale cavitation of mouse 8-cell embryos cultured in vitro. ER? specific antagonist MPP(5?M and 10?M) and agonist PPT(10?M) could respectively inhibit and promote the cavitation ratio of mouse embryos(P<0.05). After 5?M MPP treatment for 24 h, followed by washing and transferring to MPP-free KOSM media, most morula continued to develop into blastocyst stage and maintained blastocyst cavity expansion. However, embryos in 10?M MPP group failed to develop further into blastocyst stage following drug-removal(P<0.001).3. Immunofluorescence results showed that 5?M MPP treatment induced the translocation of nuclear ERa, and lowered the nuclear expression of CDX2 and GATA3 in outer cells, but the nuclear localization of OCT4 was not affected. And the m RNA levels of Tead4, Gata3 and Cdx2 were obviously reduced in MPP(5?M) group compared with control group(P<0.05). But, the expression of neither CDX2 and OCT4,nor TE-related genes, was not significantly changed in PPT(10?M) group.4. CCK-8 results showed that 10?M MPP obviously inhibited the proliferation of MCF-7 cells(P<0.01). However, Western-blot results showed that the total levels of ER? protein was not affected.5. Immunofluorescence results showed that in PPT(10?M) group, fluorescence intensity of Occludin protein was obviously strengthened and membrane-localized Occludin protein was increased compared with control group.Conclusion:ER? specific antagonist MPP treatment inhibits blastocyst formation in a dose-dependent manner, while ER? specific agonist PPT, at proper concentration, promotes the cavitation ratio of mouse embryos. This indicates that ER? probably plays an important role in mouse blastocyst formation. MPP significantly decreased the nuclear expression of CDX2, but no significant changes of OCT4 were observed. Moreover, after MPP treatment, the expression levels of the genes related to TE specification, Tead4, Gata3 and Cdx2, were significantly reduced. The results suggest that ER? might affect mouse embryo cavitation by regulating TE lineage differentiation. Although PPT treatment did not affect the m RNA and protein expression of these TE-specific factors, the TJ-related Occludin protein was indeed increased, showing that ER? may have its functions in regulating the formation of trophectoderm polarity.