The Effects Of ERα-Specific Antagonist On Mouse Preimplantation Embryo Development In Vitro And Zygotic Genome Activation

Objective:To investigate whether ERα has important effects on the activation of zygoticgenomes, we observed the affection of ER antagonist (ICI182780), ERα-specificantagonist (MPP), ERβ-specific antagonist (PHTPP) and ERα-specific antibodies onthe development of mouse Pre-implantation Embryos in vitro; we analyzed themRNA levels of ERα target gene—c-Myc, and the four related genes of ZGA toelucidate the further functions of ERα in Pre-implantation Embryos.Methods:1. KM and B6C3F11-cell embryos and2-cell embryos got from two kinds ofhybrids (♀B6C3F1×♂KM and♀KM×♂KM) were cultured in M16medium, and M16medium supplemented with different concentrations of ICI182780, MPP, PHTPP andERα-specific antibodies respectively. The different development potential wasobserved under an inverted microscope.2. KM1-cell embryos were collected and cultured in M16and MPP (25μΜ)mediums.2-cell embryos were obtained for the detections as follows:(1) The different levels of nuclear ERα were monitored by Confocal laserscanning microscopy.(2) The different protein levels of ERα and c-MYC were detected byWestern-blot.(3) The different mRNA levels of ERα, c-Myc, Jmjd2c, MuERV-L, Zscan4d,Hsp70.1and eIF1A were measured by Realtime-PCR.Results:1. Most of the mouse1-cell embryos and2-cell embryos developed in vitro wereblocked at2-cell stage by ICI182780, MPP and ERα-specific antibodies, little could be developed to4-cell embryos (P <0.01), whereas PHTPP had no inhibition on thedevelopment of mouse1-cell embryos and2-cell embryos obviously in vitro.2. The nuclear ERα fluorescence relative intensity in MPP group was lower thanit in M16group (P<0.01).3. The protein expression levels of ERα and c-MYC were no significantdifferences between MPP group and M16group (P>0.05).4. In MPP group, the MuERV-L mRNA levels were significantly lower thanM16group (P <0.01), while the mRNA levels of the other genes were no significantdifferences compared with M16group (P>0.05).Conclusion:The development of mouse pre-implantation embryos from two-cell to four-cellwas blocked by ICI182780, MPP and ERα-specific antibodies in vitro, which causedthe pre-implantation embryos were not able to develope to blastocyst stage, whereasPHTPP had no inhibition on the development of mouse1-cell embryos and2-cellembryos obviously in vitro.. The results suggest that ERα may play an important rolein the development of mouse pre-implantation embryos in vitro. ERα-SpecificAntagonist (MPP) could lower the mRNA expression levels of MuERV-L whichbelongs to ZGA related genes, showing that ERα may have its functions in zygoticgenome activation by influencing the expression levels of ZGA related genes.