The Roles Of Estrogen Receptor ? In Regulating Zygotic Gene Activation Of Mouse Preimplantation Embryos

Objective(1)To detect the expression patterns of estrogen receptor alpha(ER?)and its activated form,pER?-S118,during mouse preimplantation 1-cell and 2-cell embryos;and to investigate their correlation with the occurrence of zygotic gene activation(ZGA).(2)To study the stage-specific effects of ER? specific antagonist MPP on mouse preimplantation embryo development,and to study its possible function mechanisms.(3)To investigate the possible regulation mechanisms of ER? on the expression of microRNA and to study their possible roles on mouse ZGA through regulating Oct4 and Sox2.Methods(1)Collect mouse preimplantation embryos in an interval of 3 h from 15 h to 54 h post hCG injection,and by using immunostaining,the subcellular localization of ER?and pER?-S118 were detected.(2)Mouse 1-cell,2-cell and 4-cell embryos at different developmental stages were collected and cultured respectively in KSOM medium(control group),and KSOM medium supplemented with MPP(experimental group).After 3h treatment,embryos of each stage were transferred into fresh KSOM medium until blastocyst stage and the number of blastocysts was recorded.Collect 1-cell embryos at 21 h post hCG injection and cultured in KSOM medium containing MPP;the samples were recovered at 27 h post hCG injection,lysised and total RNA was extracted;and then the mRNA expression levels of zygotic genes,eIF1 A and MuERV-L,were detectedusing RT-qPCR.After 21~27 h MPP treatment,the expression levels of histone methylation(H3K4me3 and H3K27me3)were detected using imunostaining.Mouse preimplantation embryos were collected at 21 h post hCG injection,and cultured in KSOM+BrdU and KSOM + MPP + BrdU medium,and at 27 h post hCG injection the levels of DNA replication were detected by using anti-BrdU immunostaining.Mouse preimplantation embryos were collected at 27 h post hCG injection,and after MPP treatment,the embryos were fixed and the nuclear shapes were detected using immunostaining,and the BrdU incorporation experiment was conducted to the study the effects of MPP on DNA replication;and by using immunostaining,the expression of S phase initiation factors,c-MYC and E2F1,were detected.(3)Mouse embryos were collected at 21 h post hCG injection,the MPP treatment experimental model were established,and after extracting total RNA,Megaplex reverse transcription and preamplification,Taqman quantitive PCR based microarray were performed to detect the changes of microRNA;and by extracting the sperm microRNA expression data by literature searching and intersection analysis,the consensus parts of sperm originated microRNA with our microarray results were selected for further study;and by bioinformatics analysis,like gene ontology and signaling pathway research,the possible roles of ER? through regulating microRNA was explored;finally,the expression of bioinformatics predicted target proteins,Oct4 and Sox2,was detected using immunostaining.Results(1)The nuclear localization of ER? was first detected at mid-S phase of mouse1-cell embryos,and disappeared during the first mitosis;reappeared in the early 2-cell stage(Telophase of the first mitosis),and sustained nuclear staining until Metaphase of 2-cell embryos.pER?-S118 expression was evident on the nuclear surface of early1-cell embryos,and overexpressed in the nuclears of Prophase and Prometaphse 1-cell and 2-cell embryos,and elevated expression was also observed in the cytoplasm of Metaphase embryos,and dramatic decrease was observed at Anaphase;the nuclear localization of pER?-S118 in 2-cell embryos was first detected at S phase initiation.(2)MPP treatment significantly inhibited the development of mouse preimplantation embryos,and the stage-specific affection pattern correlated with the occurrence of ZGA.After 21 ~27 h MPP treatment,the mRNA expression levels of mouse zygotic genes,eIF1 A and MuERV-L,were declined;and the expression of H3K27me3 in the male pronucleus was decreased.MPP treatment significantly inhibited the S phase progression of both 1-cell embryos and 2-cell embryos.The subcellular expression levels of c-MYC and E2F1 in the 2-cells were not significantly affected by MPP treatment.(3)Microarray detection results showed that,the number of microRNA increased(126)was higher than that decreased(64).The sperm originated microRNAs that could be significantly affected by MPP treatment were enriched in molecular functions as “response to stimulus,expression(and post-transcription)regulation,epigenetic regulation,negative control of(macromolecular)metabolism,negative regulation of gene expression,negative regulation of mRNA translation.” The targets of sperm ogiginated microRNA were significantly enriched in “DNA dependent regulation of transcription”;and those of minor zygotic genes were more significantly enriched in diverse biological processes.The DNA dependent transcription regulation of minor zygotic gene was regulated in a signaling network directed by “Akt1 –Foxo3 – Stat3 –ER? – Oct4” axis.After MPP treatment,the expression of OCT4 in2-cell embryos elevated and that of SOX2 decreased;after Akt specific inhibitor API-2 treatment,the expression level of pER?-S118 was elevated.Conclusions1.ER? is able to regulate the DNA dependent transcription regulation of mouse minor ZGA,through affecting the S phase progression,male chromatin methylation,and expression of sperm originated microRNAs.2.The function mechanism of ER? during mouse ZGA involve the regulation of Oct4 and Sox2.3.Furthermore,the activity of ER? was affected by the phosphorylation pathway of Akt;and by regulating miR-125a-5p and miR-125a-3p,ER? is able to regulate thefunction of Akt,thus a feedback circle is formed.