| In chicken production,the efficiency of chicken follicular development and maturation are the key factor in determining the age of laying,the number of eggs produced and non-laying,and other reproductive traits.In the follicular growth and ovulation process,the estrogen produced by granulosa cells under the action of follicle stimulating hormone,interacting with receptors,induced ovarian and follicular growth and development-related gene expression.Estrogen receptors include ER? and ER? plays an important role in the development of ovaries and follicles through different distribution and content levels in ovaries,however,reports about the molecular mechanism of estrogen receptor-mediated chicken egg production are rare.In the chicken ovary and follicle,the expression of ER? was obviously higher than that of ER?.In the previous studies,the changes of ER? and ER? in the different developmental stages of hens were different,suggesting that ER? may be involved in the regulation of hens during sexual maturation,and ER? mainly played a role in the early maturation.In this study,we used immunofluorescence technique to analyze the changes of ER? in ERa at the 45 d,90d?prenatal?and 160d?postpartum?,further confirming the results of the preliminary study of our group.ER? decreased at the early stage of sexual maturity?45d-90d?,and increased at the time of sexual maturity?90d-160d?.Fluorescence intensity is mainly distributed in the granulosa cell layer of the primary follicle of the ovarian cortex.The content of estradiol?E2?in hen serum was analyzed by enzyme-linked immunosorbent assay?ELISA?,results showing that the changes of E2 in the serum of different developmental stages were consistent with the changes of their receptors.Therefore,ER? is presumed to be a key factor in the growth and development of ovarian follicles in sexual mature chickens,which can induce the expression of genes related to follicle growth and development,and play an important role in ovarian development,maturation and ovulation.Then,what causes this dynamic expression of ER? in chicken ovary?We further analyzed the differential expression mechanism of ER? in chicken ovary.1.The epigenetic modification of ER? promoter region was analyzed by bisulfite sequencing.The results showed that there were no significant differences on the whole between 90 d and160d chicken ovaries.But there were two different methylation levels in 17 CpG sites detected in ER? promoter region while the expression of ER? is inconsistent,suggesting that methylation of DNA in the promoter region may not be involved in the differential expression of ER?.2.ChIP-qPCR was used,with Anti-Histone H3?di+tri methyl K4?antibody?Anti-Histone H3?tri methyl K9??Anti-Histone H3?tri methyl K36??Anti-Histone H3?acetyl K27?and Anti-Histone H3?acetyl K9,phospho S10?aimed,to analyze the differences of histone modification of ER? in chicken ovary tissues at 90 days and 160 days.The results showed that the modification level of H3K4,trimethylation and H3K9 phosphorylation was not significantly correlated while H3K9 trimethylation,H3K27 acetylation and H3K36 trimethylation levels were significantly different at two time points in the chicken ovaries.According to the biological function of the three,it was sure that H3K27 acetylation was the key to the apparent regulation of ER? expression and showed that the expression of p300 was consistent with the change of acetylation of H3K27 in promoter region of ER? gene.To further reveal the important function of ER? in ovarian development,maturation and ovulation,ChIP-seq technique was used to screen out the ER?-induced expression of related genes with the basis of the expression of ER? in different developmental stages of hen ovary,and estrogen-induced genes were screened from Chicken ovaries of 45 d,90d and 160 d.And,the method of chicken ovarian tissue broken for ChIP was established.The results of sequencing are as follows.1.The ratioof unique alignment sequences was 82.03%,79.93% and 78.38%,respectively,in the hens of 45 d,90d and 160 d.2.The only alignment sequence was subjected to peak scan analysis,The number of binding sites identified at different developmental stages was 24886,21680 and 23348,with an average length of about 150 bp,accounting for 0.35%,0.3% and 0.34% of the genome,respectively.3.Most of the binding site functional elements are located between the gene and the intron,and the sum is more than 80%.4.The motif analysis showed that there were multiple motifs at each time point,and six of them were identified,and the first order at each point time was almost the same as 15 bp.The ranked second motif is rich in AT binding sites at three different time points,rich in C sequences at 45 d and 90 d,and 160 d rich in G sequences.5.ER?-induced expression gene at different time points was analyzed.The specific expression factors respectively were 13,20 and 9.At 45 d and 90 d The co-induced expression genes were 69 at 45 d and 90 d,The co-induced expression genes were 66 in 45 d and160d,The co-induced expression genes were 66 at 90 d and 160 d,The co-induced expression genes were 60 at 45d?90d and 160 d.6.To analyze the consistency of ER?-induced gene and ER? at different stages.The results showed that there were 13 genes,such as ATP2A3,TACC1,TAPP2,and so on,which were consistent with the expression level of ER? at 45 d and 90 d.there were 9 genes,such as cRhoB,FCHSD2,FHL1,and so on,which were consistent with the expression level of ER?at 90 d and 160 d.there were 25 genes,such as gga-mir-3534,HTRA3,uc338,and so on,which were consistent with the expression level of ER? at 45 d and 160 d.7.Analysis of ER? indured genes involved in the cohort and related pathways,compared with45 d and 90 d,there are 4 cohorts and 15 channels corresponding to the RDF value is less than0.01.compared with 90 d and 160 d,there are 9 cohorts and 14 channels corresponding to the RDF value is less than 0.01.8.Construction of protein interactions between the network structure,shown in the protein network structure map contains 47 protein nodes and 164 interaction edges.There were 18down-regulated proteins at 45 d and 90 d,but no significant difference at 90 d and 160 d.At 90 d and 160 d down but at 45 d and 90 d no significant difference in the protein were 18.There were 9 genes down-regulated at 45 d and 90 d and at 90 d and 160 d.One of the genes up-regulated at 45 d and 90 d but at 90 d and 160 d.9.The interaction of proteins was calculated based on the number of nodes in the network structure and the 10 network nodes were obtained including AKT1,ACTN2,CREB1,EPHA5,DMD,CDK6,PIK3 CG,CTNNA2,APP and CTNNA3.According to the network structure of ER?-induced gene at different developmental stages,it was shown that these target genes were located in the core region of network structure.The results showed that AKT1 gene had a biological function in adult maturation,and EPHA5 and ACTN2 played a biological function in the early stage of sexual maturity.CREB1 increased with the maturation time of hen development,whose biological function decreased.In summary,the expression level of ER? was significantly changed at different developmental stages during the maturation of hens,and decreased in the early stage of sexual maturation,but the late stage of sexual maturity increased.The expression of ER? was not affected by the methylation of promoter region CpG.H3K27 acetylation was the key modification of the apparent regulation of ER? expression,and an important factor of thisacetylation modification is the dynamic expression of p300 gene in hens ovarian tissue.ER?can induce the expression of genes such as ATK1,ACTN2,CREB1 and EPHA5 etc.The differential expression of ER? may affect the expression levels of related genes.These differences may be the key sites which mediating the ovarian maturation,affecting the sexual maturity and follicular development of the hens. |
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