Effect Of PCB118 Exposure On Methylation Mechanism Of Endometrial Receptivity And Its Influence On Embryo Implantation

Part ? Effect of Chronic PCB118 Exposure on Endometrial Receptivity and Embryo ImplantationObjective Polychlorinated biphenyls (PCBs) are one of the most common endocrine disrupting chemicals and have obvious toxicity on human reproductive development. The aim of our study was to investigate the toxicity of chronic PCB 118 exposure on embryo implantation and endometrial receptivity.Methods Virgin CD-1 female mice (3-week old) were housed and orally treated with PCB 118 (0,1,10, 100?g/kg) for a month. After mating with fertile males, the pregnant mice were sacrificed on gestation D5. The number of implantation sites on day 5 of pregnancy demarcated by distinct blue bands was recorded; The morphology of pinopodes were examined using scanning-electron microscopy. Real-time PCR, westernblot and immunohistochemistry were used to study the expression of ESR1, PGR, HOXA10, ITGB3.Results Compared with the control group, implantation failures were observed in 1?g/kg PCB118 and 100?g/kg PCB118 treated groups. Abnormal Uterine morphology with open uterine lumens and densely packed stromal cells and poor developed pinopodes were substantially in response to PCB118 doses above, as well as the significant down-regulation of implantation-associated genes (HOXA10, Integrin?3, LIF).Conclusion It was confirmed that chronic exposure to PCB 118 produced implantation defect in association with a defective uterine morphology during the implantation period. Alterations in the expression of ESR1?HOXA10?ITGB3 could explain, at least in part, the mechanism of effects of PCB118 exposure on endometrial receptivity and embryo implantation.Part ? Effect of Chronic PCB118 Exposure on Methylation of ESR1 and HOXA10Objective Epigenetics such as DNA methylation is a hotspot of recent studies, PCBs can affect the expression of genes in tumor biological behavior through regulating the methylation patterns. The purpose of this part is to investigate possible DNA methylation mechanism involved in the affect of PCB118 exposure on endometrial receptivity.Methods Virgin CD-1 female mice (3-week old) were housed and orally treated with PCB 118 (0,1,10, 100?g/kg) for a month. After mating with fertile males, the pregnant mice were sacrificed on gestation D5. The methylation status of ESR1 and HOXA10 promoter was determined by methylation specific PCR and bisulfite sequencing. Real-time PCR and westernblot were performed to measure the expression of DNMTs?MLL1?EZH2, H3K4 and H3K27?Results It showed that methylation status of HOXA10 was significantly increased, while the methylation status of ESR1 was increased but without any significant difference in PCB118 exposure group compared with the control group. Real-time PCR and westeernblot detected increased expression level of DNMTland DNMT3b. For histone methylation, The expression level of EZH2 was not varied, as well as the protein level of H3K27, but high expression level of MLL1 and H3K4 was measured in PCB118 exposure groups.Conclusion Alterations in methylation of HOXA10 could explain, at least in part, the mechanism of effects of PCB118 exposure on the implantation process. Chronic PCB118 exposure affected histone methylation during implantation period, MLL1 could be a key molecule.Part ? Genome-wide Screen of DNA Methylation Changes Induced by Chronic PCB118 ExposureObjective Endometrial receptivity is a process involving multiple genes and molecular regulation. HOXA10 is the key molecule but is not the whole determining factor. The purpose of this part was to investigate the methylation effects of chronic PCB118 exposure via genome-wide screen methylation chip and to elucidate the underlying evidences to prognosis of implantation failure in clinic, and posibility molecular targeting in drug research.Methods Virgin CD-1 female mice (3-week old) were housed and orally treated with PCB 118 (0,1,10, 100?g/kg) for a month. After mating with fertile males, the pregnant mice were sacrificed on gestation D5. DNA and RNA was extracted from endomtrial tissues. High-density methylated DNA immunoprecipitation chip was used to detected the difference of.genome-wide DNA methylation status in four groups. Gene ontology(GO) and genomes KEGG pathyway analysis were conducted to identify biological informations associated with the methylated genes. Four genes were validated byMeDIP-qPCR, BSPand real time PCR were perfromed to examied the methylation and expression level of FGF4and PCDH17in endometrial tissues.Results The methylation chip showed that PCB118 exposure resulted in different methylation changes in 2591 genes promoter, of which 385 genes showed a marked changes in promoter methylation. Most of the genes promoter belongs to the high density CpG promoter (HCP). In order to identify functions associated with these differently methylated genes, we conducted GO and KEGG pathway analysis. The differential promoter methylation involved in lots of biological process, such as cell cycle, tissue morphogenesis, cytoskeleton organization, cancer signaling pathway, MAPK signaling pathway and so on. Four genes (PCDH17 ITGB8 FREM2 and FGF4) were selected via the rule of Peak Score>3, Peak>300, promoter CpG content (HCP or ICP). They participated in cell adhesion, cell proliferation and cell cycle. The methylatoin status of the four genes were measured by MeDIP-qPCR. All of the four genes were confirmed to be hypermethylated in PCB118 exposure groups. Real-time PCR showed a downregulated mRNA expression levels of PCDH17, ITGB8, FREM2 and FGF4 genes in PCB118 exposure group.Conclusion PCB118 exposure could induce global DNA methylation changes.Tumor signaling pathway and MAPK signaling pathway is the direction of future study. We identified a panel of candidate epigenetic biomarker (PCDH17 ITGB8 FREM2 and FGF4) in PCB118 exposure groups. The genes may be promising for drug research in the future.