Targeted Genome-Wide Methylation and Gene Expression Analyses Reveal Signaling Pathways Involved in Ovarian Dysfunction after Developmental EDC Exposure in Rats

Transient exposure to methoxychlor (MXC), an environmental endocrine-disrupting chemical, during fetal and neonatal stages causes ovarian dysfunction in pubertal, adult, and aging animals. Adult animals have reduced number of ovulations and abnormal follicular composition associated with altered gene expression and DNA methylation patterns. To test the hypothesis that the ovarian epigenomic changes induced by MXC are detectable following the exposure period, leading to altered gene expression by adulthood, we conducted a targeted genome-wide methylation study using Nimblegen 3x720K CpG Island Plus RefSeq Promoter Arrays. Control (vehicle), low-dose MXC (20 mu g/kg/day), or high-dose MXC (100 mg/kg/day) treatments were administered between Embryonic Day 19 and Postnatal Day (PND) 7. Ovaries were collected at PND 7 immediately after exposure or at adulthood, PND 60. Array hybridizations were conducted with genomic DNA after methylated DNA immuno-precipitation and the array data were analyzed. DNA methylation events were functionally annotated, and candidate loci common to all the treatments or unique to some treatments were identified. Specific loci encoding signaling molecules such as the regulatory subunit p85 of phosphoinositide-3-kinase, insulin-like growth factor-1 receptor, Harvey rat sarcoma viral oncogene, insulin receptor, and forkhead box protein O3 were identified to be hypermethylated in MXC-treated ovaries at PND 7 and/or PND 60. Examination of gene expression changes with TaqMan low-density arrays revealed that nearly 25% of the genes that were assayed were downregulated. These data demonstrate that key molecules in specific signaling pathways such as PTEN signaling, IGF-1 signaling, or rapid estrogen signaling are epigenetically altered in MXC-exposed ovaries, which is associated with ovarian dysfunction and female infertility.