Targeted Genome-Wide Methylation and Gene Expression Analyses Reveal Signaling Pathways Involved in Ovarian Dysfunction after Developmental EDC Exposure in Rats1

Abstract

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 μg/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 immunoprecipitation 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.

Effects of Endocrine-disrupting Chemicals on Female Reproductive Health

“…This finding correlates with the lack of significant effects on the levels of ESR1 protein in the adult ovary [24,160]. Further analysis has shown that the DNA methylation levels in the promoter regions of these genes were unchanged in neonatal ovaries (PND 7) immediately after the exposure [25]. These data demonstrate the age-dependence/hormone responsiveness of the epigenetic changes, which has also been shown in other tissues (e.g., uteri) with other compounds (e.g., DES, genistein) [161].…”

“…A more recent targeted genome-wide methylation array study has revealed that members of essential signaling pathways are hypermethylated and their gene expression down-regulated in MXC-treated ovaries. IGF-1 signaling was the most significantly affected pathway wherein several members of the family -Igf1r, insulin receptor (Insr), Pik3r1, Hras, and Foxo3 -were hypermethylated [25]. These data suggested that the initial DNA methylation patterns were representative of the gene expression patterns responsive to the EDC exposure and not the adult hypermethylation events.…”

“…In contrast, although ESR1 plays a role in the regulation of theca cell steroidogenesis in the ovary, its main function is to mediate estrogen-regulated feedback in the hypothalamus and pituitary [98,99]. On the other hand, very little is known about non-genomic estrogen signaling that is mediated by membrane bound ESR1 (mESR1) in the ovary but recent evidence has demonstrated a role for PI3K/AKT signaling downstream of potential mESR1 activation in the ovary [25,100].…”

PI3K/PTEN/Akt and TSC/mTOR signaling pathways, ovarian dysfunction, and infertility: an update

“…From activated murine follicles, mature oocytes could be retrieved for IVF and embryo transfer, followed by delivery of healthy pups with proven fertility. Recently, Zama & Uzumcu (2013) have demonstrated an age-dependent effect on epigenetic alterations in key molecules of specific signaling pathways such as PTEN, and IGF1, or rapid estrogen signaling on developmental exposure of rats to methoxychlor, an environmental endocrine-disrupting chemical. Hypermethylation of Igf1r, Pi3kr, and Foxo3 had an effect on follicular maturation supported by reduced ovulations observed in treated ovaries.…”

Environmental Epigenomics: Applications of Epigenetic Biomarkers to Investigate Epigenetic Alterations from Environmental Exposures

“…In animal models, environmental infl uences on epigenetic mechanisms have been transmitted trans-generationally. A targeted genome-wide methylation study in rats using Nimblegen 3x720K CpG Island Plus RefSeq Promoter Arrays found that key molecules in specifi c pathways, including PTEN, IGF-1, or rapid estrogen signaling were epigenetically altered in methoxychlor-exposed ovaries (Zama and Uzumcu 2013 ). It has also been reported that early life exposures to endocrine disruptors may alter gene expression in hypothalamic nuclei via DNA methylation and histone acetylation (De Coster and van Larebeke 2012 ;Gore 2008 ).…”