Date of Award

January 2013

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Hugh S. Taylor

Subject Area(s)

Obstetrics and gynecology

Abstract

Bisphenol-A (BPA) is an environmentally ubiquitous estrogen-like endocrine-disrupting compound. Exposure to BPA in utero has been linked to female reproductive disorders including endometrial hyperplasia, endometriosis, fibroids and breast cancer. Estrogens have a role in the etiology of each of these conditions. Here we hypothesize that BPA exposure in utero leads to changes in developmental programming of estrogen responsive gene expression. Additionally, we examine methylation status of several altered genes with known estrogen response elements.

8 pregnant CD-1 mice were continuously treated with BPA (5 mg/kg/day) or vehicle via osmotic minipump on days 9-18 of gestation. 2 wks after birth, uteri of half the female offspring were isolated, and RNA was extracted. At 6 wks, remaining female offspring were oophorectomized, then at 8 wks were treated with a single IP injection of 300 ng estradiol (E2) or vehicle, and the uteri were removed for RNA and DNA isolation. Total RNA was labeled and hybridized to a mouse BeadChip WG-6 expression microarray (Illumina). Genes showing statistically significant change (> 2-fold) versus control were verified using real time RT-PCR. Genomic DNA was enriched using MeDIP, then labeled and hybridized to a mouse Roche/NimbleGen 720K CpG Promoter Methylation Array.

At 2 weeks, global gene expression was remarkably similar among the control and BPA exposed groups. Of a total 45,000 genes examined, only 18 (10 upregulated and 8 downregulated) showed changes in expression of 2-fold or greater. After estrogen exposure at puberty (8 weeks), the expression profile was markedly changed. At baseline, a total of 365 genes (77 upregulated and 288 downregulated) showed altered expression in BPA-exposed offspring. With E2 treatment, expression of another 316 genes (90 up and 226 down) showed altered E2 response in BPA-treated offspring; this included several genes that were not previously regulated by estrogen (e.g. Fzd10, Gdf10) or that demonstrated an exaggerated response to estrogen treatment (Ret, Wif1, S100a8). Expression changes of those genes with the greatest fold change were verified by real-time RT-PCR. Of these genes with significantly altered expression at puberty, 208 also showed aberrations to the normal pattern of methylation, including changes to at least 14 genes with known ERE's (e.g. G6pdx, Stat5a, Scd1).

Significant changes in gene expression were observed in the uteri of mice exposed to BPA as a fetus; however, these differences became apparent only after endogenous estrogen exposure at puberty or with estrogen treatment. Gene-environment interactions driven by BPA alter the normal developmental programming of estrogen responsive gene expression in the uterus. Hyperresponsiveness to estrogens is a potential mechanism to explain the increased incidence of estrogen related disorders seen after exposure to endocrine disruptors. Many of these changes may be mediated by alterations to genome-wide methylation patterns.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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