Bisphenol a exposure disrupts genomic imprinting in the mouse

Abstract

Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.

Figure 1. Representative LC-SRM/MS chromatograms demonstrating significantly higher BPA level in serum from treated mice.

Top panels represent the chromatograms for unlabeled BPA and lower panels labeled BPA ([¹³C₁₂]-BPA) spiked prior to sample extraction to quantify BPA levels in serum from representative control (B) and upper dose treated mice (C). Blank sample used as control for storage and extraction is indicated in (A). Y-axis represents relative abundance of signal intensity and X-axis retention time in minute. Differences in BPA concentrations were determined based on peak heights (see Materials and Methods). NL = normalized levels of intensity; m/z = mass to charge ratio.

Figure 2. Map of mouse chromosome 7 showing imprinted domains analyzed in the current work is illustrated.

Black = paternally expressed genes; dark grey = maternally expressed genes; and light grey = differentially methylated regions (DMRs). Arrows indicated direction of gene transcription. C = centromere and T = telomere. Not drawn to scale.

Figure 3. Allele-specific expression studies showed increased proportion of tissues with loss of imprinting following BPA exposure.

(A–D) Each black circle represents an individual E9.5 embryo (Igf2) or placenta (Snrpn, Kcnq1ot1 and Ascl2). Y-axis indicates percentage of total mRNA expression derived from the repressed allele. Loss of imprinting (LOI) or biallelic expression was called when the repressed allele exhibited ≥10% of total expression. Data from control (n = 23), lower dose (n = 22) and upper dose (n = 28) exposure groups are shown for (A) Snrpn, (B) Igf2, (C) Kcnq1ot1 and (D) Ascl2; a = P<0.05; b = P<0.01 and c = P<0.001. Number of tissues (out of total examined) showing LOI is indicated below each exposure group. In (E), percentages of placentas (Snrpn and Kcnq1ot1) and embryos (Igf2) showing LOI at E9.5 and E12.5 are compared.

Figure 4. BPA exposure altered total mRNA expression of imprinted genes relative to reference genes.

Samples from control, lower dose and upper dose exposure groups were analyzed for total expression of the imprinted (A) Snrpn, (B) Igf2, (C) Kcnq1ot1, (D) Cdkn1c and (E and F) Ube3a genes with sample sizes (n) indicated. Assayed samples included embryos (Igf2), placentas (Snrpn, Kcnq1ot1, Cdkn1c, and Ube3a) or brains (Ube3a) showing normal, monoallelic (black circle) or biallelic expression (red circle). Average total expression in each exposure group is indicated with a black horizontal line; a = P<0.05; b = P<0.01 and c = P<0.001.

Figure 5. Analysis in control and upper dose BPA exposed placentas indicated that exposure significantly reduced methylation.

The Snrpn ICR is depicted in (A), including the sequences analyzed in the (B) pyrosequencing and (C) bisulfite sequencing assays. A. 16 CpG sites (highlighted in red and bold) located in a 451 bp region of the Snrpn ICR were assayed by bisulfite sequencing. The underlined genomic sequence containing 7 CpGs represents the region analyzed by pyrosequencing. B. Pyrosequencing data in control, lower dose and upper dose BPA exposure are shown with samples exhibiting monoallelic (black circles) or biallelic expression of Snrpn (red circles). Y-axis represents percentage of total methylation. Black horizontal line in each exposure group indicates average methylation. Sample sizes analyzed in each exposure group are shown; a = P<0.05 when analyzed through ANOVA but not significant through Kruskal-Wallis. C. Three placentas from both control and upper dose BPA exposure groups were analyzed by bisulfite sequencing and shown here is the methylation status of maternal ICR. Each circle represents a CpG site with black = methylated and white = unmethylated. Percentages of methylation at all CpGs are indicated. Average CpG methylation levels are 82.0±4.3% in controls and 53.8±2.4% in upper dose (P<0.01).

Figure 6. BPA exposure altered DNA methylation at the Igf2 DMR1 and H19/Igf2 ICR in the embryos.

A. Pyrosequencing assays tested methylation of 4 and 6 CpGs (highlighted in bold) in 457 bp and 221 bp genomic regions at the Igf2 DMR1 and H19/Igf2 ICR, respectively. Methylation at the (B) Igf2 DMR1 and (C) H19/Igf2 ICR was analyzed in embryos from controls, lower dose and upper dose exposure groups. Analysis in the upper dose exposure group included embryos that showed monoallelic (black circles) or biallelic (red circles) expression of the Igf2 gene. Sample sizes analyzed in each exposure group are indicated with a = P<0.05 and b = P<0.01. Black horizontal line indicates average methylation level in each exposure group.

Figure 7. Impact of BPA exposure is shown as number of genes exhibiting imprinting perturbations.

Effects of BPA exposure were more significant in the (B) placentas compared to (A) embryos. For each exposure group (control [blue], lower dose [pink] or upper dose [red]), percentage of tissues showing LOI (Y-axis) of at least 1 gene (≥1), 2 genes (≥2) or 3 genes (≥3) is indicated; a = P<0.05; b = P<0.01; c = P<0.001.

Figure 8. BPA exposure reduced genome-wide DNA methylation in the placenta but not the embryo.

Results of LUMA studies showed genome-wide DNA methylation levels in the (A) embryos and (B) placentas from control, lower dose and upper dose BPA exposure groups. Black circles indicate tissues with normal, monoallelic expression of all imprinted genes tested; red circles are tissues that showed LOI of at least 1 imprinted gene. Black horizontal lines represent average total methylation in each exposure group; a = P<0.05.

Figure 9. BPA exposure was associated with defective placental development.

Hematoxylin and eosin-stained histological sections of representative E12.5 conceptuses from (A) control and (B) upper dose BPA exposure groups are shown. F = fetus; DE = maternal decidua; L = labyrinth zone and JZ = junctional zone. Dotted line indicates the boundary between JZ and L, continuous line the boundary between JZ and DE, and arrows the accumulation of red blood cells. Total placenta area (C) and the ratio of area occupied by the labyrinth zone and total placenta area (D) were measured in both control (blue) and upper dose BPA exposed (red) placentas; a = P<0.05; b = P<0.001.