Varying Susceptibility of the Female Mammary Gland to In Utero Windows of BPA Exposure

Abstract

In utero exposure to the endocrine disrupting compound bisphenol A (BPA) is known to disrupt mammary gland development and increase tumor susceptibility in rodents. It is unclear whether different periods of in utero development might be more susceptible to BPA exposure. We exposed pregnant CD-1 mice to BPA at different times during gestation that correspond to specific milestones of in utero mammary gland development. The mammary glands of early-life and adult female mice, exposed in utero to BPA, were morphologically and molecularly (estrogen receptor-α and Ki67) evaluated for developmental abnormalities. We found that BPA treatment occurring before mammary bud invasion into the mesenchyme [embryonic day (E)12.5] incompletely resulted in the measured phenotypes of mammary gland defects. Exposing mice up to the point at which the epithelium extends into the precursor fat pad (E16.5) resulted in a nearly complete BPA phenotype and exposure during epithelial extension (E15.5 to E18.5) resulted in a partial phenotype. Furthermore, the relative differences in phenotypes between exposure windows highlight the substantial correlations between early-life molecular changes (estrogen receptor-α and Ki67) in the stroma and the epithelial elongation defects in mammary development. These data further implicate BPA action in the stroma as a critical mediator of epithelial phenotypes.

Figure 1.

ERα expression is strictly mesenchymal throughout perinatal murine mammary gland development. Immunohistochemistry at representative time points of fetal ERα expression at E13.5, E16.5, and E18.5 of in utero mice treated with oil control and BPA. Arrowheads indicate exterior of the fetus and origin of invaginating epithelial cells from the epidermis, nipple sheath. At no embryonic time point was ERα expressed in the mammary epithelium. Scale bars at ×20 magnification and ×10 magnification = 100 μM.

Figure 2.

BPA causes early-life defects in epithelial elongation within the mammary gland. (a) Schematic time line of varied in utero treatment model with oil control or windows of in utero treatment with 25 μg/kg body weight BPA daily, beginning at E8.5 and varied through E18.5. Dissection light-microscope images representative of mammary glands from 4.5-week-old mice were harvested and whole mounts stained with carmine aluminum. (b) Epithelial elongation indicated by direction of growth plus or minus from the leading edge of the lymph node (dashed line) to the most distal rudiment of the epithelial tree (solid line). Graph shows the comparison of epithelial elongation in millimeters between treatments; each dot represents separate biological replicates (n ≥ 7). (c) The numbers of TDEs are indicated by white and black arrowheads combined, and the numbers of TEBs meeting a size threshold of ≥150 pixels in width are indicated by just black arrowheads. (d) The number of TDEs, including TEBs, was quantified among the various BPA treatments. (e) Plot of the number of TEBs normalized to the total epithelial area. Each dot represents a separate biological replicate (n ≥ 7). Open circles indicate the carmine-stained mammary gland image representative of each treatment mean in (b) and (c). *P < 0.05.

Figure 3.

Early-life epithelial dysfunction of the mammary gland by in utero BPA significantly correlated to the stromal compartment. Representative IHC of an oil-treated in utero mammary gland from 4.5-week-old mice, dual-stained with SMA and (a) ERα or (b) Ki67 (magnification ×20). (Inset) The same oil-treated specimen to show tissue compartment segmentation by InForm Advanced Image Analysis Software used to differentiate the stromal (red) and epithelial (green) tissue compartments. The in utero treatment windows with BPA were derived similarly, as indicated. Graph of (a) ERα or (b) Ki67 positivity in the (left) stroma and (right) epithelium of TDEs, including the TEBs. Each dot represents separate biological replicates (n ≥ 7), and open circles indicate representative images of the mean shown for each treatment. Correlation of epithelial elongation across treatments [Fig. 2(b)] to the (c) stromal compartment surrounding the TDEs [(left) ERα and (right) Ki67 positivity] and (d) epithelial compartment of TDEs [(left) ERα and (right) Ki67 positivity]. *P < 0.05; **P < 0.001. Dotted lines indicate 95% confidence intervals.

Figure 4.

In utero BPA changes later-life ERα expression of mammary tissue compartments. Representative IHC of oil and varied with BPA-treated in utero mammary glands from adult 14-week-old mice, dual-stained (as in Fig. 3) with SMA and ERα focused on alveolar structures (magnification ×20). Graphs present percentage of ERα positivity quantified in each tissue compartment of the alveolar structures of the epithelium (left) and surrounding stroma (right). Each dot represents separate biological replicates (n ≥ 4), and open circles indicate representative images for each treatment. *P < 0.05.

Figure 5.

In utero BPA significantly alters the density and complexity of the mammary epithelium. Epithelium quantitation shown by color thresholding in ImageJ as pixels in epithelium as a percentage of total pixels in field of view. Representative dissection light-microscope images of carmine-stained mammary gland whole mounts from adult mice at (a) 14 weeks old and (b) 20 weeks old treated with oil and varied in utero BPA windows (as described in Fig. 2). Graphs present percentage of area of epithelium as quantified by ImageJ. Each dot represents separate biological replicates: (a) n ≥ 9 and (b) n ≥ 3. Open circles indicate mammary gland representing that treatment, as shown. *P < 0.05.