2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits fibroblast growth factor 10-induced prostatic bud formation in mouse urogenital sinus

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dorsalizes the pattern of prostatic buds developing from the urogenital sinus (UGS) of male fetal mice, causing some buds to form in inappropriate positions while blocking formation of others. This teratogenic TCDD action significantly reduces prostate main duct number and causes ventral prostate agenesis in exposed males. The purpose of this study was to determine whether inhibition of fibroblast growth factor 10 (FGF10) signaling is mechanistically linked to mouse prostatic budding impairment by TCDD. In utero TCDD exposure induced aryl hydrocarbon receptor-responsive cytochrome P450 1b1 messenger RNA (mRNA) in ventral UGS regions where Fgf10 and fibroblast growth factor receptor 2 (Fgfr2) mRNA were expressed and where budding was most severely inhibited by TCDD. However, TCDD exposure did not reduce Fgf10 or Fgfr2 mRNA abundance in the UGS or alter their distribution. Addition of FGF10 protein to UGS organ culture media increased the abundance of UGS basal epithelial cells immunopositive for phosphorylated extracellular signal-regulated kinase (ERK). FGF10 also increased the number of 5-bromo-2'-deoxyuridine (BrdU)-labeled UGS epithelial cells and increased the number of prostatic buds formed per UGS. Addition of TCDD to UGS organ culture media did not alter FGF10-induced ERK activation in UGS basal epithelium but prevented FGF10-induced BrdU incorporation and blocked FGF10-induced prostatic bud formation. These results identify basal urogenital sinus epithelium cells as the key site of FGF10 action during fetal prostate development and suggest that TCDD likely acts downstream of FGFR2 and ERK to restrict UGS epithelial cell proliferation and prevent prostatic bud formation.

FIG. 1.

In utero TCDD exposure increases Cyp1b1 mRNA abundance in ventral UGM but does not alter Fgf10 mRNA abundance or distribution in this region. Male fetal mice were exposed in utero to corn oil (vehicle) or TCDD (5 μg/kg maternal dose) on E15.5. (A) Cyp1b1 and Fgf10 mRNA abundance was determined by real-time RT-PCR on E16.5 and normalized to Ppia mRNA abundance. Results are mean ± SE of n ≥ 3 independent samples per group from at least three separate litters. Significant differences between groups (p < 0.05) are indicated by an asterisk. (B) ISH was used to visualize the pattern of AHR-responsive Cyp1b1 mRNA expression and Fgf10 expression in sagittal UGS sections on E16.5. Dashed lines were added to demarcate the boundary between UGE and UGM, thin gray lines were added to show the outside edge of each UGS section, and a thin black oval indicates the approximate position of the VMP. BL. bladder; E, UGE; M, UGM; and PU, pelvic urethra.

FIG. 2.

Recombinant FGF10 protein significantly increases prostatic budding in UGS organ culture, and TCDD blocks this effect. UGS from E14.5 male C57BL/6J mouse fetuses were incubated for 3 days in organ culture medium containing vehicle, recombinant FGF10 protein (300 ng/ml), TCDD (1nM), or FGF10 + TCDD. Media and supplements were replenished after the second day of culture. At the end of the incubation, UGE was separated from UGM and visualized by scanning electron microscopy. (A) Micrographs of one UGE from each exposure group are shown. Prostatic buds are pseudocolored red. (B) Prostatic buds were counted using micrographs of each UGE that were taken from four separate angles in order to ensure that all prostatic buds present were counted. Results for the total number of prostatic buds per UGS are mean ± SE of n ≥ 6 independent samples per group from at least three separate litters. The presence of an asterisk indicates a mean that is significantly different from the vehicle-treated group (control, p < 0.05). The presence of a dagger indicates it is significantly different from 1nM TCDD (control, p < 0.05).

FIG. 3.

In utero TCDD exposure does not alter Fgfr2 mRNA abundance or distribution in UGE. Male fetal mice were exposed in utero to corn oil (vehicle) or TCDD (5 μg/kg maternal dose) on E15.5. (A) Fgfr2 mRNA abundance was determined by real-time RT-PCR on E16.5 and normalized to Ppia mRNA abundance. Results are mean ± SE of n ≥ 3 independent samples per group from at least three separate litters. (B) ISH was used to visualize the pattern of Fgfr2 mRNA expression in midsagittal UGS sections on E16.5. Dashed lines were added to demarcate the boundary between UGE and UGM. Thin gray lines were added to show the outside edge of the UGS sections. BL, bladder; E, UGE; M, UGM; and PU, pelvic urethra.

FIG. 4.

In utero TCDD exposure significantly decreases ERK activation in UGE cells. Male fetal mice were exposed in utero to corn oil (vehicle) or TCDD (5 μg/kg maternal dose) on E15.5. (A) IHC was used to visualize the pattern of activated phosphorylated ERK protein expression (red) in sagittal UGS sections on E16.5. Results are representative of at least three UGSs per group. Dashed lines were added to demarcate the boundary between UGE and UGM. BL, bladder; E, UGE; M, UGM; and PU, pelvic urethra; S, sinus space. (B) The percentage of ventral UGE (vUGE) cells expressing active phosphorylated ERK (red) was determined in the ventral prostatic budding region (inside black rectangle) by counting separately all the vUGE cells and all the red-stained vUGE cells on three sections per UGS. Results are mean ± SE of n ≥ 3 UGSs per group, each from a different litter. Significant differences among groups (p < 0.05) are indicated by an asterisk.

FIG. 5.

TCDD does not prevent ERK activation by recombinant FGF10 protein in the prostatic budding region of cultured UGS. UGS from E14.5 male C57BL/6J mouse fetuses were incubated for 3 days in organ culture medium containing vehicle, recombinant FGF10 protein (300 ng/ml), TCDD (1nM), or FGF10 + TCDD. (A) IHC was then used to visualize the pattern of activated phosphorylated ERK protein expression (red) in the prostatic budding region of sagittal UGS sections. Dashed lines demarcate the boundary between UGE and UGM in the prostatic budding region. E, UGE; M, UGM; PU, pelvic urethra; BL, bladder; and S, sinus space. (B) The percentage of UGE cells expressing active phosphorylated ERK (red) was determined in the prostatic budding region by counting all the UGE cells and all the red-stained UGE cells in a six-cell-thick layer of UGE cells adjacent to the UGE-UGM interface (dashed line). Three sections per UGS were analyzed. Results are mean ± SE of n ≥ 3 UGSs per group, each from a different litter. Significant differences among groups (p < 0.05) are indicated by an asterisk.

FIG. 6.

TCDD inhibits FGF10-dependent cell proliferation in UGS organ culture. UGS from E15.5 male C57BL/6J mouse fetuses were incubated for 4 h in organ culture medium containing vehicle or 1nM TCDD. Vehicle or 300 ng/ml recombinant FGF10 protein was then added to organ culture medium, and tissues were cultured for an additional 16 h. BrdU was added to the culture medium after 12 h. (A) Sections from cultured UGSs were then examined by IHC and (B) BrdU-positive UGE cells (red) were quantified as a percentage of the total number of prostatic UGE cells (nuclei are blue) in tissue sections from three UGSs per treatment group. Three sections per UGS were examined. Dashed lines were added to demarcate the boundary between UGE and UGM in the UGS proper. The percentage of BrdU-positive UGE cells was determined in the UGE region circumscribed by these dashed lines. E, UGE; M, UGM; PU, pelvic urethra; and S, sinus space. Results are mean ± SE of n ≥ 3 UGSs per group, each from a different litter. Significant differences among groups (p < 0.05) are indicated by an asterisk.