Estrogen receptor alpha and imprinting of the neonatal mouse ventral prostate by estrogen

Abstract

Exposure to estrogen in the neonatal period affects prostatic growth and leads to an increased incidence of prostatic intraepithelial neoplasia in later life. The effects of neonatal estrogen are clearly dependent on estrogen receptor (ER) alpha because they do not occur in ERalpha-knockout mice. Because ERalpha is expressed in the stroma, but not in the epithelium, of the adult ventral prostate, the concept of indirect estrogen effects through stromal signaling has been proposed. Here, we show that during the first 4 weeks of life, there are profound and rapid changes in the ER profile in the mouse ventral prostate. ERalpha is abundant in the stroma during week 1, but by week 2 it is exclusively epithelial, and then by week 4, ERalpha is lost and ERbeta is dominant in the prostatic epithelium. The presence of ERalpha is associated with a high proliferation index, and ERbeta is associated with quiescence. Branching morphogenesis was altered in ERalpha-/-, but not in ERbeta-/-, mice. We conclude that imprinting and branching morphogenesis of the ventral prostate are mediated by estrogen acting directly on epithelial and stromal ERalpha during the first 2 weeks of life.

Fig. 1.

Expression of nuclear receptors and cell proliferation in neonatal VP. (A) Immunohistochemical staining of ERα,ERβ, and AR in neonatal VP. VPs of WT mice aged 1, 2, 3, 4, and 5 weeks were collected. ERα staining was observed in the nuclei of epithelial cells from 2- and 3-week-old mice (black arrow) but not in 1-week-old mice or in mice older than 4 weeks. ERα staining also could be seen in a certain number of stromal cells in all samples, particularly in those from 1-week-old mice (red arrow). ERβ staining was seen in epithelial cells of mice older than 2 weeks. AR staining was observed in nuclei of ductal epithelial cells of mice of all ages studied; only 1-week-old mice showed positive staining in the stroma (red arrow). Antibodies and dilutions used are as follows: anti-ERα MC-20 (1:120), anti-ERβ (1:100), and anti-AR N-20 (1:300). (B and C) Examination of cell proliferation in neonatal VP. At the age of 1, 2, and 4 weeks, WT mice were killed 2 h after s.c. injection of BrdUrd (100 mg/kg). (B) Dissected VP was fixed and sectioned for immunostaining with an anti-BrdUrd antibody. Red filled arrows show positive staining in epithelial cells. Red open arrows show positive staining in stroma. Many proliferating cells in stroma can be seen in 1-week-old mice, whereas almost no staining can be observed after 2 weeks of age. (C) The BrdUrd staining ratio in epithelial cells was 12.4 ± 2.5% in 1-week-old mice, 8.1 ± 1.5% in 2-week-old mice, and 5.0 ± 2.0% in 4-week-old mice.

Fig. 2.

Expression of ERα in VP of 2-week-old mice. (A)ERα mRNA detection by RT-PCR. RNA was prepared from uterus, ovary, and VP of 2-month-old female (n = 2) and male (n = 2) mice. RNA was extracted from pooled AP and VP from three 2-week-old mice. RT-PCR conditions are described in Materials and Methods. M, DNA marker; Ut, uterus; Ov, ovary. (B) Immunohistochemical staining of ERα in an ERα^-/- mouse. No positive signal was detectable in 2-week-old ERα^-/- VP. (C) Specificity of the ERα antibody. The positive staining obtained with the ERα antibody (Left) was completely extinguished after adsorption of ERα antibody with high salt extracts from adult uterus (Right). (D)ERα detection by Western blotting. The fractions corresponding to the 4S and 8S peaks obtained by sucrose gradients were used for Western blotting. Both 2-week-old VP and 8-month-old uterus showed a specific ERα band. The antibody used was anti-ERα MC-20 (1:1,000 dilution).

Fig. 3.

Effect of 3βAdiol on cell proliferation and AR in VP. (A) The effect of 3βAdiol on proliferation in the neonatal VP. Two-week-old WT mice were treated with 3βAdiol and killed 2 h after s.c. injection of BrdUrd (100 mg/kg). Dissected VP was fixed and sectioned for immunostaining with anti-BrdUrd antibody. The percentage of BrdUrd-positive cells in the epithelium was 2.32 ± 0.65% in WT mice and was increased 2-fold to 4.43 ± 1.34% after treatment with 3βAdiol. (B) Immunohistochemical staining for AR in 2-week-old ERα^-/-, CYP7B1^-/-, and WT mice. Intensity of AR staining in the epithelial cells of CYP7B1^-/- mice was higher than it was in WT mice. Antibodies used were anti-AR N-20 (1:300 dilution).

Fig. 4.

Role of ER in morphology of the VP. The VPs of 4-week-old ERα^-/- and WT mice and 9-week-old ERα^-/-,ERβ^-/-, and WT mice were examined under a light microscope. (A–D) VP from 4-week-old ERα^-/- and WT littermates. (A) The lobes of VP were dissected and photographed under a dissecting microscope. (B) A view of the whole ductal tree from WT and ERα^-/- mice. VPs were separated into two parts, digested with collagenase, and examined for branching morphology. (Scale bars: 500 μm.) (C) Higher magnification to show primary branches from WT and ERα^-/- mice with tracing of the primary and secondary branches shown in red. (D) The total number of primary and secondary branches in WT and ERα^-/- mice were counted and compared. In ERα^-/- mice there was significantly less branching than WT mice. (E and F)VP from 9-week-old ERα^-/- mice and WT littermates. (E) VPs were dissected, separated into two lobes, and photographed under a dissecting microscope. (F) Branching morphology of WT and ERα^-/- mice. (G and H) VP from 9-week-old ERβ^-/- and WT littermate mice. (G) VPs were dissected, and pictures were taken under microscope. (H) Branching morphology of WT and ERβ^-/- mice.

Fig. 5.

Summary of nuclear receptor expression and proliferation index in the epithelium and stroma of VP from 1- to 5-week-old mice. In epithelial cells, ERα is dominant at 2–3 weeks of age and disappears after 4 weeks. ERβ and AR are detectable from 2 weeks of age and dominate by 4 weeks. In the stroma, ERα and AR, but not ERβ, are already present in 1-week-old mice. After 2 weeks of age, AR is lost and expression of ERα is reduced. ERβ is more abundant in stroma after 2 weeks of age.