Role of estrogen receptor beta in uterine stroma and epithelium: Insights from estrogen receptor beta-/- mice

Abstract

In this study, we compared the uterine tissue of estrogen receptor (ER)beta(-/-) mice and their WT littermates for differences in morphology, proliferation [the percentage of labeled cells 2 h after BrdUrd injection and EGF receptor (EGFR) expression], and differentiation (expression of progesterone receptor, E-cadherin, and cytokeratins). In ovariectomized mice, progesterone receptor expression in the uterine epithelium was similar in WT and ERbeta(-/-) mice, but E-cadherin and cytokeratin 18 expression was lower in ERbeta(-/-) mice. The percentage of cells in S phase was 1.5% in WT mice and 8% in ERbeta(-/-) mice. Sixteen hours after injection of 17beta-estradiol (E(2)), the number of BrdUrd-labeled cells increased 20-fold in WT mice and 80-fold in ERbeta(-/-) mice. Although ERalpha was abundant in intact mice, after ovariectomy, ERalpha could not be detected in the luminal epithelium of either WT or ERbeta(-/-) mice. In both untreated and E(2)-treated mice, ERalpha and ERbeta were colocalized in the nuclei of many stromal and glandular epithelial cells. However, upon E(2) + progesterone treatment, ERalpha and ERbeta were not coexpressed in any cells. In WT mice, EGFR was located on the membranes and in the cytoplasm of luminal epithelium, but not in the stroma. In ERbeta(-/-) mice, there was a marked expression of EGFR in the nuclei of epithelial and stromal cells. Upon E(2) treatment, EGFR on cell membranes was down-regulated in WT but not in ERbeta(-/-) mice. These findings reveal an important role for ERbeta in response to E(2) and in the organization, growth, and differentiation of the uterine epithelium.

Fig. 1.

Immunohistochemical detection of E-cadherin. Nine-week-old mice received a single s.c. injection of 150 μl of olive oil containing no hormone (A and D), 100 ng of E₂ (B and E), or 100 ng of E₂ + 1 mg of P₄ (C and F), and 16 h later, expression of E-cadherin in the uterus was examined. As expected, E-cadherin immunoreactivity was confined to the epithelial compartment. Its expression was markedly lower in vehicle-treated ERβ^−/− than in WT mice (A and D). (Insets) Outlined areas at 4 times greater magnification. Treatment with E₂ restored E-cadherin levels in ERβ^−/− mice.

Fig. 2.

Detection of S-phase cells in uteri of WT and ERβ^−/− littermates. Sixteen hours before being killed, mice received a single injection of vehicle (A and C) or 100 ng of E₂ (B and D). Two hours before being killed, mice were injected i.p. with BrdUrd at 30 mg/kg. In vehicle-treated WT mice, 1.5% of epithelial cells were labeled, whereas 8% were labeled in ERβ^−/− mice. After administration of E₂, 23% of luminal epithelial cells were labeled in WT mice, and 82% were labeled in ERβ^−/− mice (B, D, and E).

Fig. 3.

Immunohistochemical detection of CK18 (epithelial marker) and CK13 (uterine cervical marker) in the uteri of WT and ERβ^−/− littermates. Mice received a single injection with vehicle (A, D, G, and J), 100 ng of E₂ (B, E, H, and K), or 100 ng of E₂ + 1 mg of P₄ (C, F, I, and L). The differentiation marker CK18 is confined to luminal and glandular cells in the uteri of WT (A–C) and ERβ^−/− mice (D–F). In vehicle-treated mice, CK18 expression was lower in ERβ^−/− than in WT mice (A and D). Treatment with E₂ or E₂ + P₄ resulted in reduction of CK18 expression in WT mice (B and C) but complete loss CK18 in ERβ^−/− mice (E and F). Expression of CK13 in the uterine cervix was similar in WT (G–I) and ERβ^−/− mice (J–L).

Fig. 4.

Colocalization of ERα and ERβ in uteri of WT mice. In intact WT mice, ERα, as expected, was abundantly expressed in both epithelium and stroma (M). However, after ovariectomy, ERα could not be detected in the luminal epithelium of either WT or ERβ^−/− mice (G–L). Ovariectomized mice received a single injection of vehicle (A, D, G, and J), 100 ng of E₂ (B, E, H, and K), or 100 ng of E₂ + 1 mg of P₄ (C, F, I, and L). Colocalization of ERα (green) and ERβ (red) in the stroma was seldom seen in mice treated with vehicle or E₂ + P₄ (A–C) but was seen in mice treated with E₂ alone. In the glandular epithelium (D–F) receptors were colocalized (yellow and arrows) in vehicle-treated (D) and E₂-treated (E, arrows) mice. Upon administration of E₂ + P₄ (F), expression of ERβ was extinguished, whereas ERα expression remained.

Fig. 5.

Immunohistochemical detection of PR. PR was abundant in the luminal and glandular epithelium of both WT and ERβ^−/− mice. Stromal PR immunoreactivity was faint in vehicle-treated WT and ERβ^−/− mice (A, D, and G). It was markedly induced in both WT and ERβ^−/− mice by the administration of 100 ng of E₂ (B, E, and G), or 100 ng of E₂ + 1 mg of P₄ (C, F, and G).

Fig. 6.

Immunohistochemical analysis of EGFR protein expression. Mice received a single injection of vehicle (A and D), 100 ng of E₂ (B and E), or 100 ng of E₂ + 1 mg of P₄ (C and F). In vehicle-treated mice, there was strong membrane EGFR immunoreactivity in the luminal epithelium of WT mice (A) but no membrane or cytoplasmic staining in ERβ^−/− mice (D). E₂ treatment resulted in enhanced EGFR expression in the cytoplasm of WT mice (B) and increased nuclear staining in ERβ^−/− mice. In ERβ^−/− mice treated with E₂ + P₄, there was a marked increase in nuclear localization of EGFR in the stroma (F), whereas in WT mice, this treatment resulted in nuclear localization of EGFR in the epithelium but not in the stroma. (Insets) Outlined areas at 3 times greater magnification.