Estrogen receptor-α mediates diethylstilbestrol-induced feminization of the seminal vesicle in male mice

Abstract

Background: Studies have shown that perinatal exposure to the synthetic estrogen diethylstilbestrol (DES) leads to feminization of the seminal vesicle (SV) in male mice, as illustrated by tissue hyperplasia, ectopic expression of the major estrogen-inducible uterine secretory protein lactoferrin (LF), and reduced expression of SV secretory protein IV (SVS IV).

Objectives: The present study was designed to evaluate the role of the estrogen receptor (ER) in this action by using ER-knockout (ERKO) mice.

Methods: Wild-type (WT), ERα-null (αERKO), and ERβ-null (βERKO) male mice were treated with either vehicle or DES (2 μg/day) on neonatal days 1-5. These mice were divided into two groups: In the first group, intact mice were sacrificed at 10 weeks of age; in the second group, mice were castrated at 10 weeks of age, allowed to recover for 10 days, treated with dihydrotestosterone (DHT) or placebo, and sacrificed 2 weeks later. Body weights and SV weights were recorded, and mRNA expression levels of Ltf (lactoferrin), Svs4, and androgen receptor (Ar) were assessed.

Results: In DES-treated intact mice, SV weights were reduced in WT and βERKO mice but not in αERKO mice. DES-treated WT and βERKO males, but not αERKO males, exhibited ectopic expression of LF in the SV. DES treatment resulted in decreased SVS IV protein and mRNA expression in WT males, but no effect was seen in αERKO mice. In addition, DES-treated βERKO mice exhibited reduced Svs4 mRNA expression but maintained control levels of SVS IV protein. In DES-treated castrated mice, DHT implants restored SV weights to normal levels in αERKO mice but not in WT mice, suggesting full androgen responsiveness in αERKO mice.

Conclusions: These data suggest that DES-induced SV toxicity and feminization are primarily mediated by ERα; however, some aspects of androgen response may require the action of ERβ.

Figure 1

SV weight (wt) measured at 10 weeks of age in intact male WT, αERKO, and βERKO mice treated neonatally with vehicle (control) or DES (2 μg/day on days 1–5). Treatment groups: WT control, n = 19; WT DES, n = 18; αERKO control, n = 11; αERKO DES, n = 20; βERKO control, n = 14; βERKO DES, n = 20). Data are mean ± SE. *p < 0.05 compared with the corresponding control, by parametric test.

Figure 2

Pathology of SVs of male WT (A,B), αERKO (C,D), and βERKO (E,F) mice treated neonatally with vehicle (control; A,C,E) or DES (2 μg/day on days 1–5; B,D,F). SVs are stained with hematoxylin and eosin. Bar = 100 µm.

Figure 3

SVS IV expression in SVs of adult intact male mice (all three genotypes) treated neonatally on days 1–5 with vehicle (control) or 2 μg/day DES. Treatment groups: WT control, n = 12; WT DES, n = 12; αERKO control, n = 7; αERKO DES, n = 15); βERKO control, n = 8; βERKO DES, n = 15). (A) Svs4 expression in the SV by real-time RT-PCR; expression levels were normalized to the Ppia housekeeping gene. (B) SVS IV protein expression in SVs; Equal protein loading was confirmed with MEMCode total protein stain before immunoblotting. *p < 0.05 compared with the corresponding control, by ANOVA followed by Tukey’s test.

Figure 4

SV weight (wt) of male mice (WT and αERKO) treated neonatally on days 1–5 with vehicle (control) or 2 μg/day DES, castrated at 10 weeks of age, and exposed to DHT in Silastic tubing or Silastic tubing alone for an additional 2 weeks. Treatment groups: without (–) DHT: WT control, n = 11; WT DES, n = 6; αERKO control, n = 4; αERKO DES, n = 10; with (+) DHT: WT control + DHT, n = 8; WT DES + DHT, n = 6; αERKO control + DHT, n = 7; αERKO DES + DHT, n = 8). *p < 0.05 compared with the corresponding control, by parametric tests.

Figure 5

LF expression in SVs of adult intact male mice (all three genotypes) treated neonatally on days 1–5 with vehicle (control) or 2 μg/day DES. Treatment groups: WT control, n = 12; WT DES, n = 12; αERKO vehicle, n = 7; αERKO DES, n = 15; βERKO control, n = 8; βERKO DES, n = 15. (A) Ltf expression in the SV by real-time RT-PCR. All expression levels were normalized to the Ppia housekeeping gene. (B) LF protein expression in SVs; equal protein loading was confirmed with MEMCode total protein stain before immunoblotting (data not shown). *p< 0.05 compared with the corresponding control, by ANOVA followed by Tukey’s test.