Estrogen receptor-alpha expression in the mammary epithelium is required for ductal and alveolar morphogenesis in mice

Abstract

The estrogen receptor-alpha (ERalpha) is a critical transcription factor that regulates epithelial cell proliferation and ductal morphogenesis during postnatal mammary gland development. Tissue recombination and transplantation studies using the first generation of ERalpha knockout (ERKO) mice suggested that this steroid hormone receptor is required in the mammary stroma that subsequently exerts its effect on the epithelium through additional paracrine signaling events. A more detailed analysis revealed that ERKO mice produce a truncated ERalpha protein with detectable transactivation activity, and it is likely that this functional ERalpha variant has masked the biological significance of this steroid receptor in the mammary epithelium. In this article, we describe the generation a Cre-lox-based conditional knockout of the ERalpha gene to study the biological function of this steroid receptor in the epithelial compartment at defined stages of mammary gland development. The mouse mammary tumor virus (MMTV)-Cre-mediated, epithelial-specific ablation of exon 3 of the ERalpha gene in virgin mice severely impaired ductal elongation and side branching. The conditional knockout resulted in ablation of the ERalpha protein, and the progesterone receptor (PR), whose expression is under the control of ERalpha, was largely absent. The whey acidic protein (WAP)-Cre-mediated deletion of ERalpha during successive gestation cycles resulted in a loss of ductal side-branching and lobuloalveolar structures, ductal dilation, and decreased proliferation of alveolar progenitors. These abnormalities compromised milk production and led to malnourishment of the offspring by the second lactation. These observations suggest that ERalpha expression in the mammary epithelium is essential for normal ductal morphogenesis during puberty and alveologenesis during pregnancy and lactation.

Fig. 1.

Targeted and conditional disruption of the mouse ERα gene, using the Cre-loxP recombination system. (A) Components of the ERα exon 3 WT allele, the targeted allele after homologous recombination in ES cells, the floxed allele after deletion of the neomycin resistance gene (pGK-Neo) in ES cells, and the deleted ERα allele in Cre-recombinase transgenic mice. B, BamHI; N, NheI; E, Eco47III; H, HpaI; P, PmlI. (B) Genotyping PCR of tail clips of MMTV-Cre/ERα^fl/fl mice (lane 1), virgin ERα^fl/fl mice (lane 2), and WAP-Cre/ERα^fl/fl mice (lane 3). Lane 4 contains extract of a mammary gland from a parous WAP-Cre/ERα^fl/fl mouse. The MMTV promoter is active in skin, explaining the presence of the KO allele in the tail clip in lane 1, whereas WAP is specific to the mammary gland during late pregnancy, accounting for the 0.60-kb knockout allele in lane 4 but not lane 3.

Fig. 2.

ERα is required for mammary gland development. Whole mounts of mammary glands (A–D) and H&E staining (E and F) from mature virgin MMTV-ERKO mice (A, C, and E) and ERα^fl/fl mice (B, D, and F). In ERα^fl/fl, but not MMTV-ERKO mice, the no. 4 inguinal mammary fatpad was fully occupied by the developed epithelial network.

Fig. 3.

Dilation and inadequate branching of ducts in WAP-ERKO mice during second lactation. Whole mounts (A and C) and H&E staining (B and D) were used to analyze the morphology of the mammary glands at second lactation day 1 of the WAP-ERKO females (C and D) and ERα^fl/fl controls (A and B). Note different magnifications in B and D, emphasizing grossly enlarged ducts in mutant mice.

Fig. 4.

Growth retardation in WAP-ERKO pups from the second litter is attributable to mother's genotype. (A) WAP-Cre/ERα^fl/fl pups nursed by their WAP-ERKO mothers (WAP^fl/fl–WAP^fl/fl, n = 5) were significantly smaller than control ERα^fl/fl pups nursed by ERα^fl/fl mothers (fl/fl–fl/fl). Beginning on day 4 of lactation, a subset of WAP-Cre/ERα^fl/fl pups was fostered with lactating ERα^fl/fl mothers (WAP fl/fl-fl/fl). After 4 days of fostering by an ERα^fl/fl dam (lactation day 8), there was no longer a statistically significant difference between WAP-Cre/ERα^fl/fl and ERα^fl/fl pup mass (n = 4). Data are presented as the mean ± 1 standard deviation. (B) Pairs of representative pups nursed for 10 days by birth mother or for 4 days by birth mother followed by 6 days of nursing by foster dam as in A.

Fig. 5.

Loss of ERα and PR immunohistochemical staining in WAP-ERKO (WAP fl/fl) but not ERα^fl/fl (fl/fl) mammary glands of the second lactation, day 1. Nuclei of multiple luminal epithelial cells in control mammary glands were positive for ERα and PR (C and D), which were absent in the enlarged ducts of WAP-ERKO mice (A and B).