Postnatal mammary ductal growth: three-dimensional imaging of cell proliferation,effects of estrogen treatment,and expression of steroid receptors in prepubertal calves

Cows may provide insights into mammary development that are not easily obtained using mouse models. Mammary growth in control and estrogen-treated calves was investigated to evaluate general patterns of proliferation and relationship to estrogen receptor (ER) expression. After in vivo labeling with bromodeoxyuridine (BrdU), serial histological sections of mammary tissue were used to generate three-dimensional reconstructions. BrdU-labeled cells were present throughout the highly branched terminal ducts. ER and progesterone receptors (PR) were colocalized in nuclei of ductal epithelial cells. However, basal cells and epithelial cells that were located in the central region of epithelial cords and those that lined the lumen of patent ducts were ER- and PR-negative, as were stromal cells. Cells along the basal portion of the epithelium were not myoepithelial. ER in mammary epithelial cells but not stromal cells is analogous to patterns in human breast but contrasts with localization in murine mammary gland. After estrogen stimulation, 99% of BrdU-labeled (and Ki67-labeled) epithelial cells were ER-negative. Data suggest that proliferation in response to estrogen treatment was initiated within ER-positive epithelial cells of the developing mammary gland and the signal was propagated in paracrine fashion to stromal elements and ER-negative epithelial cells.     

In situ localization of progesterone receptors in normal mouse mammary glands: Absence of receptors in the connective and adipose stroma and a heterogeneous distribution in the epithelium

In normal mammary glands of both rodents and humans, progesterone promotes the proliferation of epithelial cells and several lines of evidence suggest that this action of progesterone may be mediated by progesterone receptor (PR). It is well established that normal mammary development involves a complex interplay between the epithelial cells and the surrounding fatty stroma. Furthermore, during mammary development, there is a change in both the relative proportion of epithelial cells and the steady-state levels of PR. Therefore, towards understanding the precise role of PR in mammary development, we have generated a highly sensitive antibody against mouse PR and examined its pattern of localization. Immunoreactive PR was detected only in the epithelial cells of the ducts while both the adipose and fibrous stroma surrounding these ducts were receptor negative. Similarly, PR mRNA was also associated only with the ductal epithelial cells. Approximately only 45–50% of the ductal cells were receptor positive and this distribution remained unchanged whether or not the tissues had been exposed to estrogen, suggesting that they may represent a distinct subpopulation. The potential significance of these findings to mammary development is discussed.     

Vitamin D(3) receptor ablation alters mammary gland morphogenesis

Postnatal mammary gland morphogenesis is achieved through coordination of signaling networks in both the epithelial and stromal cells of the developing gland. While the major proliferative hormones driving pubertal mammary gland development are estrogen and progesterone, studies in transgenic and knockout mice have successfully identified other steroid and peptide hormones that impact on mammary gland development. The vitamin D(3) receptor (VDR), whose ligand 1,25-dihydroxyvitamin D(3) is the biologically active form of vitamin D(3), has been implicated in control of differentiation, cell cycle and apoptosis of mammary cells in culture, but little is known about the physiological relevance of the vitamin D(3) endocrine system in the developing gland. In these studies, we report the expression of the VDR in epithelial cells of the terminal end bud and subtending ducts, in stromal cells and in a subset of lymphocytes within the lymph node. In the terminal end bud, a distinct gradient of VDR expression is observed, with weak VDR staining in proliferative populations and strong VDR staining in differentiated populations. The role of the VDR in ductal morphogenesis was examined in Vdr knockout mice fed high dietary Ca(2+) which normalizes fertility, serum estrogen and neonatal growth. Our results indicate that mammary glands from virgin Vdr knockout mice are heavier and exhibit enhanced growth, as evidenced by higher numbers of terminal end buds, greater ductal outgrowth and enhanced secondary branch points, compared with glands from age- and weight-matched wild-type mice. In addition, glands from Vdr knockout mice exhibit enhanced growth in response to exogenous estrogen and progesterone, both in vivo and in organ culture, compared with glands from wild-type mice. Our data provide the first in vivo evidence that 1,25-dihydroxyvitamin D(3) and the VDR impact on ductal elongation and branching morphogenesis during pubertal development of the mammary gland. Collectively, these results suggest that the vitamin D(3) signaling pathway participates in negative growth regulation of the mammary gland.     

Identification of putative bovine mammary epithelial stem cells by their retention of labeled DNA strands

Stem cells appear to retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. In this study, proliferating cells in the prepubertal bovine mammary gland were labeled using five daily injections of 5-bromo-2-deoxyuridine (BrdU). Five weeks later, BrdU-labeled mammary epithelial cells were still evident. The percentage of BrdU-labeled epithelial cells was greatest in the lower region of the mammary gland, near the gland cistern, and was decreased toward the periphery of the parenchymal region, where the ducts were invading the mammary fat pad. Increased numbers of BrdU-labeled epithelial cells in basal regions of the gland are likely a consequence of decreased proliferation rates and increased cell cycle arrest in this area. In peripheral regions of mammary parenchyma, the percentage of heavily labeled epithelial cells averaged 0.24%, a number that is consistent with estimates of the frequency of stem cells in the mouse mammary gland. Epithelial label-retaining cells seemingly represent a slowly proliferating population of cells, as 5.4% of heavily labeled cells were positive for the nuclear proliferation antigen Ki67. Because epithelial label-retaining cells contain estrogen receptor (ER)-negative and ER-positive cells, they apparently comprise a mixed population, which I suggest is composed of ER-negative stem cells and ER-positive progenitors. Continuing studies will address the usefulness of this technique to identify bovine mammary stem cells and to facilitate studies of stem cell biology.     

Regulation of progesterone receptors and decidualization in uterine stroma of the estrogen receptor-alpha knockout mouse

Regulation of progesterone receptor (PR) in uterine stroma (endometrial stroma plus myometrium) by estrogen was investigated in estrogen receptor-alpha (ERalpha) knockout (alphaERKO) mice. 17 beta-Estradiol (E(2)) increased PR levels in uterine stroma of ovariectomized alphaERKO mice, and ICI 182 780 (ICI) inhibited this E(2)-induced PR expression. Estrogen receptor-beta(ER beta) was detected in both uterine epithelium and stroma of wild-type and alphaERKO mice by immunohistochemistry. In organ cultures of alphaERKO uterus, both E(2) and diethylstilbestrol induced stromal PR, and ICI inhibited this induction. These findings suggest that estrogen induces stromal PR via ERbeta in alphaERKO uterus. However, this process is not mediated exclusively by ERbeta+, because in ERbeta knockout mice, which express ERalpha, PR was up-regulated by E(2) in uterine stroma. In both wild-type and alphaERKO mice, progesterone and mechanical traumatization were essential and sufficient to induce decidual cells, even though E(2) and ERalpha were also required for increase in uterine weight. Progesterone receptor was strongly expressed in decidual cells in alphaERKO mice, and ICI did not inhibit decidualization or PR expression. This study suggests that up-regulation of PR in endometrial stroma is mediated through at least three mechanisms: 1) classical estrogen signaling through ERalpha, 2) estrogen signaling through ERbeta, and 3) as a result of mechanical stimulation plus progesterone, which induces stromal cells to differentiate into decidual cells. Each of these pathways can function independently of the others.     

Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland

The role of estrogen in promoting mammary stem cell proliferation remains controversial. It is unclear if estrogen receptor (ER)-expressing cells have stem/progenitor activity themselves or if they act in a paracrine fashion to stimulate stem cell proliferation. We have used flow cytometry to prospectively isolate mouse mammary ER-expressing epithelial cells and shown, using analysis of gene expression patterns and cell type-specific markers, that they form a distinct luminal epithelial cell subpopulation that expresses not only the ER but also the progesterone and prolactin receptors. Furthermore, we have used an in vivo functional transplantation assay to directly demonstrate that the ER-expressing luminal epithelial subpopulation contains little in vivo stem cell activity. Rather, the mammary stem cell activity is found within the basal mammary epithelial cell population. Therefore, ER-expressing cells of the mammary epithelium are distinct from the mammary stem cell population, and the effects of estrogen on mammary stem cells are likely to be mediated indirectly. These results are important for our understanding of cellular responses to hormonal stimulation in the normal breast and in breast cancer.     

Hormone-dependent changes in A and B forms of progesterone receptor

The influence of different estrogen and/or progesterone treatments on concentrations of A and B forms of progesterone receptor (PR-A and PR-B) in the different cell types of chick oviduct was studied. A semiquantitative immunohistochemical assay for cellular PR concentrations was developed using a computer-assisted image analysis system. The staining intensity of nuclear PR in the basal layer of epithelial cells, glandular, smooth muscle and mesothelial cells was analysed separately using two monoclonal antibodies, PR6 and PR22. The measured concentrations of PR varied between different cell types and from cell to cell. A significant decrease in PR concentration, as noted by a decrease in staining intensity, was observed in all cell types studied 2 or 6 h after a single injection of progesterone with or without simultaneous estrogen administration. The decrease was also verified with immunoblotting and an immunoenzymometric assay (IEMA) for chicken PR. After down-regulation the concentration of PR recovered to the control level within 48 h after progesterone or estrogen administration. Estrogen administration alone was observed to cause changes in the concentration of PR-A only, having little or no effect on PR-B concentration depending on the cell type studied.

These findings indicate that estrogen and progesterone cause cell-specific changes not only to the total concentration of PR but also to the cellular ratio of PR-A and PR-B.     

Immunophenotypic properties and estrogen dependency of budding cell structures in the developing mouse mammary gland

Abstract. The initial phase of growth of the parenchymal component of the mouse mammary gland is ductal clongation, which is mainly accomplished by proliferating cells in a specialized structure termed end bud. End buds are composed of multiple layers of epithelial cells (so called body cells) which are capped by a single layer of morphologically unique cells termed cap cells.
We sought to examine the interrelationship between cap cells and other epithelial cell subclasses using a variety of antibodies to different keratin proteins and also antibodies to vimentin, actin and collagen IV. An extensive immunohistochemical characterization of the epithelial components of the developing and differentiating mammary gland demonstrated that cap cells were devoid of any immunohistochemically - detectable keratins but were positive for collagen IV. In contrast, the majority of cells in the end bud along with the luminal epithelial and myoepithelial cells were keratin positive. The body cells of the end bud were the only cells which were positive for antibody to keratin 6, a keratin which previously has been reported to be expressed in proliferating mammary epithelial cells. In addition, estrogen receptor was localized only to epithelial cells of ducts, alvcoli and body cells of end buds, but not to cap cells or myoepithelial cells. We interpret these results to suggest that cap cells are not totpotent stem cells but rather cells specialized in paving the way for ductal elongation as well as serving as precursors to myoepithelial cells.     

Steroid receptors and proliferation in the human breast

Despite recent gains in our knowledge of the hormonal control of proliferation and differentiation in the rodent mammary gland, the factors regulating these processes in the human are poorly understood. We have developed a model in which intact normal human breast tissue is grafted subcutaneously into adult female athymic nude mice and treated with oestrogen (E) and/or progesterone (P) at human physiological serum levels. We have shown that (i) E and not P is the major epithelial cell mitogen in the adult non-pregnant, non-lactating breast, (ii) E induces progesterone receptor (PR) expression and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E is required to stimulate proliferation. These data raised the question of whether one cell type demonstrated two different responses to the two different E concentrations or whether PR expression and proliferation occurred in separate cell populations. Using dual label immunofluorescence, we showed that steroid receptor expression and proliferation (Ki67 antigen) are detected in separate cell populations in normal human breast epithelium, and that cells expressing the oestrogen receptor-alpha (ERalpha) invariably contained the PR. We also reported that this separation between steroid receptor expression and proliferation observed in the normal human epithelium is disrupted at an early stage in breast tumourigenesis. One interpretation supported by our recent findings is that some ERalpha/PR-positive epithelial cells are quiescent breast stem cells that act as "steroid hormone sensors". Such hormone sensor cells might secrete positive or negative paracrine/juxtacrine factors dependent on the prevailing E or P concentration to influence the proliferative activity of adjacent ERalpha/PR-negative epithelial cells.     

A novel LacZ reporter mouse reveals complex regulation of the progesterone receptor promoter during mammary gland development

To further our understanding of progesterone (P) as an endocrine mammogen, a PR(lacz) knockin mouse was generated in which the endogenous progesterone receptor (PR) promoter directly regulated lacZ reporter expression. The PR(lacz) mouse revealed PR promoter activity was restricted to the epithelial compartment during the prenatal and postnatal stages of mammary gland development. At puberty, PR promoter activity was unexpectedly robust and restricted to the body cells within the terminal end buds and to the luminal epithelial cells in the subtending ducts. In the adult, the preferential localization of PR(lacz) positive cells to the distal regions of ductal side branches provided a cellular context to the recognized mandatory role of P in ductal side-branching, and segregation of these cells from cells that undergo proliferation supported an intraepithelial paracrine mode of action for P in branching morphogenesis. Toward the end of pregnancy, the PR(lacz) mouse disclosed a progressive attenuation in PR promoter activity, supporting the postulate that the preparturient removal of the proliferative signal of P is a prerequisite for the emergence of a functional lactating mammary gland. The data suggest that PR expression before pregnancy is to ensure the specification and spatial organization of ductal and alveolar progenitor cell lineages, whereas abrogation of PR expression before lactation is required to enable terminal differentiation of the mammary gland.     

Atbf1 Regulates Pubertal Mammary Gland Development Likely by Inhibiting the Pro-Proliferative Function of Estrogen-ER Signaling

ATBF1 is a candidate tumor suppressor that interacts with estrogen receptor (ER) to inhibit the function of estrogen-ER signaling in gene regulation and cell proliferation control in human breast cancer cells. We therefore tested whether Atbf1 and its interaction with ER modulate the development of pubertal mammary gland, where estrogen is the predominant steroid hormone. In an in vitro model of cell differentiation, i.e., MCF10A cells cultured in Matrigel, ATBF1 expression was significantly increased, and knockdown of ATBF1 inhibited acinus formation. During mouse mammary gland development, Atbf1 was expressed at varying levels at different stages, with higher levels during puberty, lower during pregnancy, and the highest during lactation. Knockout of Atbf1 at the onset of puberty enhanced ductal elongation and bifurcation and promoted cell proliferation in both ducts and terminal end buds of pubertal mammary glands. Enhanced cell proliferation primarily occurred in ER-positive cells and was accompanied by increased expression of ER target genes. Furthermore, inactivation of Atbf1 reduced the expression of basal cell markers (CK5, CK14 and CD44) but not luminal cell markers. These findings indicate that Atbf1 plays a role in the development of pubertal mammary gland likely by modulating the function of estrogen-ER signaling in luminal cells and by modulating gene expression in basal cells.     

Role of epidermal growth factor in the acquisition of ovarian steroid hormone responsiveness in the normal mouse mammary gland

The purpose of the present studies was to investigate the role of epidermal growth factor (EGF) in the acquisition of estrogen (E) and progestin (P) responsiveness in the mouse mammary gland in vivo. Using the Elvax 40P implant technique to introduce bioactive molecules directly into the mammary gland to produce a localized effect, we have made the novel observation that EGF implanted into glands of pubertal mice followed by E treatment resulted in the precocious acquisition of E-inducible progesterone receptors (PR). In sexually mature mice, EGF implants alone were able to increase PR. A neutralizing antibody specific for EGF blocked E-dependent stimulation of end-bud development and PR induction. Furthermore, the antiestrogen ICI 182,780 blocked the EGF-induced stimulation end-buds and PR induction, indicating that these EGF effects are mediated via estrogen receptors (ER). Immunohistochemical analysis showed that the endogenous EGF content of mammary glands of mature mice was higher than pubertal mice, that E implants caused a localized increase in mammary gland EGF content in both pubertal and mature mice, and that in mature mice E caused an increase in stromal cell EGF content. We have previously shown that the acquisition of E-inducible PR can be modulated by mammary stroma, and the present results indicate that mammary stroma could modulate hormonal responsiveness through control of local growth factor concentration. Taken together, these results provide evidence that E-dependent responses of mouse mammary gland in vivo, such as end-bud proliferation and PR regulation, may be mediated by EGF through an ER-dependent mechanism. J. Cell. Physiol. 174:251–260, 1998. © 1998 Wiley-Liss, Inc.     

A putative human breast stem cell population is enriched for steroid receptor-positive cells

Breast epithelial stem cells are thought to be the primary targets in the etiology of breast cancer. Since breast cancers mostly express estrogen and progesterone receptor (ERalpha and PR), we examined the biology of these ERalpha/PR-positive cells and their relationship to stem cells in normal human breast epithelium. We employed several complementary approaches to identify putative stem cell markers, to characterise an isolated stem cell population and to relate these to cells expressing the steroid receptors ERalpha and PR. Using DNA radiolabelling in human tissue implanted into athymic nude mice, a population of label-retaining cells were shown to be enriched for the putative stem cell markers p21(CIP1) and Msi-1, the human homolog of Drosophila Musashi. Steroid receptor-positive cells were found to co-express these stem cell markers together with cytokeratin 19, another putative stem cell marker in the breast. Human breast epithelial cells with Hoechst dye-effluxing "side population" (SP) properties characteristic of mammary stem cells in mice were demonstrated to be undifferentiated "intermediate" cells by lack of expression of myoepithelial and luminal apical membrane markers. These SP cells were 6-fold enriched for ERalpha-positive cells and expressed several fold higher levels of the ERalpha, p21(CIP1) and Msi1 genes than non-SP cells. In contrast to non-SP cells, SP cells formed branching structures in matrigel which included cells of both luminal and myoepithelial lineages. The data suggest a model where scattered steroid receptor-positive cells are stem cells that self-renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells.     

Immunocytochemical Localization of Sex Steroid Hormone Receptors in Normal Human Mammary Gland

The sex steroids, estrogens, progesterone, and androgens, all play a role in mammary development and function. To precisely identify the sites of action of these steroids, we studied the localization of the estrogen receptor α (ERα) and ERβ, the progesterone receptor A (PRA) and PRB, and androgen receptors (AR) in the normal human mammary gland. Immunocytochemical localization of ERα, ERβ, PRA, PRB, and AR was performed with reduction mammoplasty specimens from premenopausal women. ERα, PRA, PRB, and AR were localized mostly to the inner layer of epithelial cells lining acini and intralobular ducts, as well as to myoepithelial cells scattered in the external layer of interlobular ducts. AR was also found in some stromal cells. ERβ staining was more widespread, resulting in epithelial and myoepithelial cells being labeled in acini and ducts as well as stromal cells. These results suggest that all sex steroids can directly act on epithelial cells to modulate development and function of the human mammary gland. Estrogens and androgens can also indirectly influence epithelial cell activity by an action on stromal cells. (J Histochem Cytochem 58:509–515, 2010)