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Endocrinology Vol. 148, No. 5 2290-2300
Copyright © 2007 by The Endocrine Society

Differential Hormonal Regulation and Function of Progesterone Receptor Isoforms in Normal Adult Mouse Mammary Gland

Mark D. Aupperlee and Sandra Z. Haslam

Department of Physiology (S.Z.H.) and Cell and Molecular Biology Program (M.D.A.), Michigan State University, East Lansing, Michigan 48824

Address all correspondence and requests for reprints to: Sandra Z. Haslam, Ph.D., Department of Physiology, 2201 Biomedical and Physical Sciences Building, Michigan State University, East Lansing, Michigan 48824. E-mail: shaslam{at}msu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In normal mouse mammary gland, the mitogenic action of progesterone (P) is mediated by two P receptor (PR) isoforms, PRA and PRB. PRA is predominantly expressed in the adult virgin, and PRB is predominantly expressed during pregnancy. To investigate hormonal regulation of PR isoform expression and isoform-specific functions in vivo, adult ovariectomized BALB/c mice were treated for 3, 5, or 10 d with estrogen (E), P, or estrogen plus progesterone (E+P). Using an immunohistochemical approach with isoform-specific antibodies, we investigated hormonal regulation of PRA and PRB and their functional roles in proliferation and morphogenesis. Significant E-induced proliferation was only observed after 5 d at the distal tips of ducts; there was no sidebranching or alveologenesis. P induced proliferation that resulted in sidebranching and alveologenesis, but E+P treatment produced more proliferation sooner and more extensive sidebranching and alveologenesis. PRA levels were increased by E and decreased by P. Increased PRB levels were induced by treatment with P or E+P and coincided with the formation of alveoli. PRA was the predominant PR isoform expressed during sidebranching, and colocalization of PRA with 5-bromo-2'-deoxyuridine revealed that proliferation of PRA-positive and -negative cells was responsible for P-induced sidebranching. PRB was the predominant PR isoform expressed during alveologenesis, and colocalization of PRB with 5-bromo-2'-deoxyuridine showed that both PRB-positive and -negative cells proliferated during alveolar expansion. These results demonstrate different hormonal regulation of PRA and PRB levels in vivo and suggest that P can induce proliferation through either PRA or PRB via direct and paracrine mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGESTERONE (P) PLAYS an important role in regulating proliferation and differentiation in the normal mammary gland (1). Proliferation is regulated by P in the adult human breast (2) as well as in rodent and monkey models (1, 3). P acts through binding to its cognate nuclear receptor, the P receptor (PR), and PR exists as two isoforms, PRA and PRB, which are identical except for a 164-amino-acid N-terminal extension on PRB. PRA and PRB can regulate different genes and have different functions (4). Studies using PRB knockout mice, PRB transgenic mice, and immunohistochemical analysis of PRB expression in wild-type mice indicate a role for PRB in alveologenesis (5, 6, 7). The role of PRA in the mammary gland is less clear, but studies with PRA transgenic mice and immunohistochemical analysis of PRA expression in wild-type mice indicate that PRA may be involved in ductal development and sidebranching (7, 8).

To date, PR expression and P action in the normal mammary gland have been most extensively studied in the mouse model. PRA and PRB expression are temporally and spatially separated during murine mammary gland development from puberty through pregnancy, lactation, and involution (7). Our previous studies of developmental expression of PR isoforms found that PRA is predominantly expressed in the virgin mouse mammary gland, which is primarily composed of ducts, whereas expression of PRB is induced in alveolar structures upon lobuloalveolar development during pregnancy along with a concomitant decrease in PRA expression (7). Additionally, PRA and PRB are infrequently expressed in the same cell. In contrast, in the normal adult premenopausal human breast, PRA and PRB are coexpressed in the same cells and the ratio of PRA to PRB in individual PR-positive cells is 1:1 (9). The pattern and level of PRA and PRB expression at other stages of human mammary gland development are not known. However, breast cancers exhibit an altered PRA to PRB ratio, with a higher PRA to PRB ratio associated with less differentiated and more aggressive tumors (9). The mechanism(s) that regulate the relative expression of PRA and PRB in breast cancer is not known.

Little is currently known about the hormonal regulation of PRA and PRB expression in the normal breast. Studies using human breast cancer cell lines have shown that estrogen (E) induces expression of PR, and P has been shown to down-regulate expression of PR (10, 11). However, it is not clear how individual PR isoforms are regulated. It has been reported that ovariectomy reduces PR expression in the mouse mammary gland (12). Although a role for E in regulation of PR has been determined (12, 13), isoform-specific regulation of PRA and PRB has not been examined. The effects of E and/or P on PR isoforms in vivo in the normal human breast have not been well studied. Because alterations in PRA to PRB ratios are associated with breast cancer progression (9), understanding the normal regulation of PRA and PRB may provide insight into the deregulation that occurs in breast cancer.

Biochemical analyses of PR expression, such as immunoblots, can provide useful information about the molecular sizes of protein isoforms or protein posttranslational modifications. However, with regard to analysis of whole mammary gland extracts, immunoblots can be limited in their sensitivity and accuracy for PR detection and quantitation due to the dilution of epithelial proteins by stroma-derived proteins. This occurs because PR is expressed in the epithelial compartment of the gland, whereas the stromal compartment is PR negative (4, 7). This is particularly relevant for quantitation of relative expression of PRA or PRB in mammary tissues that exhibit changes in overall epithelial content, such as after ovariectomy vs. treatment with pregnancy levels of estrogen plus progesterone (E+P). In the first case, the gland exists as a rudimentary ductal system with a predominance of stroma, whereas after E+P treatment there is a proliferative expansion of the epithelium in the form of sidebranches and alveoli and an overall increase in the ratio of epithelium to stroma. Thus, the same amount of protein from mammary gland extracts obtained at different physiological states represents different amounts of mammary epithelium.

In the present study, we used an immunohistochemical approach with antibodies specific for PRA or PRB (7, 14) to examine the hormonal regulation of PRA and PRB and the roles of PRA and PRB in mediating proliferative and morphological responses in the adult mouse mammary gland. One advantage of this approach is that it allowed analysis and quantitation of the cellular distribution of PR isoforms and their colocalization with proliferation markers. We found that PRA expression was increased by E and decreased by P. The initial proliferative response to P, leading to sidebranching, was mediated by PRA. Proliferation and PRB expression were induced by P alone, but were accelerated and enhanced by the combination of E+P. Induction of PRB expression coincided with decreased PRA levels and the onset of alveologenesis. Analysis of ER{alpha} expression revealed that only PRA was extensively colocalized with ER{alpha}. These studies demonstrate differences in the hormonal regulation of PRA and PRB and isoform-specific roles in mediating proliferation and differentiation in the normal mouse mammary gland.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mammary glands were obtained from adult (19–22 wk old) BALB/c female mice purchased from Harlan (Indianapolis, IN). Hormone-treated adult virgin mice were ovariectomized (OVX) and 1 wk after OVX animals were injected for 3, 5, or 10 d with saline control (C), 17-ß-estradiol (E) (1 µg/injection), P (1 mg/injection), or E+P (1 µg + 1 mg, respectively, per injection) administered sc. Two hours before they were killed, mice were injected with 5-bromo-2'-deoxyuridine (BrdU) (70 µg/g of body weight) to label proliferating cells. All animal experimentation was conducted in accord with accepted standards of humane animal care and approved by the All University Committee on Animal Use and Care at Michigan State University. Mammary tissues were fixed and processed as whole mounts (15) or formalin-fixed and paraffin-embedded for immunohistochemistry as previously described (7).

Immunohistochemistry with anti-PR isoform-specific antibodies
The protocol used to detect PRA and PRB was the same as previously described (7). Tissue sections were treated with a combination of heat and pressure for antigen retrieval and then blocked with goat antimouse IgG Fab fragments (Jackson ImmunoResearch Laboratories, West Grove, PA) [1:100 in PBS containing 1% BSA (1% PBSA), 60 min], blocked with normal goat serum (Vector Laboratories, Burlingame, CA) (1:1 in PBS, 30 min), and then incubated with primary antibody against PRA (hPRa7; Neomarkers, Fremont, CA) (1:50 in PBS/0.5% Triton X-100, overnight, 4 C) or against PRB (hPRa6; Neomarkers) (1:50 in PBS/0.5% Triton X-100, overnight). Sections were rinsed with PBS/0.5% Triton X-100, and the primary antibody was recognized by goat antimouse antibody conjugated to Alexa 488 (Molecular Probes, Eugene, OR) (1:200 in PBS, 30 min).

When immunostaining to recognize PRA, nuclei were counterstained with 4',6-diamidino-2-phenylindole, dilactate (DAPI) (Molecular Probes) (1:10,000 in H2O), and sections were visualized and images captured using a Nikon inverted epifluorescence microscope (Mager Scientific, Dexter, MI) with MetaMorph software (Molecular Devices Corp., Downington, PA). When immunostaining to recognize PRB, nuclei were counterstained with TOPRO-3 Iodide (Molecular Probes) (1:1000 in fluorescent mounting media) and sections were visualized and images were captured using a Zeiss Pascal laser scanning confocal microscope (Zeiss, Thornwood, NY).

Colocalization of PRA or PRB with BrdU, cyclin D1, or ER{alpha}
Double labeling of PRA or PRB with BrdU and cyclin D1 was performed as previously described (7). Briefly, after antigen retrieval, sections were blocked and incubated with anti-PRA or PRB antibody (overnight, 4 C) as described above.

For colocalization with BrdU, sections were first stained for PRA or PRB, and then PRA or PRB localization was detected with a goat antimouse secondary conjugated to Alexa 488 (Molecular Probes) (1:200 in PBS, 30 min). Next, sections were blocked with goat antimouse IgG Fab fragments (Jackson ImmunoResearch Laboratories) (1:100 in 1% PBSA, 60 min), blocked with normal goat serum (Vector Laboratories) (1:1 in PBS, 30 min), and incubated for 60 min at room temperature with anti-BrdU antibody (kit from Amersham Biosciences, Piscataway, NJ). BrdU localization was detected with a biotinylated goat antimouse secondary (Dako, Carpinteria, CA) (1:400 in PBS, 30 min), which was recognized by streptavidin-conjugated Alexa 546 (Molecular Probes) (1:100 in PBS, 30 min).

For colocalization with cyclin D1, sections were first stained for PRA or PRB, and then PRA or PRB localization was detected with a goat antimouse secondary conjugated to Alexa 546 (Molecular Probes) (1:200 in PBS, 30 min). Sections were then blocked with 2% PBSA for 30 min and incubated overnight at 4 C with rabbit polyclonal anti-cyclin D1 antibody (Biosource, Camarillo, CA) (1:100 in 2% PBSA). Cyclin D1 localization was detected with a goat antirabbit antibody conjugated to Alexa 488 (Molecular Probes) (1:200 in PBS, 30 min).

For colocalization with ER{alpha}, sections were first stained for PRA or PRB, and then PRA or PRB localization was detected with a goat antimouse secondary conjugated to Alexa 488 (Molecular Probes) (1:200 in PBS, 30 min). Next, sections were blocked with goat antimouse IgG Fab fragments (Jackson ImmunoResearch Laboratories) (1:100 in 1% PBSA, 60 min), blocked with normal goat serum (Vector Laboratories) (1:1 in PBS, 30 min), and incubated overnight at 4 C with mouse monoclonal anti-ER{alpha} antibody (NCL-L-ER-6F11) (Novocastra, Newcastle, UK). ER{alpha} localization was detected with a biotinylated goat antimouse secondary (Dako) (1:400 in PBS, 30 min), which was recognized by streptavidin-conjugated Alexa 546 (Molecular Probes) (1:100 in PBS, 30 min).

For all dual-immunofluorescence labeling, nuclei were counterstained with DAPI (Molecular Probes) (1:10,000 in H2O), and sections were visualized and images were captured using a Nikon inverted epifluorescence microscope (Mager Scientific) with MetaMorph software (Molecular Devices Corp.).

Quantitation of fluorescence and statistical analyses
Sections treated for detection of PRA, PRB, BrdU, or cyclin D1 by immunofluorescence methods were quantitated for the number of positive luminal epithelial cell nuclei from captured images using MetaMorph software. Positive nuclei displayed staining above luminal epithelial cytoplasmic background. To analyze fluorescence intensity, the average pixel intensity of all positively stained nuclei within the ductal epithelium was determined. Images were thresholded to exclude background fluorescence and gated to include intensity measurements only from positively staining epithelial cells. Six mice per treatment group were analyzed; a minimum of 1000 total cells and three independent sections per mouse were analyzed. Results are expressed as mean ± SEM, and differences are considered significant at P < 0.05 by using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The most dramatic changes in mammary gland morphology, proliferation, and PRA and PRB expression occur in the adult mammary gland in response to pregnancy (7). Thus, we treated adult OVX mice with pregnancy levels of E, P, or E+P for a total of 3, 5, or 10 d, and studied the hormonal regulation of PRA and PRB expression and their relationship to hormonal regulation of proliferation and alveolar morphogenesis.

Morphological responses of the mammary gland to hormone treatments
Morphological responses to ovariectomy and hormonal treatments are shown in Fig. 1Go. Ovariectomy and control treatment resulted in a reduction in the size of the ducts and duct ends, and mammary gland morphology was similar after ovariectomy and 3, 5, or 10 d of control treatment. Treatment with E produced a transient enlargement of the distal tips of ducts and dilation of ducts; this response was maximal after 5 d and decreased by 10 d. Treatment with E+P produced morphological changes that increased with treatment length. After 3 d of E+P, sidebranching and some dilation of the ducts were observed. Treatment for 5 d with E+P produced more extensive sidebranching and the start of alveologenesis, as defined by the presence of multiluminal structures at the ends of sidebranches. A close examination of sidebranches revealed bulb-shaped structures that appeared to pinch off into alveolar units. After 10 d of E+P treatment, there was more extensive alveolar development; all the sidebranches produced lobular structures with multiple alveoli. After 3 d of treatment with P alone, no change in morphology was visible. However, after 5 d of P treatment, there was extensive sidebranching throughout the mammary gland. Treatment with P for 10 d produced the start of alveologenesis. These results show that treatment with P alone caused sidebranching and the initiation of alveologenesis. Sidebranching and alveologenesis were accelerated and enhanced by the addition of E in the E+P-treated mice.


Figure 1
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  		FIG. 1. Morphological response of the mammary gland to hormone treatment. Mammary gland whole mounts were prepared from adult BALB/c OVX mice treated for 3, 5, or 10 d with saline (control, C), E, P, or E+P. Proliferation in response to E occurred after 5 d at the distal tips of ducts (black arrow indicates the distal tip of a duct), but the distal tips regressed by 10 d. A higher magnification inset of stimulation of the distal tips at the ends of ducts is shown for 5 d E. After 3 d of E+P or 5 d of P, sidebranching was present (black arrowheads). Alveologenesis started after 5 d of E+P or 10 d of P (open arrowheads). A higher magnification inset of alveologenesis is shown for 5 d E+P. Expansion of alveoli occurred after 10 d of E+P (open arrowhead) (scale bar, 1 mm).

 
Proliferative responses to hormone treatments
The morphological changes described above are associated with proliferation. To determine the relationships among hormone treatments, proliferation, and changes in morphology, mice were treated with a pulse of BrdU 2 h before they were killed. Because the mammary epithelium is composed of two cell types, luminal epithelial cells and myoepithelial cells, double labeling with BrdU and {alpha}-smooth muscle actin (SMA), a specific marker of myoepithelial cells, was carried out to determine the proliferative response in the two mammary cell types.

Analysis of proliferation in response to the various hormone treatments is shown in Fig. 2Go, A and B. No proliferation was observed in luminal or myoepithelial cells in OVX, control-treated animals. Treatment with E produced a significant but transient increase in proliferation after 5 d; proliferation occurred in luminal epithelial cells and myoepithelial cells and was specifically localized to the distal tips of ducts.


Figure 2
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  		FIG. 2. Cell type-specific proliferation in response to hormone treatment. Dual immunofluorescence detection of BrdU and {alpha}-SMA was performed on mammary gland sections from adult OVX mice treated for 3, 5, or 10 d with saline (control, C), E, P, or E+P. A, Quantitation of the percent BrdU-positive myoepithelial cells and luminal epithelial cells was determined. Proliferation after 5 d of E treatment was significantly increased only in the distal tips of ducts. Proliferation was highest after 3 or 10 d of E+P treatment. Treatment with P produced sustained proliferation after 3, 5, or 10 d of treatment. Proliferation was induced in both luminal and myoepithelial cells. The values represent the mean ± SEM from three to five mice per group with a minimum of 1000 cells per mouse analyzed. B, After 3 d of E+P treatment, proliferating cells were recognized using an anti-BrdU antibody (teal), myoepithelial cells were distinguished by anti-{alpha}-SMA staining (red), and nuclei were counterstained with DAPI (blue). Proliferation occurred in both myoepithelial (solid white arrowheads) and luminal epithelial cells (open arrowheads) (scale bar, 25 µm).

 
Surprisingly, 3 d of treatment with P alone also increased proliferation of luminal and myoepithelial cells relative to OVX controls. Luminal epithelial cell and myoepithelial cell proliferation were also observed in ducts, sidebranches and alveoli after 5 and 10 d of P treatment. From 7–11% in luminal epithelial cells were BrdU positive (BrdU+) and 4–12% myoepithelial cells were BrdU+ between 3 and 10 d of treatment.

Overall, the greatest proliferation throughout the gland was observed in response to E+P treatment. Proliferation occurred in ducts, sidebranches, and alveoli. After 3 d of E+P treatment, 26% of luminal epithelial cells were BrdU+ and 23% of myoepithelial cells were BrdU+. Proliferation decreased after 5 d of E+P treatment to 7% in luminal epithelial cells and 6% in myoepithelial cells. After 10 d of E+P treatment, proliferation increased and 17% of luminal epithelial cells in both ducts and alveolar structures were BrdU+; a smaller percentage of myoepithelial cells (7%) were BrdU+.

These results demonstrate that treatment with E alone produced a transient burst of proliferation in luminal and myoepithelial cells that was restricted to duct ends. In contrast, treatment with P alone produced a low level of sustained proliferation in myoepithelial and luminal epithelial cells throughout the 10-d treatment period. Thus, P by itself, in the absence of E, was capable of inducing proliferation in both cell types. Treatment with E+P resulted in a biphasic proliferative response that was high at d 3, decreased at d 5, and was high again at 10 d of treatment. Notably, E enhanced overall proliferation when combined with P.

Hormonal regulation of PRA
The hormonal regulation of PRA was investigated by immunofluorescence staining with antibody specific for PRA. The effects of ovariectomy and hormone treatments on the percentage of PRA-positive (PRA+) cells are shown in Fig. 3AGo. Ovariectomy did not change the percentage of PRA+ cells compared with the ovary-intact control. Similarly, no effect of treatment with E on the percentage of PRA+ cells was observed. To determine whether the cellular content of PRA was affected by the various treatments, analysis of immunofluorescence staining intensity was performed using software that measures pixel intensity in positive cells (Fig. 3Go, B and C). This analysis revealed that the level of PRA protein was significantly decreased by ovariectomy compared with ovary-intact controls. Treatment with E increased PRA content in PRA+ cells relative to OVX controls, but did not restore PRA content to the level of the ovary-intact control. Treatment with P for 5 or 10 d significantly reduced the percentage of PRA+ cells and significantly decreased PRA levels below that of OVX controls. Treatment for 10 d with E+P produced the largest decrease in the percentage of PRA+ cells. Treatment with E+P did not increase PRA content above that in OVX controls. These results demonstrate that E up-regulates the levels of PRA, whereas P down-regulates PRA levels and blunts up-regulation by E.


Figure 3
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  		FIG. 3. Hormonal regulation of PRA expression in the adult mouse mammary gland. PRA was detected by immunofluorescence with an anti-PRA antibody on mammary gland sections from adult intact or OVX mice treated for 3, 5, or 10 d with control (C), E, P, or E+P. A, Quantitation of the percent PRA-positive luminal epithelial cells. The values represent the mean ± SEM from three to five mice per group with a minimum of 1000 cells per mouse analyzed. *, P < 0.05, 10 d E+P was significantly less than all other groups. #, P < 0.05, 5 and 10 d P were significantly less than intact, all C- and E-treated groups and 3 d E+P group (P < 0.05). B, Representative sections of PRA immunofluorescence staining from adult intact or OVX mice treated for 10 d with C, E, P, and E+P. The percent PRA-positive cells decreased in the E+P- or P-treated group. PRA intensity was highest in the intact mouse and following E treatment of OVX mice and lowest in the P-treated OVX mice (scale bar, 25 µm). C, Quantitation of PRA immunofluorescence staining intensity. Values represent the mean pixel intensity in PRA-positive cells ± SEM from three to five mice per group with a minimum of 1000 cells per mouse analyzed. Intensity of PRA staining was significantly decreased after ovariectomy, and treatment with E significantly increased the intensity of PRA staining compared with OVX controls. Treatment with E+P did not increase PRA staining, and treatment with P alone further decreased the intensity of PRA staining compared with the OVX control groups.

 
The relationship between PRA level and proliferation during morphogenesis
PRA and PRB are detected only in luminal epithelial cells (7); therefore, analysis of the relationship between PR isoforms and proliferation was determined in luminal epithelial cells. The relationship between PRA, P, and proliferation was examined in E+P- or P-treated mice by studying the colocalization of PRA with the proliferation marker BrdU by dual immunofluorescence (Fig. 4Go). After 3 d of E+P treatment, 41% of luminal epithelial cells were PRA+, 25% were BrdU+, and 4% were positive for both PRA and BrdU. Of the total proliferating cells, 16% expressed PRA (Fig. 4Go, A and B). Thus, a subpopulation of PRA+ cells, in addition to PRA-negative (PRA–) cells, was proliferating coincident with the formation of sidebranches (Fig. 1Go). After 5 d of E+P treatment, only 5% of the luminal epithelial cells were proliferating and now only 5% of proliferating cells were PRA+. The lower percentage of cells positive for both PRA and BrdU was not due to a decrease in the percentage of PRA+ cells because 42% of luminal epithelial cells were still PRA+. After 10 d of treatment with E+P, 13% of luminal epithelial cells were proliferating. Because there was a significant increase in the total number of epithelial cells after 10 d of E+P treatment (Fig. 1Go), the total number of proliferating cells was likely higher at 10 d compared with 5 d of E+P. The percentage of PRA+ cells was significantly reduced to 20%, and only 3% of BrdU+ cells were PRA+. The reduced percentage of PRA+ cells and reduced colocalization of PRA and BrdU coincided with increased alveologenesis.


Figure 4
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  		FIG. 4. Localization of PRA in proliferating cells. A, Dual immunofluorescence detection of PRA and BrdU was performed on mammary gland sections from adult OVX mice treated for 3, 5, or 10 d with E+P or P alone. In the representative images shown, PRA+ cells are teal, BrdU+ cells are pink, PRA and BrdU+ cells are white, and nuclei counterstained with DAPI are blue. After 3 d of E+P, a small population of proliferating cells express PRA (open arrowhead), whereas after 5 or 10 d of E+P most proliferating cells are PRA negative (yellow arrowhead). Examples of cells expressing PRA only are indicated with white arrowheads (scale bar, 25 µm). B, Quantitation of the percent PRA+ cells, BrdU+ cells, and colocalization of PRA and BrdU in E+P-treated mammary glands. C, Quantitation of the percent PRA+ cells, BrdU+ cells, and colocalization of PRA and BrdU in P-treated mammary glands. The values represent the mean ± SEM from three to five mice per group with a minimum of 1000 cells per mouse analyzed.

 
Overall, proliferation induced by P alone was lower than that induced by E+P (Fig. 4CGo). The percentage of BrdU+ cells was 9, 7, and 4% after 3, 5, and 10 d of P treatment, respectively. In P-treated mice, the development of sidebranches was delayed and observed only after 5 d of treatment compared with after 3 d of E+P treatment. This is likely due to the lower amount of proliferation induced by P alone. BrdU incorporation in PRA+ cells was lower than observed with E+P treatment. After 3 or 5 d of P treatment, only 2% of proliferating cells were PRA+. After 10 d of P treatment, when alveolar development was observed, no PRA+ cells were proliferating. Taken together, the results obtained in P- and E+P-treated mice suggest that alveolar development and expansion are not associated with the proliferation of PRA+ cells.

Nuclear localization of cyclin D1 is associated with the induction of cell cycle progression toward S-phase and cyclin D1 expression is believed to be regulated by P (16). Additionally, cyclin D1 expression is believed to be required for alveologenesis during pregnancy (17). Thus, we also examined colocalization of PRA with cyclin D1 after treatment with E+P or P by dual immunofluorescent labeling (Fig. 5Go). After 3 d of E+P treatment, nuclear cyclin D1 was expressed in 51% of luminal cells, 42% of cells were PRA+, and 42% of cyclin D1-positive (cyclin D1+) cells were also PRA+ (Fig. 5BGo). After 5 d of E+P treatment, the percentage of cells expressing nuclear cyclin D1 was reduced to 34%, and only 13% of these cyclin D1+ cells were PRA+. Thus, although the percentage of PRA+ cells after 3- or 5-d E+P treatment was similar, colocalization of PRA with cyclin D1 significantly decreased after 5 d of treatment. After 10 d of E+P treatment, nuclear cyclin D1 was expressed in 37% of luminal epithelial cells, and only 3% of cyclin D1+ cells were PRA+. Thus, PRA colocalized with cyclin D1 during sidebranching after 3 d of E+P treatment, but not during maximal alveologenesis observed after 10 d of E+P treatment.


Figure 5
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  		FIG. 5. Colocalization of PRA and cyclin D1. A, Dual immunofluorescence detection of PRA and cyclin D1 was performed on mammary gland sections from adult OVX mice treated for 3, 5, or 10 d with E+P or P alone. In the representative merged images, PRA+ cells are teal, cyclin D1+ cells are pink, PRA and cyclin D1+ cells are white, and nuclei counterstained with DAPI are blue. Specific examples of PRA+ cells (white arrowhead), cyclin D1+ nuclei (yellow arrowhead), and colocalization of PRA and cyclin D1 (open arrowhead) are shown. Colocalization of PRA with cyclin D1 was greatest after 3 d E+P and decreased after 5 and 10 d of E+P treatment (scale bar, 25 µm). B, Quantitation of percent PRA+ cells, cyclin D1+ cells, and colocalization of PRA and cyclin D1 in E+P-treated mammary glands. The percent cyclin D1+ cells increases in E+P-treated mammary glands relative to saline control treatment. C, Quantitation of percent PRA+ cells, cyclin D1+ cells, and colocalization of PRA and cyclin D1 in P-treated mammary glands. The percent cyclin D1+-positive cells increases in E+P-treated mammary glands relative to saline control treatment. The values represent the mean ± SEM from three to five mice per group with a minimum of 1000 cells per mouse analyzed.

 
In mice treated with P alone for 3 d, the percentage of cyclin D1+ cells (37%) was lower than observed with E+P treatment, and 29% of cyclin D1+ cells were PRA+ (Fig. 5CGo). The percentage of cyclin D1+ cells and colocalization with PRA did not change significantly after 5 or 10 d of treatment, and PRA colocalized with cyclin D1 with a similar frequency at 3, 5, and 10 d of P treatment.

Hormonal regulation of PRB
High levels of PRB are detected mainly during pregnancy and after involution (7). PRB expression during pregnancy is primarily associated with the formation of alveolar structures, and PRB levels are lower in ducts (7). During pregnancy, levels of E and P are much higher than during normal estrus cycles (18, 19). Additionally, these high hormone levels are maintained during pregnancy, so there is greater continuous exposure to hormones during pregnancy than during the estrus cycle in the adult virgin (18). Based on these previous findings, we hypothesized that regulation of PRB would differ from that of PRA and would be up-regulated by P.

The regulation of PRB expression was examined in OVX mice by immunofluorescence using an antibody specific for PRB (Figs. 6Go and 7Go). PRB was not detected after ovariectomy or after treatment with E (data not shown). PRB levels were increased by 5 d of E+P or by 10 d of P treatment, but at a very low level, so accurate determination of the percent positive cells was not feasible (Fig. 6Go). PRB levels rose to a clearly detectable level in 25% of epithelial cells only after 10 d of E+P treatment (Fig. 7Go). Thus, PRB was only detected after prolonged treatment with P or E+P. Additionally, the increase in PRB levels coincided with the appearance of alveolar structures (Fig. 1Go).


Figure 6
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  		FIG. 6. Hormonal regulation of PRB expression in the adult mouse mammary gland. Immunofluorescence detection of PRB was performed on mammary gland sections from adult OVX mice treated for 3, 5, or 10 d with C, E, P, or E+P. PRB was only detected after 5 d E+P or 10 d of P treatment. PRB (green nuclei, white arrowheads) was faintly detected after 5 d of E+P or 10 d of P treatment, but was most strongly expressed after 10 d of E+P treatment. Nuclei were counterstained with DAPI (blue) (scale bar, 20 µm).

 

Figure 7
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  		FIG. 7. Colocalization of PRB with BrdU or cyclin D1. Dual immunofluorescence detection of PRB colocalization with BrdU or with cyclin D1 was performed on mammary gland sections from adult OVX mice treated for 10 d with E+P. A, Quantitation of percent PRB+ cells, BrdU+ cells, and colocalization of PRB and BrdU. B, Quantitation of percent PRB+ cells, cyclin D1+ cells, and colocalization of PRB and cyclin D1. The values represent the mean ± SEM from three to five mice per group with a minimum of 1000 cells per mouse analyzed.

 
The relationship between PRB level and proliferation during morphogenesis
Colocalization of PRB with proliferation was determined by dual immunofluorescence with antibodies specific for PRB and BrdU. This analysis was carried out only in the 10-d E+P treatment group because this was the only treatment that resulted in clearly detectable and quantifiable levels of PRB (Fig. 7AGo). After 10 d of E+P treatment, 10% of luminal epithelial cells were BrdU+, 30% were PRB+, and 49% of the BrdU+ cells were PRB+. Thus, about half of the proliferating cells were PRB+, and because increased PRB levels coincided with the development of alveoli, this suggests that alveoli are formed by proliferating PRB+ cells. This is in contrast to the finding that only 3% of proliferating cells were PRA+ during the time of maximal alveolar development at 10 d of E+P treatment (Fig. 4BGo)

Dual immunofluorescence detection of PRB and cyclin D1 was also performed in the 10-d E+P-treated mammary gland (Fig. 7BGo). Nuclear cyclin D1 expression was detected in 35% of luminal epithelial cells and 30% of cyclin D1+ cells were PRB+. This is in contrast to the 7% of cyclin D1+ cells that were PRA+ (Fig. 5BGo). Thus, after 10 d of E+P treatment, PRB was the predominant isoform expressed and was more frequently colocalized with BrdU and cyclin D1 than PRA. Taken together, the frequent colocalization of PRB with BrdU and with cyclin D1 at the time of extensive alveolar development further suggests that PRB+ cells proliferate to form alveoli.

Colocalization of PRA and PRB with ER
As shown above, the levels of both PRA and PRB were regulated by E, which acts through binding to the E receptor (ER). E increased PRA levels and enhanced P-induced increase in PRB levels. ER{alpha}, and not ERß, is required for ductal development in the mammary gland (20, 21). ERß appears to play a role during lactation, when neither PRA nor PRB are expressed (21). Thus, to further address the role of E in the regulation of PR isoforms, we used dual immunofluorescence to analyze ER{alpha} expression with PRA or PRB expression.

ER{alpha} was expressed in all the treatment groups through d 10 (Fig. 8AGo). However, intensity of staining with anti-ER{alpha} antibody was lower in E- and E+P-treated mammary glands than in control or P-treated glands, indicating that E down-regulates ER{alpha} levels (Fig. 8AGo). Notably, PRA and ER{alpha} were coexpressed in the same cell under all treatment conditions (Fig. 8BGo). Increased PRB levels coincided with decreased ER{alpha} levels and the majority of PRB+ cells were ER{alpha} negative (Fig. 8BGo). The coexpression of ER{alpha} and PRA suggests that E acting through ER{alpha} may directly regulate PRA expression. Conversely, the lack of significant coexpression of PRB with ER{alpha} suggests that E acts indirectly to enhance P-induced up-regulation of PRB.


Figure 8
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  		FIG. 8. Colocalization of PRA and PRB with ER{alpha}. Dual immunofluorescence detection of ER{alpha} and ER{alpha} colocalization with PRA or with PRB was performed on mammary gland sections from adult OVX mice treated for 3, 5, or 10 d with saline control (C), E, P, or E+P. A, Representative immunofluorescence images of ER{alpha} staining. ER{alpha} (red) expression was detected in all groups, but was decreased by treatment with E or E+P. Nuclei were counterstained with DAPI (blue) (scale bar, 25 µm). B, Representative images of PRA (green) or PRB (green) colocalization with ER{alpha} (red) are shown. In merged images, colocalized cells are yellow. There was a high degree of PRA and ER{alpha} expression colocalization. The majority of PRB-positive cells (white arrowheads) did not colocalize with ER{alpha}. Instances of PRB and ER{alpha} colocalization are shown with yellow arrowheads (scale bar, 20 µm).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have reported previously that PRA is predominantly expressed in the virgin gland, whereas PRB is predominantly expressed during pregnancy. In this report, we have examined the hormonal basis for the differential expression of the two PR isoforms. Because the greatest proliferative and morphological responses to P occur during pregnancy, we have focused our study on the mature, adult mammary gland and the effect of pregnancy levels of E and P on PR isoform levels. Additionally, we have analyzed the relationship between regulation of PR isoform expression, proliferation, and alveolar development.

PRA is up-regulated by E and down-regulated by P
We found that although ovariectomy did not affect the percentage of PRA+ cells, it dramatically reduced the level of PRA protein. PRA levels could be restored by treatment with E. In contrast, treatment with P alone or E+P caused a reduction in the percentage of PRA+ cells. Additionally, P treatment decreased PRA levels below that observed after ovariectomy. Furthermore, when P was combined with E, it blunted the up-regulation of PRA by E. These results are in agreement with previous studies that have shown estrogenic regulation of PR in the adult virgin mouse mammary gland (12), and our results demonstrate that PRA is the predominant isoform that is regulated by E. Based on in vitro studies, progestins are reported to down-regulate PR (11). Our studies confirm down-regulation of PR levels by P and show that this effect is specific for the PRA isoform in the adult virgin mouse mammary gland.

PRA mediates sidebranching
Alveolar development proceeds through a specific sequence of proliferative and morphological events. During pregnancy, the earliest event in alveolar development is the production of ductal sidebranches. Our studies indicate that ductal sidebranching can be effectively induced by P in OVX mice and does not require E. We have previously shown that PRA is the predominant isoform expressed in the nulliparous mouse mammary gland (7), and a recent study using the same anti-PRA antibody confirmed this result (22). Because ductal sidebranching is induced at a time when PRA is the predominant isoform expressed, we conclude that this process is mediated by P acting through PRA.

Interestingly, sidebranching can be induced by E+P treatment in the PRA gene-deleted mouse (PRAKO) (23). The PRAKO mouse studies were carried out in the C57BL/6 genetic background. C57BL/6 adult wild-type mice have less developed mammary glands when compared with other strains, such as BALB/c (24). Additionally, the C57BL/6 strain is less responsive to hormones than the BALB/c strain in which our studies were carried out (25). In particular, we have found C57BL/6 mice to be much less responsive to P than BALB/c mice and exhibit delayed sidebranching during pregnancy (our unpublished observations). Therefore, it is possible that additional mechanisms that promote ductal sidebranching may be operative in the C57BL/6 strain and might explain the lack of a phenotype in the C57BL/6 PRAKO mouse.

Ductal sidebranching is accelerated in mice treated with E+P and because E increases PRA levels, it is likely that E contributes to ductal sidebranching through its positive effect on the level of PRA. Three days of E+P treatment produced the greatest colocalization of PRA with BrdU and PRA with nuclear cyclin D1, indicating that a subset of PRA+ cells was proliferating in response to E+P treatment at this time. However, in both P- and E+P-treated mice, the majority of cells that proliferate and form sidebranches were PRA–. The delayed development of sidebranches in P-treated mice most likely reflects the overall lower proliferation observed after P treatment compared with E+P treatment. Interestingly, although a significant percentage of PRA+ cells colocalized with nuclear cyclin D1 after P treatment, only a small percentage of PRA+ cells were BrdU+. This suggests that overall fewer PRA+ cells proliferated after P treatment. Alternatively, it is possible that treatment with P alone leads to slower progress through the G1 phase of the cell cycle, which is reflected by the difference in colocalization with PRA and nuclear cyclin D1, a G1 phase marker, or BrdU, an S phase marker.

PRB up-regulation by P
We found that PRB was expressed at a detectable level only after sustained exposure to P. E alone did not result in up-regulation of PRB; however, E accelerated the up-regulation by P. The earliest detection of significant PRB levels coincided with the development of alveoli at 5 d of E+P or 10 d of P treatment. Why prolonged treatment with P was required to obtain increased PRB levels is not entirely clear. The coincident timing of the decreased levels of PRA, the initiation of alveologenesis, and PRB up-regulation suggest that these events are linked.

One possible explanation for the requirement of prolonged P treatment to up-regulate PRB is that PRA inhibits PRB expression. In this case, one would expect that the up-regulation of PRB by P would occur when PRA levels are at their lowest. However, this explanation is not compatible with the observation that PRA levels are decreased faster and to a lower level after treatment with P alone (Fig. 1AGo), yet PRB expression is up-regulated later and less robustly in P-treated glands compared with E+P-treated glands (Fig. 6Go).

There are two significant differences between P- and E+P-treated glands that may affect the up-regulation of PRB: 1) the overall lower amount of proliferation and 2) the longer time required for ductal sidebranching and the development of alveoli to occur in the P-treated glands compared with E+P-treated glands. It has been hypothesized by others that the adult virgin mammary gland contains progenitor cells that give rise to three different cell lineages: ductal luminal cells, alveolar luminal cells, and myoepithelial cells (26). It is possible that the cells that proliferate to form the sidebranches in response to P are derived from progenitor cells committed to the alveolar luminal cell lineage and that it is a property of these cells to express PRB and form alveoli. Once PRB expression is induced, then P acting through PRB may form a positive regulatory loop to further increase PRB expression and the expansion of alveolar cells.

The role of E and ER{alpha}
E up-regulation of PRA was correlated with the coexpression of ER{alpha} and PRA within the same cells. Based on this observation, it is likely that E acting though ER{alpha} regulates PRA through a direct mechanism in PRA+ cells. We also observed that E down-regulated ER{alpha}, which may indicate that decreases in PRA after prolonged E+P treatment are partially due to a loss of ER{alpha}. Surprisingly, we found that ER{alpha} was not expressed in the majority of PRB+ cells. Although E enhanced the up-regulation of PRB, it does not appear to be due to a direct, ER-mediated effect in PRB+ cells. We propose that E may facilitate the up-regulation of PRB through the maintenance of PRA and lead to the enhancement of sidebranching and the expansion of the putative alveolar cell lineage in which PRB is then induced. Additionally, we considered the possibility that E could enhance PRB up-regulation indirectly through a systemic effect by increasing plasma prolactin (Prl) levels. However, treatment of OVX adult mice for 5 d with Prl alone had no stimulatory effect on mammary gland morphology. Also, the morphology of the mammary gland after treatment for 5 d with P+Prl was not different from that observed after treatment with P alone (our unpublished observations).

P induces sidebranching and alveologenesis through direct and paracrine mechanisms
It has been reported previously that, in the virgin mouse, mammary gland proliferating cells are PR negative (5, 27). This has been interpreted to mean that P induces proliferation through a paracrine mechanism(s) (5, 27). In the present study, at least 84% of the cells proliferating at the time of ductal sidebranching were PRA– (Fig. 4BGo). These results suggest that P acting on PRA+ cells may induce a paracrine factor that stimulates the proliferation of PRA– cells. Studies by others have implicated Wnt4 as a paracrine mediator of P-induced sidebranching (28). Thus, our study provides further evidence for a paracrine mechanism of P action and indicates that this mechanism is operative in PRA+ cells during sidebranching. We also have made the novel observation that, in addition to increasing proliferation in epithelial cells, P also increased proliferation in myoepithelial cells. Proliferation of myoepithelial cells was observed during both ductal sidebranching and alveologenesis. Because myoepithelial cells lack both PRA and PRB, this indicates that their proliferation is mediated through an indirect effect of P. The mechanisms operative in P-induced proliferation of PR– luminal epithelial cells and myoepithelial cells are currently under investigation.

Cyclin D1 expression has been shown to be regulated by P (16) and is believed to be essential for alveolar development leading to lactation (17). We have shown that cyclin D1 levels were increased by treatment with P or E+P. The highest percentage of cyclin D1+ cells was observed after 3 d of E+P treatment and coincided with the development of sidebranches. Colocalization of cyclin D1 with PRA and colocalization of PRA with BrdU were also highest at this time point. However, 60% of cyclin D1+ cells were PR–. These results also indicate that cyclin D1 may be regulated by P in PRA– cells through a paracrine mechanism(s) and that the up-regulation of cyclin D1 by P in both PRA+ and PR– cells promotes the proliferation that produces sidebranching.

During alveologenesis and alveolar expansion at 5 or 10 d of E+P treatment, colocalization of PRA with cyclin D1 or BrdU was significantly decreased. The decrease in colocalization of PRA with cyclin D1 after 5 or 10 d of E+P treatment is similar to the decreased colocalization during extensive alveolar expansion at d 14 of pregnancy (7). In contrast, cyclin D1 and PRB were highly colocalized after 10 d of E+P during alveolar expansion and similar to the level of colocalization at d 14 of pregnancy (7). Of the total cyclin D1+ cells, 40% were PRB+, less than 5% were PRA+, and about 55% of cyclin D1+ cells were PR–. This suggests that the up-regulation of cyclin D1 by P in both PRB+ and PR– cells likely promotes the proliferation required for alveolar expansion. Thus, P acting on either PRA+ or PRB+ cells appears to produce indirect effects on PR– cells that promote sidebranching and alveologenesis, respectively.

E enhances P-induced sidebranching and alveologenesis
We propose the following two models that integrate the effects of E and P to explain the observed differences in PRA and PRB expression, proliferation, ductal sidebranching, and alveologenesis in P- vs. E+P-treated mice. The first model describes the sequence of events resulting from E+P treatment. In this case, we propose that the high proliferation index observed after 3 d of E+P treatment is due to the combination of 1) robust stimulation of proliferation of PR– cells by paracrine factors induced by P in PRA+ cells, and 2) proliferation of a subpopulation of PRA+ cells, both facilitated by maintenance of PRA levels by E. Longer treatment with E+P, after 5 and 10 d, results in the down-regulation of ER{alpha} by E that, together with P, leads to down-regulation of PRA. Proliferation of putative alveolar progenitor cells in sidebranches and sustained P exposure leads to the increase in PRB levels. An earlier increase in PRB, by 5 d, results in earlier and more extensive alveolar development.

The second model describes the sequence of events resulting from treatment with P alone. The lower amount of proliferation after 3 d of P treatment is due to 1) a less robust paracrine induction of proliferation in PRA– cells, and 2) reduced proliferation of PRA+ cells, both due to the lower level of PRA in the absence of the E. Thus, in the absence of E, 5 d of P treatment are required to produce an amount of ductal sidebranching comparable to that observed by 3 d of E+P. Subsequent to the reduced proliferation of the putative alveolar progenitor cells during ductal sidebranching, up-regulation of PRB and alveologenesis are delayed.

In summary, we have shown that PRA and PRB are differentially regulated. PRA is up-regulated by E and down-regulated by P, whereas PRB is up-regulated by P. ER{alpha} colocalizes with PRA, and this suggests that E directly up-regulates PRA through an ER mediated mechanism. PRB does not colocalize significantly with ER, and if E has a role in PRB regulation, it likely occurs through an indirect mechanism. The proliferative and morphological changes in the mammary gland that occur during pregnancy were mimicked in our experiments by continuous treatment with either P or E+P. We have shown that P acting on PRA+ cells caused ductal sidebranching by promoting proliferation of both PRA+ and PRA– cells. This was followed temporally by the induction of PRB and P action in PRB+ and PRB– cells to cause the formation and expansion of alveoli.


   Footnotes
 
This work was supported by the Breast Cancer and the Environment Research Centers Grant U01 ES/CA 012800 from the National Institute of Environment Health Science and the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Environment Health Science or National Cancer Institute, National Institutes of Health. This work was also supported by Department of Defense Breast Cancer Research Program Fellowship DAMD17-03-1-0605 to M.D.A.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 22, 2007

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; DAPI, 4',6-diamidino-2-phenylindole, dilactate; E, estrogen; E+P, estrogen plus progesterone; ER, E receptor; OVX, ovariectomized; P, progesterone; PR, P receptor; Prl, prolactin; {alpha}-SMA, {alpha}-smooth muscle actin.

Received December 20, 2006.

Accepted for publication February 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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