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Endocrinology Vol. 143, No. 10 4074-4083
Copyright © 2002 by The Endocrine Society

ARTICLE

Mammary Gland Development in Transgenic Male Mice Expressing Human P450 Aromatase

Xiangdong Li, Anni Wärri, Sari Mäkelä, Tommi Ahonen, Tomi Streng, Risto Santti and Matti Poutanen

Departments of Physiology (X.L., M.P.) and Anatomy (X.L., A.W., S.M., T.A., T.S., R.S.), Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland; and Department of Medical Nutrition, Karolinska Institute NOVUM (S.M.), S-14157 Huddinge, Sweden

Address all correspondence and requests for reprints to: Matti Poutanen, Ph.D., Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: matti.poutanen{at}utu.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently generated a transgenic mouse strain that expresses the human aromatase gene under the ubiquitin C promoter (AROM+). We have previously shown that in these mice the serum estradiol concentration is highly elevated, whereas the testosterone concentration is decreased. In the present study we examined mammary gland development in AROM+ male mice at different ages and found that the mammary glands of AROM+ males undergo ductal and alveolar development morphologically resembling that of terminally differentiated female mammary glands, expressing mRNA for a milk protein gene (ß-casein). The male mammary glands also express multiple hormone receptors typical for female mammary gland: estrogen receptor {alpha} and ß, progesterone receptor, and PRL receptor. Furthermore, data showed activation of the Stat5 (signal transducer and activator of transcription 5) signaling pathway in the AROM+ male mammary gland. Interestingly, the phenotype observed is in part reversible. Treatment with finrozole, a specific aromatase inhibitor, caused an involution of the differentiated phenotype of the mammary gland, marked by the disappearance of alveolar structures and the majority of the tertiary side branches of the ducts. The present animal model is a valuable tool for better understanding the cellular and molecular mechanisms involved in the development of gynecomastia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS AND estrogens have opposing actions on the mammary gland during embryogenesis; estrogens have stimulatory, and androgens have inhibitory effects on its growth. In adults the effect of androgens has been less studied, and the results are still controversial. However, an enlargement of the male breast, gynecomastia, may result from an unbalanced action of androgens and estrogens, either from decreased testosterone production and/or action or from increased estrogen production and/or action. In situations when the serum concentrations of androgens and estrogens do not display any alterations, gynecomastia is suggested to be due to excessive local estrogen formation in the mammary gland (1). Gynecomastia often develops as a consequence of antiandrogen therapy, which further supports the role of androgens in the development of gynecomastia. However, the mechanism of an antiandrogen action is not fully understood (2). Antiandrogens such as flutamide interfere with the testicular-hypothalamic feedback mechanism and subsequently stimulate androgen production, but they simultaneously block the receptor binding of androgens in the mammary gland.

The majority of the recent studies have focused on the prevalence and clinical treatment of gynecomastia, but the hormonal causes are still poorly understood, especially those of the direct and possible antagonistic actions of androgens on estrogen action or the androgen-independent action of estrogens in the male mammary gland. An animal model with a condition resembling gynecomastia would be a valuable tool for better understanding the cellular and molecular mechanisms involved in the development of gynecomastia as well as for implementation of preclinical studies on effective hormonal treatments affecting the androgen-estrogen balance, such as aromatase inhibitors. Transgenic male mice overexpressing P450 aromatase [mouse mammary tumor virus (MMTV)-arom+] have been reported to develop gynecomastia-like changes at the age of 3 months (3). Under the control of a complete MMTV-long terminal repeat, the aromatase transgene is reported to be activated at the onset of puberty and expressed in the mammary gland, resulting in the local overproduction of estrogens. The peripheral estrogen concentrations were only slightly elevated in these transgenic mice. We have recently generated another transgenic mouse strain that expresses the human P450 aromatase gene under the human ubiquitin C promoter (AROM+) (4). The ubiquitin C promoter is known to be activated on prenatal d 15 (5). Transgene expression was demonstrated in several adult tissues, such as testis and liver (4), and serum estradiol concentrations are highly elevated in adults (4 months of age), whereas testosterone concentrations are reduced (4). Furthermore, the PRL concentration in these mice is elevated.

In the present study we examined mammary gland development in AROM+ male mice at different ages. The data showed that these mammary glands undergo ductal and alveolar development morphologically resembling that of age-matched female mammary glands and express mRNA for a milk protein (ß-casein). Interestingly, mammary gland phenotype in AROM+ mice was in part reversible. These findings considerably differ from those described for MMTV-arom+ mice, which show much less developed mammary structures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the transgene
Generation of transgenic mice expressing human aromatase cDNA under control of the ubiquitin C promoter (AROM+) has been described previously (4). AROM+ female mice were maintained in a standard colony and used as breeders. They had free access to soy-free food pellets (SDS, Witham, UK) and tap water. All mice were handled in accordance with the institutional animal care policies of University of Turku (Turku, Finland). The genotyping of the AROM+ mice was carried out as described previously (4).

Morphological and histological assessment of mammary gland
For the whole mount preparations, the fourth inguinal mammary glands were removed from AROM+ male mice at the ages of 20 and 45 d and 4–9 months and from age-matched wild-type (WT) females. The mammary glands of the female mice were also removed at early and late gestation. Mammary gland whole mounts were prepared as described previously (6). The isolated glands were spread on glass slides and fixed with Carnoy’s fixative (acetic acid-ethanol). The slides with the specimen were rinsed in distilled water and stained with carmine alum overnight. The glands were destained using a series of washes with 70–100% ethanol, cleared in xylene, and mounted in Permount. For histological analyses, the fourth mammary gland removed from AROM+ male mice at the age of 4 months was fixed with 4% paraformaldehyde for at least 24 h and embedded in paraffin. Five-micrometer sections of the mammary specimens were prepared and stained with hematoxylin-eosin.

RT-PCR
Total RNA was isolated from mammary fat pad with the parenchyma using the acid phenol method, and RT-PCR was carried out. Four micrograms of total RNA were incubated with 10 IU avian myeloblastosis virus reverse transcriptase (Finnzymes, Espoo, Finland) at 43 C for 20 min. The cDNAs were then denatured at 95 C for 5 min and amplified by 25 cycles of PCR using the following conditions: 94 C for 1 min, 50 C for 1 min, and 72 C for 1 min, and an aliquot of the RT-PCR product was subjected to agarose gel electrophoresis and visualized by ethidium bromide staining. To control the total amount of RNA used, a 200-bp fragment of the ß-actin ribosomal protein gene was amplified. The primers used for the various RT-PCR analyses are described in Table 1Go (7, 8, 9, 10).


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  		Table 1. Primer pairs used in RT-PCR analyses in AROM+ mouse model

 
Immunohistochemistry
Five-micrometer thick sections were cut from paraffin-embedded mammary tissues and mounted on slides. After deparaffinization and rehydration in xylene and ethanol, they were placed in 10 mM citrate buffer (pH 6.0), followed by heating in a microwave oven for antigen retrieval. For this, three periods of 5 min each were used, after which the sections were treated with 3% H2O2 in PBS (pH 7.6) for 20 min. The sections were then incubated overnight at 4 C with in PBS containing 3% BSA and one of the following antibodies: 1) antibody for estrogen receptor {alpha} (ER{alpha}; rabbit polyclonal IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) used at a 1:200 dilution, 2) antibody for ERß (chicken polyclonal IgG; provided by Prof. J. Å. Gustafsson, Department of Medical Nutrition, Karolinska Institute NOVUM, Huddinge, Sweden) used at a 1:500 dilution, 3) antibody for progesterone receptor (PR; rabbit polyclonal IgG; Santa Cruz Biotechnology, Inc.) used at a 1:500 dilution, 4) antibody for proliferating cell nuclear antigen (PCNA; mouse monoclonal IgG; NovoCastra, Newcastle, UK) used at a 1:200 dilution, 5) antibody for activated signal transducer and activator of transcription 5 (Stat5; mouse monoclonal IgG; Advantex Bioreagents, Conroe, TX) used at a 1:500 dilution, and 6) antibody for PRL (rabbit polyclonal antiserum; NIDDK, NIH, Bethesda, MD) used at a 1:1000 dilution. The primary antibody bound was detected by using biotinylated goat antirabbit, antimouse, or antichicken IgG, followed by incubation with avidin-biotin-peroxidase complex (Vector Laboratories, Inc., Burlingame, CA), and was visualized using 3',3'-diaminobenzidine tetrahydrochloride. Sections were slightly counterstained with Mayer’s hematoxylin and mounted.

For immunostaining Npt2b and NKCC1, 7-µm-thick frozen sections were fixed with methanol (5 min) and acetone (2 min) at -20 C. Thereafter, the sections were incubated for 1 h with rabbit polyclonal antisera for Npt2b (1:200 dilution; provided by Dr. Jurg Biber, Department of Physiology, University of Zurich, Zurich, Switzerland) and for NKCC1 (1:1000 dilution; provided by Dr. Jim Turner, National Institute of Dental and Craniofacial Research, NIH). The antigen-antibody complexes were visualized using biotinylated antirabbit antibody (Vector Laboratories, Inc.) combined with streptavidin-fluorescein isothiocyanate (DAKO Corp., Glostrup, Denmark) complex. A confocal laser microscope setup (model TCS SP scanner and DMRE microscope, Lasertechnik, Leica Corp., Heidelberg, Germany) was used to detect emission light emitted from fluorescein isothiocyanate. The sections were exposed to a 488-nm excitation wavelength, and the emission was obtained at 495–540 nm. Four images at 2-µm intervals on the z-axis were collected with a confocal scanner equipped with an argon-krypton ion laser system (Omnichrome, Chino, CA) coupled with the SCANware 4.2a program (Leica Corp.).

Progesterone measurement
The mouse sera and standard tubes were extracted twice with 2 ml diethyl ether and evaporated under nitrogen. The residues were resuspended in 3% PBS-BSA, and progesterone was measured by RIA as described previously (11).

Investigational drug and control substances
The vehicle was prepared as follows: 0.25 g (carboxylmethyl-cellulose; lot 939512, Tamro Ltd., Vantaa, Finland) was weighed and solubilized in 50 ml deionized (Milli-Q, Millipore, Bedford, MA) water. The solution was prepared once a week and was stored at 4 C. An appropriate amount of an aromatase inhibitor, finrozole (MPV-2213ad, Hormos Medical Ltd., Turku, Finland), was weighed in a transparent glass mortar. A few drops of the vehicle were added, and the mixture was thoroughly mixed. Thereafter, one third of the final volume of the vehicle was added to the mortar and placed into an ultrasonic incubator for 5 min. This procedure was completed a total of three times to reach the final volume. The dose of finrozole was 10 mg/kg body weight, and it was daily given to mice by gavage in 0.2 ml for 6 wk.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of the mammary gland structure with whole mounts
No differences were detected in the structures of the mammary glands between WT and AROM+ female mice during the 4-month follow-up period (data not shown). However, analyzing the whole mounts of the mammary fat pads containing the parenchyma from transgenic and nontransgenic littermates revealed significant morphological changes in mammary glands of the AROM+ male mice, induced by the hormonal imbalance present in the transgenic male mice. As expected, mammary fat pads of WT males were totally devoid of ductal structures (Fig. 1AGo). In contrast, well organized ductal and lobulo-alveolar structures were present in the AROM+ males. Mammary ducts were seen prepubertally as early as d 20 after birth (Fig. 1FGo). In the pubertal AROM+ male mice (at 40 d of age; Fig. 1GGo), mammary ducts had started to grow and were well organized; the ductal tree resembled that of a normal age-matched virgin female mouse (Fig. 1BGo). The primary ducts of the AROM+ males contained approximately 10–15 side branches. Terminal end buds (TEBs) were clearly visible at the tips of the mammary ducts, indicating active ductal elongation.



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  		Figure 1. Whole mount analysis of mammary glands of WT and AROM+ male mice at different stages of development. The lymph node serves as a reference point to evaluate ductal outgrowth. A, The mammary fat pad of a WT male mouse is devoid of ductal structures. B and C, The mammary gland of a virgin WT female mouse at the age of 40 d (B) and at the age of 4 months (C). D and E, The mammary gland of a WT female mouse on d 12.5 of gestation (D) and on d 16.5 of gestation (E). F, Mammary gland of an AROM+ male mouse. At the age of 20 d, few rudimentary ducts emerging from the nipple are present proximal to the lymph node. G, Prominent TEBs are visible at the age of 40 d (arrow). H, At the age of 4 months, the ducts have reached the border of the fat pad. At this stage the TEBs have disappeared. I, At the age of 6 months, the gland displayed increased alveolar development (arrows) resembling structures present in the mammary glands of the female mice on d 12.5 of gestation. J, At the age of 9 months, extensive lobulo-alveolar development occurs that resembles the structures present in female mammary gland in late gestation (on d 16.5 of pregnancy). At this stage, the mammary fat pad is completely filled with secretory lobulo-alveolar structures.

 
The mammary ducts had completed their elongation in mature AROM+ male mice at the age of 4 months (Fig. 1HGo) and totally filled the mammary fat pads, similarly to those in age-matched virgin WT female mice (Fig. 1CGo). At this age, lobulo-alveolar structures were seen, and TEBs were no longer detectable, indicating termination of the ductal elongation at the age when it occurs in normal female mice. Histological sections indicated that ducts had a single layer of epithelial cells surrounded by stromal cells (Fig. 2AGo). Stainable material was frequently seen in the lumen of the ducts, suggesting that the epithelial cells had differentiated and were actively secreting (Fig. 2BGo). At the age of 6 months, increased tertiary side-branching and lobulo-alveolar development could be detected in AROM+ mammary glands (Fig. 1IGo), and the glands structurally resembled mammary glands of WT female mice at the second week of gestation (Fig. 1DGo).



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  		Figure 2. Histological analysis of mammary gland of the AROM+ male at the age of 4 months. A, Ducts and lobulo-alveolar structures are observable in AROM+ male mice. B, An alveolus with higher magnification. Lipid-rich secretory product is present in the alveolus (arrow). C–E, RT-PCR revealed that mRNA for ß-casein, NKCC1, and Npt2b, respectively, is expressed in mammary tissue of AROM+ male mice. F, Immunohistochemical staining for Npt2b in mammary epithelium of AROM+ mice. G, Lactating d 1 female used as a positive control for Npt2b staining. H, Immunohistochemical staining for NKCC1 in mammary epithelium of AROM+ mice. I, Lactating d 1 female used as a negative control for NKCC1 staining.

 
At the age of 9 months, the AROM+ male mammary glands (Fig. 1JGo) had differentiated further and resembled terminally differentiated female glands typically present in late gestation (16.5 d; Fig. 1EGo). The glands contained secretory products in lumen. To further determine the secretory activity of the mammary epithelium, the expression of ß-casein mRNA was analyzed in the mammary glands of AROM+ male mice at the age of 4 months. The data revealed that ß-casein mRNA was expressed in the AROM+ males, whereas no expression was detected in WT male mice (Fig. 2CGo). The expression of a milk protein is a further indication of female-like mammary gland development in AROM+ males. The differentiation stage of the AROM+ mammary gland was further studied by analyzing the expression of proteins characteristic for ductal epithelium (Na-K-Cl cotransporter, NKCCl) and for the secretory function of lactating females (sodium phosphate cotransporter isoform, Npt2b). Immunohistochemistry and RT-PCR data indicated that both of these proteins were expressed at a low level in a few epithelial structures of AROM+ male mammary gland at the age of 4 months (Fig. 2Go, D–I). However, most of the epithelial structures did not stain for either of these markers.

Immunocytochemical staining of ER{alpha}, ERß, PR, PCNA and epithelial markers in the mammary gland of AROM+ males
To analyze hormonal factors putatively involved in mammary gland development of AROM+ males, we performed an immunohistochemical analysis of the key steroid hormone receptors. Immunohistochemical staining for ER{alpha} in the AROM+ mammary gland revealed positive staining of the nuclei of the epithelial cells in both ducts and alveoli at the age of 4 months (Fig. 3AGo). A similar cellular distribution was observed for ERß. However, ERß-positive cells were more abundant than those positive for ER{alpha} (Fig. 3BGo).



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  		Figure 3. Immunohistochemical staining of steroid receptors and a proliferation marker in AROM+ male mammary glands. Sections from 4-month-old AROM+ male mice were stained with an antibody against ER{alpha} (A), ERß (B), PR (C), and PCNA (D). Brown color in the nuclei indicates positive staining for the corresponding protein. Arrows indicate densely stained nuclei.

 
As shown in Fig. 3CGo, PR was also frequently detected in the nuclei of alveolar and ductal epithelial cells. Positive and negative cells could be found adjacent to each other. Serum concentrations of progesterone in the AROM+ male mice were slightly elevated (mean ± SEM, 1.156 ± 0.161 and 0.660 ± 0.167 nM/liter in AROM+ and WT male mice, respectively), but the difference between the two groups was not statistically significant.

Staining of the mammary epithelium of AROM+ males with a cell proliferation marker (PCNA) at the age of 4 months showed positive cells not only in the developing alveoli, but in the ducts as well (Fig. 3DGo). The majority of the epithelial cells in alveoli and a few epithelial cells in the ducts were undergoing proliferation, as assessed by PCNA immunostaining. In line with the proliferative status of the mammary glands, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling analysis revealed that only a few epithelial cells were undergoing apoptosis (data not shown).

PRL-producing pituitary cells and PRL action in AROM+ mammary gland
AROM+ males were shown to have highly elevated concentrations of PRL in serum (4). To verify the origin of the circulating PRL, pituitary lactotrope cells were stained with a specific anti-PRL antibody. The number and density of PRL-producing cells was strikingly increased in AROM+ males compared with WT animals, and the positive cells were distributed throughout the anterior pituitary (Fig. 4Go, A and B). Furthermore, detectable expression of PRL receptor (PRLR) mRNA was found in mammary glands of AROM+ males (Fig. 4EGo). Hence, it is likely that the high serum PRL concentration present in AROM+ male mice directly contributes to the mammary gland differentiation. This was further tested by analyzing the presence and activation of Stat5a and Stat5b, known mediators of PRL action in the target cells. The analyses revealed that mRNA for both Stat5a and 5b were present in AROM+ male mammary gland (Fig. 4FGo), and based on the RT-PCR analyses, the expression of Stat5b was especially abundant. To further demonstrate that these PRL signal transducers were activated upon stimulation with PRL, the phosphorylation of Stat5 proteins was demonstrated immunohistochemically in AROM+ mammary glands at the age of 4 months. The data showed intense positive staining in the nuclei of epithelial cells, especially in the alveoli (Fig. 4CGo), similar to that found in the mammary glands of gestating WT female mice used as positive controls (Fig. 4DGo).



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  		Figure 4. Localization of PRL-producing cells in the pituitary and detection of PRL signaling pathway in mammary gland of AROM+ mice. Pituitary sections obtained from AROM+ male mice (A) and WT mice (B) at the age of 4 months were stained with an antibody to rat PRL. Brown staining indicates PRL-producing cells. C and D, Paraffin-embedded tissue sections were immunostained with a monoclonal antiphosphotyrosine-Stat5 antibody, and intensive positive nuclear staining was detected in epithelial cells of the mammary glands of AROM+ males (C). Lactating mouse mammary gland was used as a positive control (D). Arrows indicate densely stained nuclei. RT-PCR analyses showed the expression of PRLR (E) and Stat5a and Stat5b (F) expression in the AROM+ male mammary gland.

 
Reversal of the mammary gland phenotype in AROM+ mice treated with aromatase inhibitor (finrozole)
Interestingly, a 6-wk treatment of AROM+ male mice with finrozole, a specific aromatase inhibitor, at the age of 4–6 months caused involution of the differentiated phenotype of the mammary gland (Fig. 5Go). The alveolar structures and the majority of the tertiary side branches of the ducts disappeared. This provides evidence that the increased P450 aromatase activity in AROM+ male mice was the primary cause of the mammary gland phenotype observed.



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  		Figure 5. Whole mount analysis of mammary glands in the placebo-treated (A and C) and finrozole-treated (B and D) AROM+ male mice at the age of 4 months. The lymph node serves as a reference point to evaluate ductal outgrowth. The AROM+ male mice were treated orally with finrozole (10 mg/kg body weight) for 6 wk before autopsy. The alveolar structures and the majority of the tertiary side branches of the ducts have disappeared in finrozole-treated AROM+ mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Two different P450 aromatase overexpressing mouse strains have recently been described (4, 12). Male mice of both strains have changes in mammary gland development, but the changes seen in the two strains differ considerably from each other, as shown in the present study. Overexpression of the human P450 aromatase under the ubiquitin C (AROM+) promoter resulted in ductal proliferation, side-branching, lobulo-alveolar development, and expression of ß-casein mRNA, all characteristics for the development of the female mammary gland and consistent with the involvement of estrogen in mammary gland development in male also (13, 14). Mammary ducts were seen as early as on d 20 after birth. In addition to having elevated estrogen levels and decreased androgen levels, the AROM+ male mice were hyperprolactinemic, which is likely to contribute to the mammary gland phenotype in AROM+ male mice by promoting terminal differentiation (15). MMTV-arom+ transgenic male mice (3), however, displayed solitary ducts with hyperplastic mammary epithelium at the age of 3 months. They had slightly elevated circulating estradiol and unaltered PRL levels. The differences in the glandular structures between the two aromatase-overexpressing mouse strains could primarily be due to an earlier onset of excessive estrogen production in the AROM+ mice, whereas the MMTV promoter is not activated before puberty.

Estrogens are apparently not required for normal prenatal mammary gland development. In contrast, testosterone produced by the developing gonads of the male fetus is known to cause a condensation of mesenchyma around the neck of the gland, and hence, the mammary bud in the male starts to regress on gestational d 13 (16). Excessive estrogen production, starting presumably on gestational d 15 in AROM+ male embryos, would thus interfere with the normal testosterone action required for regression. In the absence of testosterone-induced regression, the mammary anlage is known to maintain its full competence for mammary gland development in male mice (17, 18). Because of the late onset of estrogen overproduction in MMTV-arom+ mice, they lack this counteraction by estrogen. It is well known that the mammary gland reaches its full differentiation, as defined by the expression of milk-specific genes, in the adult female after progression through puberty and gestation. The critical period of transition from incompetence to lactogenic competence occurs approximately 4 wk after birth (16). Even though the factors accounting for lactogenic competence are not known, this period could also render mammary gland development different in the two aromatase-overexpressing strains.

Our results are consistent with the concept that estrogens play a major role in promoting elongation of the mammary ducts (19, 20). Together with progesterone, estrogens are required for the development of tertiary ducts and the maintenance of ducts and lobulo-alveolar structures (21). A whole mount analysis revealed that the mammary glands of AROM+ male mice underwent a morphogenesis similar to that which normally takes place in females. Both ER subtypes were present in the alveolar and ductal epithelium of the AROM+ male mammary gland, in line with previous findings in female mice (22). However, immunohistochemically ERß-positive epithelial cells were more frequently detected than those positive for ER{alpha}. Interestingly, the mammary phenotype of AROM+ male mice was reversible, and a 6-wk treatment with finrozole, a novel and specific inhibitor of aromatization (23), caused mammary gland involution, i.e. disappearance of the alveolar structures and the majority of the tertiary side branches of the ducts. This further substantiates the primary role of estrogen in the development of the mammary gland in male mice.

Mammary glands of AROM+ male mice also expressed PRLR and PR, which are required for the normal differentiation and function of female mammary glands. PR expression in male AROM+ mammary epithelium is in agreement with the demonstrated induction of PR gene expression by estrogens in the female mammary gland epithelium after 7 wk of age (24, 25). This suggests that estradiol could have a stimulatory role for the action of progesterone in the AROM+ mammary gland as well. A number of studies have provided evidence implying that the responses attributed to estrogen could conceivably be due to the combined effects of estrogen and progesterone in the mammary gland (26, 27). For instance, the absence of PR has a profound effect on mammary gland development. In PR-deficient mice, the ductal structure has less extensive side branching and acinar development, and lacks interductal lobulo-alveolar structures (28, 29, 30). Progesterone concentrations of AROM+ male mice were not significantly elevated compared with those of WT male mice, but they were sufficient for efficient activation of the PR-mediated signaling systems in the AROM+ mammary gland.

There is ample evidence that chronic estrogen treatment causes hyperprolactinemia and the development of prolactinomas in rodents (31, 32). This was also seen in AROM+ male mice, which had high circulating PRL concentrations and pituitary glands with increased number of pituitary lactotropes. By using RT-PCR, we demonstrated that PRLR was also expressed in the mammary gland of AROM+ male mice. Hence, it is likely that similar to the female mice, PRL-dependent signaling systems are involved in terminal differentiation of the mammary gland in AROM+ male mice.

Janus kinase 2-Stat5 is the major signaling pathway for PRL action (33). Stat5 was initially described as a mammary gland factor stimulated by PRL (34), and development of ductal branching and lobulo-alveolar structures was shown to involve activated Stat5. The two Stat5 proteins, Stat5a and Stat5b, show high sequence conservation, suggesting that they execute related or identical functions (33). Stat5a has been reported to be particularly important for mammary gland differentiation and lactation (35). In Stat5a-null mice, outgrowth of lobulo-alveolar structures of the mammary gland was arrested during pregnancy, and the females were unable to lactate after parturition because of a failure in mammary gland terminal differentiation (35). However, mammary gland development is impaired in Stat5b null mice, and although milk protein genes are expressed, the mice produce an insufficient amount of milk to feed their pups (35). The present study showed that PRL induced Stat5 (Stat5a and Stat5b) phosphorylation and nuclear translocation in the mammary gland of AROM+ male mice, which is in line with previous observations in females (9, 10, 36, 37). Similarly, the Stat5-mediated action of PRL is mandatory for fully differentiating mammary glands in females (35, 38). Our data, furthermore, indicate that especially Stat5b mRNA is abundantly expressed in the AROM+ male mammary gland. Interestingly, at the age of 4 months a milk protein, ß-casein, mRNA was also expressed in AROM+ mammary glands. Recent findings have shown that ß-casein is not expressed in female PRLR- and Stat5-null mammary glands (10), further indicating the activation of a PRLR-dependent signaling system in AROM+ males. Two marker proteins have been shown to discriminate between the ductal and alveolar epithelium (10). NKCC1 is expressed at high levels in virgin mice, but is dramatically decreased in developing alveoli, whereas Npt2b is not expressed in virgin or early pregnant mice, but is detected during late pregnancy and through midlactation (d 5). In 4-month-old AROM+ mammary glands, both Npt2b and NKCC1 were expressed in some of the epithelial cells forming ducts and alveoli-like structures. This is further proof of the female-like differentiation of the mammary gland epithelium in AROM+ males. However, more analyses need to be carried out to conclude how closely the ductal development resembles that found in the females.

Remnants of mammary ducts with atrophic epithelial cells can be detected microscopically in men. At early stages of human idiopathic gynecomastia, the mammary gland is characterized by a proliferation of both stroma and ductal epithelium, when the ducts elongate and branch (39). ER and PR as well as PRLR are present in the mammary glands of men with gynecomastia (40, 41, 42). During the progression of the condition, the stroma becomes more collagenous and less edematous, and the epithelium regresses (39). MMTV-arom+ mice showed solitary ducts with epithelial hyperplasia (3), but no alveolar structures were described. Morphologically, these structures resemble those seen in idiopathic human gynecomastia. It has been demonstrated that a slight increase in the ratio of plasma estrogen to androgen concentrations has little influence on the intraglandular levels of hormones and eventually on the induction of acinar and lobular formation in the male breast (43). This is in line with the histological changes seen in MMTV-arom+ mice, which have less profound changes in the serum estrogen/androgen ratio compared with AROM+ male mice. The overall structure of the mammary gland in AROM+ mice closely resembles that in normal age- and stage-matched females. Interestingly, a prolonged intake of progestins and estrogens will result in a similar full development and maintenance of the female-type mammary gland in male to female transsexuals (43). In addition to an earlier activation of ubiquitin C promoter, the more extensive aromatization in AROM+ mice may account for the differences between the two aromatase-overexpressing mouse strains.

Several of the hormones involved in normal mammary gland development, including estrogens, progesterone, and PRL, also participate in mammary gland carcinogenesis in females. Female sex steroids especially are known to be associated with increased breast cancer risk in women (19), and a cross-talk between the steroids and the PRL-dependent pathways has been proposed to act in a synergistic way to activate mammary gland carcinogenesis (26). However, although transgenic female mice overexpressing PRL develop mammary tumors at the age of 11–15 months (44), the putative role of PRL in the development of human breast cancer is still under debate (15). Even though the signaling pathways for both the female sex steroids and PRL are present in the AROM+ male mammary gland, no sign of carcinogenesis was detected during the 9-month-long follow-up period, suggesting that the proliferative pressure of the altered hormonal environment was not sufficient for initiation of immortalization of the ductal epithelium in the AROM+ male mammary gland.

In conclusion, the transgenic mouse model gives us a novel tool for a better understanding of normal mammary gland development and for a detailed analysis of the pathological events occurring in the mammary glands of men who develop gynecomastia. Our studies with AROM+ male mice indicate that chronic stimulation by estrogen activates a series of hormonal changes leading to an altered pattern of gene expression, including genes involved in the morphogenesis of the mammary gland. The precise role for these factors in the development of gynecomastia remains to be elucidated. The crossing of AROM+ mice with knockout mice deficient in the signaling systems for various hormones will be essential in further studies. Studies using specific receptor antagonists should also be carried out. However, the AROM+ mammary gland provides a sensitive model for preclinical studies of effective hormonal treatments affecting androgen/estrogen balance, such as aromatase inhibitors.


   Acknowledgments
 
We thank Ms. Johanna Vesa and Ms. Tuula Tanner for technical assistance with the microscopic studies, Ms. Kati Raepalo for technical assistance with genotyping the mice, and Mr. Ramin Shariatmadari for helping with the confocal microscopic analyses. Ansa Ojanlatva made linguistic suggestions.


   Footnotes
 
This work was supported by the National Technology Agency of Finland (Contract 40021/00) and the Academy of Finland.

Abbreviations: ER, Estrogen receptor; MMTV, mouse mammary tumor virus; PCNA, proliferating cell nuclear antigen; PR, progesterone receptor; PRLR, PRL receptor; Stat5, signal transducer and activator of transcription 5; TEB, terminal end bud; WT, wild-type.

Received February 13, 2002.

Accepted for publication June 10, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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