ARTICLE |
Correspondence to: Hugh J.S. Dawkins, Bill & Rhonda Wyllie Laboratory, Urological Research Centre & Clinic, Univ. of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Perth, Western Australia 6009.
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Summary |
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The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor- (ER
). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [3H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [3H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERß, suggesting that the role of ERß in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:13231330, 1999)
Key Words: mammary epithelium, proliferation, progesterone receptor
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Introduction |
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The mammary gland is unusual in that most of its growth and development occurs after birth. In the mouse, the mammary gland undergoes extensive growth during puberty (46 weeks of age), resulting in the formation of a characteristic network of branching ducts that fills the adult mammary fat pad (
In a previous study ( in both pubertal and adult mice. Progesterone has also been ascribed a significant role in stimulating the proliferation and differentiation of mammary epithelial cells during the normal development and function of the female mouse mammary gland (
-negative cells. Several lines of evidence indicate that the actions of progesterone are directly mediated by the progesterone receptor (PR), a member of the steroid receptor superfamily of nuclear transcription factors (
PR expression has been reported in the highly mitotic cap cell population of the TEBs during puberty and in the majority of the luminal cells of mammary ducts during lobuloalveolar development in pregnant adult mice (-negative, ERß was detected in low levels in this cell population, introducing the possibility that estrogen-induced PR expression or proliferation might be mediated through this receptor.
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Materials and Methods |
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Animals and Tissues
This study was performed in compliance with Australian State and Federal animal protection laws under a permit issued by the Animal Experimentation and Ethics Committee of the University of Western Australia. Mammary glands from virgin female Balb/c mice (Animal Resource Centre; Perth, Western Australia) 5 weeks of age (pubertal) and 1012 weeks of age (adult) were used in these studies. To investigate the role of ovarian hormones during mammary gland growth in adult mice, animals between 10 and 12 weeks of age were staged for estrus by vaginal smear over several days (
Immunohistochemistry
Sections were dewaxed in xylene, rehydrated, and washed in Tris-buffered saline (TBS, pH 7.4). Slides were immersed in 5 mM ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA), pH 8.0 (Sigma), and boiled for 15 min in a pressure cooker in a microwave. After antigen unmasking, sections were incubated for 20 min each in avidin and biotin blocking solutions (DAKO Biotin blocking kit; Carpinteria, CA). They were then incubated in 1.5% H2O2 (BDH, Merck; Victoria, Australia) in methanol (BDH) for 10 min, then blocked with 20% normal horse serum (NHS) in TBS for 30 min.
Sections were incubated overnight in one of the following antisera: rabbit polyclonal anti-PR (2 µg/ml in blocking solution; SC-538, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-ERß (5 µg/ml in blocking solution; Affinity Bioreagents, Golden, CO), rabbit polyclonal anti-ER (1 µg/ml in blocking solution; SC-542, Santa Cruz Biotechnology). Sections were incubated in swine anti-rabbit biotinylated secondary antibody (Dako) diluted 1:200 in blocking solution, followed by streptavidinperoxidase (Silenius; Melbourne, Australia) diluted 1:200 in TBS. All incubations were carried out at RT for 50 min. Between incubations, sections were washed three times for 5 min in PBS containing 0.2% polyoxyethylene sorbitan monolaurate (Tween-20). Immunoreactivity was detected by incubation of sections in metal-enhanced 3,3'-diaminobenzidine tetrahydrochloride (DAB; Pierce, Perth, Australia) for 1 min. The sections were washed in dH2O for 10 min, then transferred to 70% ethanol overnight before being dipped in Kodak NTB-2 auto-radiographic emulsion as described previously (
Data Collection and Analysis
At least 15 randomly chosen TEBs and/or ducts were counted per animal in serial sections of the mammary glands, representing >2000 epithelial cells for each mouse, using an Olympus BX-40 light microscope at x1000 (oil immersion). Cells were scored according to location, receptor expression (+ or -), and [3H]-Tdr labeling [background grain counts were low in all sections and nuclei with three or more silver grains above them were counted as positive (
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Results |
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Progesterone Receptor Expression in Pubertal Mice (5 Weeks)
PR-positive cells were recognizable by a well-defined brown nuclear stain (Figure 1A) and were present in mammary epithelial cells during all stages of pubertal development. The PR and proliferation status in the terminal end buds (TEBs) and the ducts of 5-week-old mice are presented separately below. Similarly, ER immunoreactivity was detected as a nuclear stain in a proportion of mammary epithelial cells, as previously described (
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Terminal End Buds
PR immunoreactivity was observed in all TEBs and was heterogeneously distributed in both the cap and body cells (Figure 1A). Of the 63 TEBs scored (representing 7435 cells), the majority of both the outer cap cell layer (90 ± 2.9%) and the inner population of body cells (83 ± 0.5%) were PR-positive (Figure 2A and Figure 2B, respectively). All cap cells were ER-negative (Figure 2A), and in the body of the TEBs 62 ± 0.2% of epithelial cells were ER
-positive (Figure 2B).
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When cells were scored for S-phase, 95 ± 1.6% of the [3H]-Tdr-positive cap cells were PR-positive and all of these cells were ER-negative (Figure 2C). In the body of TEBs, 83 ± 0.5% of proliferating cells were PR-positive compared to 63 ± 1.3% ER
-positive proliferating cells (Figure 2D). The percentage of body cells labeled with [3H]-Tdr (thymidine labeling index, TLI) was 13 ± 0.7%, significantly less (p=0.03) than that of the cap cells, which had a TLI of 24 ± 2.9%. Nevertheless, despite the relatively lower TLI of the body cells, because they constitute the majority of cells in TEBs (76 ± 1.1%) they also comprised the majority of the proliferative compartment (64 ± 0.7%).
Ducts
PR was expressed in both the basal and the luminal cells of mammary ducts in 5-week-old mice (Figure 1B). A total of 55 ducts were examined representing 4302 cells. PR was expressed in a minority (43 ± 1.9%) of basal cells (Figure 3A), all of which were ER-negative. In the luminal cell population of the ducts, 86 ± 4.1% of cells were PR-positive (Figure 3B), and only 55 ± 0.4% of luminal cells were ER
-positive (Figure 3B). The proportion of ER
-positive luminal cells in the ducts was significantly less than the proportion of ER
-positive body cells in the TEBs (p=0.004).
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The majority (78 ± 8.6%) of proliferating basal cells in the ducts were PR-positive, and all were ER-negative (Figure 3C). Almost all proliferating luminal cells (94 ± 2.0%) were PR-positive (Figure 3D); only 54 ± 5.0% of luminal cells labeled with [3H]-Tdr expressed ER
. Although the relative TLI in the luminal cell population (TLI = 7 ± 0.4) appeared to be less than that in the basal cell population (TLI = 10 ± 2.6%) this difference was not statistically significant (p=0.19). However, like the body cells of the TEBs, the inner luminal cells comprise the largest proportion of the duct epithelium (80 ± 1.9%) and hence contain the majority of duct proliferating cells (73 ± 4.4%).
Progesterone Receptor Expression in Adult Mice (1012 Weeks Old)
Staining of adult mammary tissues revealed that both basal and luminal cells expressed detectable levels of PR protein (Figure 4). In these ducts, as in those from pubertal animals, the majority of basal cells do not express PR either during pro-estrus, when 25 ± 1.4% of cells were PR-positive, or during estrus, when 32 ± 8.3% of basal cells were PR-positive (Figure 5A and Figure 5B). The majority of luminal cells have detectable levels of PR during both pro-estrus (83 ± 0.8%) and estrus (80 ± 4.1%) (Figure 5B). All basal cells were ER-negative, but approximately half of the luminal cells expressed ER
during both pro-estrus (50.6 ± 3.9%) and estrus (43.0 ± 4.4%) (Figure 5A). There was no significant difference in the proportions of PR-positive basal (p=0.47) or luminal cells (p=0.43), nor of ER
-positive luminal cells (p=0.21) during the different stages of estrus.
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Although proliferation of mammary epithelial cells is mainly observed during the estrous phase of the murine estrous cycle, cells in S-phase, detected by [3H]-Tdr labeling, were also observed during pro-estrus. During the latter, only 10 of 981 basal cells counted were labeled with [3H]-Tdr, and seven of these were stained positively for PR (Figure 6A). In the luminal cell population, 34 of 36 [3H]-Tdr-labeled cells were PR-positive (from 4735 cells counted), compared to only seven of 40 ER-positive [3H]-Tdr-labeled cells (from 6258 luminal cells counted) (Figure 6B).
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During estrus, both basal and luminal cells were stimulated to undergo DNA synthesis. As in pro-estrus, the majority of [3H]-Tdr-labeled basal cells were PR-positive (34 of 49 basal cells labeled with [3H]-Tdr from 1007 counted). During estrus there was an approximately ninefold increase in the number of proliferating luminal cells, the majority of which were PR-positive (281 of 316 luminal cells labeled with [3H]-Tdr from 5323 counted). The proportion of ER- positive proliferating luminal cells also increased during estrus to comprise approximately 60% of all proliferating cells observed compared with approximately 20% during pro-estrus.
Estrogen Receptor-ß (ERß) Expression
ERß mRNA was detected in the mammary glands of both pubertal and adult mice in pro-estrus and estrus using RT-PCR (not shown) (
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Discussion |
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Progesterone can stimulate mammary epithelial proliferation, and its specific receptor, PR, is expressed by a proportion of mammary epithelial cells ( and ERß in mammary epithelial cells was compared with these data.
In the mammary glands of pubertal mice, the majority of cells within the TEBs expressed PR, a finding consistent with the staining patterns presented in a previous immunohistochemical study (
Proximal to the TEBs, the basal and luminal cells of the ducts are continuous with and regarded as being developmentally related to the cap and body cell layers of the TEBs, respectively (
Progesterone has been proposed as the principal mammary mitogen in adult mice (
Estrogen has an established role in stimulating mammary proliferation. However, in the present and in a previous study we have demonstrated that the majority of proliferating mammary epithelial cells do not express ER (
-negative. Although estrogen induces PR expression in adult mice (
expression. Furthermore, a comparison of the proportion of ER
-positive and PR-positive luminal cells reveals that approximately 30% do not co-express these two receptors in adult mammary ducts. Recently, studies in ovariectomized mice have demonstrated constitutive expression of PR in a proportion of mouse mammary epithelial cells (
and -ß) (
The identification of ERß expression in the mammary glands of pubertal and adult mice in the present study was anticipated by the finding of ERß mRNA in the mammary gland (
In summary, this study has demonstrated that epithelial cells of the pubertal and adult mammary glands in mice exhibit a characteristic expression of the steroid hormone receptors ER, ERß, and PR. Expression and co-expression of these receptors can be related to proliferation of specific cell types in the gland and are likely to direct formation of tertiary structures in the mammary gland, such as side branching in the pubertal gland and alveolar bud formation in the adult gland. Further mapping of co-expression of steroid hormone receptors in individual cells and proliferation or migration of these cells during puberty or in the adult mouse will delineate the functional roles of the steroid hormones in development and maintenance of the mammary gland and in the preparation of the mammary ducts for pregnancy and lactation.
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Acknowledgments |
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Supported by an Arnold Yeldham and Mary Raine Medical Research Foundation grant and by the Urological Research Centre, University of Western Australia. NZ was supported by a Raine/SGIO scholarship.
We thank Ms Sharon Redmond for technical advice and Mr Janni Mirosevich for help with figure layout.
Received for publication February 8, 1999; accepted April 27, 1999.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bresciani F (1971) Ovarian steroid control of cell proliferation in the mammary gland and cancer. In Hubinot PO, Leroy F, Galand P, eds. International Seminar on Reproductive Physiology and Sexual Endocrinology. Vol 3. Basel, Karger, 130-159
Chepko G, Smith GH (1997) Three division-competent, structurally-distinct cell populations contribute to murine mammary epithelial renewal. Tissue Cell 29:239-253[Medline]
Cole HA (1933) The mammary gland of the mouse, during the oestrus cycle, pregnancy and lactation. Proc R Soc Lond [B] 114:136-161
Daniel CW, Silberstein GB (1987) Postnatal development of the rodent mammary gland. In Neville MC, Daniel CW, eds. The mammary gland: Development, regulation and function. New York, Plenum Publishing, 3-36
DeOme KB, Faulkin LJ, Bern HA, Blair PB (1959) Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res 19:515-520
Fendrick JL, Raafat AM, Haslam SZ (1998) Mammary gland growth and development from the postnatal period to postmenopause: ovarian steroid receptor ontogeny and regulation in the mouse. J Mam Gland Biol Neoplasia 3:7-22
Graham JD, Clarke CL (1997) Physiological action of progesterone in target tissues. Endocrin Rev 18:502-519
Haslam SZ (1988) Progesterone effects on deoxyribonucleic acid synthesis in normal mouse mammary glands. Endocrinology 122:464-470[Abstract]
Haslam SZ, Counterman LJ (1991) Mammary stroma modulates hormonal responsiveness of mammary epithelium in vivo in the mouse. Endocrinology 129:2017-2023[Abstract]
Imagawa W, Bandyopadhyay GK, Nandi S (1990) Regulation of mammary epithelial cell growth in mice and rats. Endocrin Rev 11:494-523[Medline]
Katzenellenbogen BS, Norman MJ (1990) Multihormonal regulation of the progesterone receptor in MCF-7 human breast cancer cells: interrelationships among insulin/insulin-like growth factor-I, serum, and estrogen. Endocrinology 126:891-898[Abstract]
Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson J-Å, Smithies O (1998) Generation and reproductive phenotypes of mice lacking estrogen receptor ß. Proc Natl Acad Sci USA 95:15677-15682
Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery Ca Jr Shyamala G, Conneely OM, O'Malley BW (1995) Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 9:2266-2278[Abstract]
Nandi S (1958) Endocrine control of mammary gland development and function in the C3H/He Crgl mouse. J Natl Cancer Inst 21:1039-1063
Nandi S (1959) Hormonal control of mammogenesis and lactogenesis in the C3H/He Crgl mouse. U Cal Publications Zool 65:1-128
Shyamala G (1997) Roles of estrogen and progesterone in normal mammary gland development. Trends Enodcrinol Metab 8:34-39
Shyamala G, BarcellosHoff MH, Toft D, Yang X (1997) In situ localisation of progesterone receptors in normal mouse mammary glands: absence of receptors in the connective and adipose stroma and a heterogeneous distribution in the epithelium. J Steroid Biochem Mol Biol 63:251-259[Medline]
Shyamala G, Yang X, Silberstein G, BarcellosHoff MH, Dale E (1998) Transgenic mice carrying an imbalance in the native ratio of A to B forms of progesterone receptor exhibit developmental abnormalities in mammary glands. Proc Natl Acad Sci USA 95:696-701
Silberstein GB, Van Horn K, Shyamala G, Daniel CW (1996) Progesterone receptors in the mouse mammary duct: distribution and developmental regulation. Cell Growth Differ 7:945-952[Abstract]
Smith GH, Medina D (1988) A morphologically distinct candidate for an epithelial stem cell in mouse mammary gland. J Cell Sci 89:173-183
Topper YJ, Freeman CS (1980) Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049-1106
Tremblay A, Tremblay GB, Labrie C, Labrie F, Giguere V (1998) EM-800, a novel antiestrogen, acts as a pure antagonist of the transcriptional functions of estrogen receptors and ß. Endocrinology 139:111-118
Williams JM, Daniel CW (1983) Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis. Dev Biol 97:274-290[Medline]
Zeps N, Bentel JM, Papadimitriou JM, D'Antuono MF, Dawkins HJS (1998) Oestrogen receptor negative epithelial cells in mouse mammary gland development and growth. Differentiation 62:227-237[Medline]
Zeps N, Dawkins HJS, Papadimitriou JM, Redmond SL, Walters MN-I (1996) Detection of a population of long-lived cells in mouse mammary epithelium. Cell Tissue Res 286:525-536[Medline]