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REPRODUCTION-DEVELOPMENT |
Laboratory of Reproductive and Developmental Toxicology (S.O.M., K.S.K.) and Comparative Medicine Branch (J.A.C., P.H.M.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Kenneth S. Korach, National Institute on Environmental Health Sciences, MD B3-02, 111 T. W. Alexander Drive, P.O. Box 12233, Research Triangle Park, North Carolina 27709, E-mail: ; or Stefan O. Mueller, Institute of Toxicology, Merck KGaA, 64271 Darmstadt, Germany, E-mail: . stefan.o.mueller{at}merck.de
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Abstract |
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and the progesterone receptor (PR) and is tightly regulated by the interaction of the mammary epithelium with the stromal compartment. Studies using mammary tissues of immature mice have indicated that stromal but not epithelial ER is required for mammary gland growth. This study investigates whether these same tissue growth requirements of neonate tissue are necessary for mammary development and response in adult mice. Mammary epithelial cells were isolated from adult mice with a targeted disruption of the ER gene (ERKO) or from wild-type counterparts and injected into epithelial-free mammary fat pads of 3-wk-old female ERKO or wild-type mice. Ten weeks after cell injection, analysis of mammary gland whole mounts showed that both stromal and epithelial ER were required for complete mammary gland development in adult mice. However, when the mice were treated with high doses of estradiol (E2) and progesterone, stromal ER was sufficient to generate full mammary gland growth. Surprisingly, ER-deficient epithelial cells were able to proliferate and develop into a rudimentary mammary ductal structure in an ER-negative stroma, indicating that neither stromal nor epithelial ER are required for the mammary rudiment to form in the adult mouse, as confirmed by the phenotype of the ERKO mammary gland. Use of thisin vivo model system has demonstrated that neonatal and adult mammary tissues use a different tissue-specific role for ER in mammary response. Immunostaining for ER and PR in the mammary outgrowths supported the view that both stromal and epithelial ER, in cooperation with epithelial PR, govern mammary gland development in adult mice. |
Introduction |
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(2, 5). Further side-branching of epithelial ducts and formation of alveolar buds occurs in the mature animal under the additional influence of prolactin and progesterone (reviewed in Refs. 2 and 6, 7, 8, 9).
The mammary phenotype of female mice with a disrupted ER gene (ERKO) proved that embryonic mammary gland development is independent of ER, but ER is required for ductal elongation during puberty and complete mammary gland development in the mature mouse (5, 10). In contrast, the progesterone receptor (PR) knockout mouse (PRKO) showed that the role of the PR is to mediate full lobuloalveolar development of the mature mammary gland (11). At puberty and maturity, mammary development is tightly regulated by the interactions between the epithelial compartment with the mammary stroma (fat pad), in which the stroma provides growth factors that stimulate epithelial proliferation (reviewed in Refs. 3, 8, 12 , and 13). Mammary tissue recombinants of PRKO and wild-type mouse stroma and epithelium, respectively, showed that PR expression in the epithelium is required for lobuloalveolar development in the mature mouse (14). The question of whether stromal or epithelial ER is responsible for mammary growth was first addressed by Cunha et al. (15). In these experiments, recombined mammary stromal and epithelial tissue from neonatal Balb/c and ERKO mice were transplanted under the renal capsule of intact athymic nude mice. Their results indicated that the stromal, but not the epithelial, ER was required for mammary epithelial ductal growth (15).
In our study, we examined whether the results of Cunha et al. (15) using neonatal tissues could be extrapolated to mammary tissue from mature mice. We isolated mammary cells of mature ERKO and their wild-type littermates and injected them into the epithelial-free mammary fat pad of female ERKO and wild-type mice, respectively (16, 17). By choosing this approach we were able to recombine epithelial with stromal tissue from adult ER-positive wild-type or ER-negative mice from the same genetic background in a physiological setting. Whole-mount analysis of mammary outgrowths and immunostaining for ER, ERß, and PR enabled the elucidation of the tissue-specific requirements for these steroid hormone receptors in mammary gland development of sexually mature mice.
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Materials and Methods |
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Isolation of epithelial cells
All experiments with mice were performed according to an approved Animal Care Protocol, in compliance with the National Institute of Environmental Health Sciences guidelines for the Humane Care and Use of Animals in Research. Primary cells of the mammary gland were obtained using modified published methods (17, 18, 19). Eight-week-old ERKO and wild-type (C57BL/6) mice were treated by sc implantation of 21-d release hormone pellets (50 mg progesterone/pellet and 0.25 mg 17ß-E2/pellet; Innovative Research of America, Sarasota, FL) for 3 wk to stimulate epithelial cell proliferation. The 11-wk-old animals were then killed and whole mammary glands (inguinal glands no. 4 and 5 of each side) were collected in ice-cold DMEM/F12 [containing 10% (vol/vol) heat-inactivated FBS and antibiotics]. Glands were minced and the minced tissue was digested in DMEM/F12 containing 10% FBS, antibiotics, 25 U/ml collagenase III (Worthington, Freehold, NJ) and 1 mg/ml hyaluronidase for 3 h at 37 C. The cell suspension was centrifuged at 300 x g for 20 min and the resulting cell pellet incubated at room temperature in DMEM/F12 containing 0.5 mg/ml protease XIV (protease, E.C. 3.4.24.31). The digestion was quenched after 1.5 h with 10% FBS and filtered through a 250-µm nitex mesh (Tetko, Kansas City, MO). The filtrates were centrifuged at 300 x g, and the resulting cell pellet from ERKO mammary glands was resuspended in DMEM/F12. Cells were counted with a hemacytometer (Daigger, Vernon Hills, IL). Cell density was adjusted to 10 x 106 cells/ml. The cell pellet from wild-type mammary glands was resuspended in 2 ml PBS/calcium and magnesium free (CMF) containing 10 µl of 0.04% (wt/vol) DNase I and subjected to a discontinuous Percoll gradient to separate epithelial from stromal cells (20). The cell suspension was layered on a 90%-68%-43%-35%-20% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) gradient in 1x PBS and centrifuged at 800 x g for 30 min. The band between 43% and 68% Percoll was isolated, and cells were washed twice in DMEM/F12 containing antibiotics and 5% heat-inactivated newborn calf serum. The cell pellet from wild-type mammary glands was resuspended in DMEM/F12 and counted in a hemacytometer, and the cell density was adjusted to 10 x 106 cells/ml.
Surgical procedures and cell injection into the cleared mammary gland
All mice for this study were housed and cared for in accordance with the NIH guidelines for the Humane Care and Use of Animals in Research and all surgical procedures were approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee. The left inguinal mammary gland was cleared from epithelial cells according to the procedure by DeOme et al. (16), except that the gland was removed from the nipple region up to the lymph node by cauterization. Ten mircoliters of 1 x 105 mammary cells in DMEM/F12 medium were injected with a Hamilton syringe (Hamilton, Reno, NV) attached to a 27-gauge needle into the remaining gland-free fat pad (17). The contralateral gland was left untraumatized. One week after trauma, 60-d hormone release pellets (50 mg progesterone/pellet and 0.25 mg E2/pellet; Innovative Research of America) were implanted sc.
Whole-mounts and carmine stain of mammary glands
Mice were killed 10 wk after cell injection and both inguinal mammary fat pads were excised and fixed for a minimum of 2 h in Carnoy’s solution (60% ethanol, 30% chloroform, and 10% glacial acetic acid). The fixed glands were washed in 70% ethanol for 15 min and then rinsed in water for 5 min. The mammary glands were stained overnight at 4 C in carmine alum stain (1 g carmine and 2.5 g aluminum potassium sulfate in 500 ml water). The glands were then dehydrated progressively in 70%-95%-100% ethanol, cleared in xylene for 1 h, and mounted on glass slides with Permount (Fisher Scientific, Suwanee, GA). Mammary whole mounts were photographed using a MZ6 dissecting microscope (Leica Corp., Deerfield, IL) and an Olympus Corp. (Lake Success, NY) Oly 760 video camera.
Immunohistochemistry
Mounting medium of whole mounts was removed by immersing slides in xylene, and the mammary glands were rehydrated progressively in 100%-95%-70% ethanol. Mammary glands were then embedded in paraffin and cut in 5-µm-thick sections and placed on positively charged glass slides (Fisher Scientific). For immunostaining of ER, ERß, and PR, slides were deparaffinized in xylene and rehydrated progressively in 100%-95%-70% ethanol. For antigen retrieval, slides were pressure-cooked for 5 min in citrate buffer according to the instructions of the supplier (Biocare Medical, Walnut Creek, CA). Endogenous peroxidase was blocked by incubating slides for 15 min in 3% hydrogenperoxide. Staining for ER and PR was done using the M.O.M. kit from Vector Laboratories (Burlingame, CA) exactly as described in the provided protocol. Mouse monoclonal antibodies raised against ER (clone ER15D, Immunotech, Marseille, France) at a dilution of 1:25 and PR (clone PR10A9, Immunotech) at a dilution of 1:50 were used. Negative controls were performed with 0.02 mg/ml normal mouse IgG (DAKO Corp., Glostrup, Denmark). The diaminobenzidine-stained slides were counterstained with hematoxylin and coverslipped in aqueous mounting medium (Innovex, Richmond, CA). For ERß staining, slides were blocked with normal goat serum (Vector Laboaratories), for 20 min. Normal rabbit IgG (Oncogene, Cambridge, MA) was used as negative control. Slides were incubated with 10 µg/ml ERß antibody (Ab-1, Oncogene) or normal rabbit IgG at 4 C overnight. Slides were stained using the ABC elite kit (Vector Laboratories) with diaminobenzidine according to the manufacturer’s protocol. Slides were counterstained and coverslipped as described above. Immunostaining was analyzed with an Olympus Corp. BX-50 microscope, and pictures were taken with an Olympus Corp. Oly 760 video camera. Labeling indices (L.I.) for ER and PR staining were determined by counting at least 500 epithelial cells each from at least two different mammary gland sections per animal. Positive receptor expression was defined as distinctive nuclear stain. Epithelial cells from mammary outgrowths as well as from the contralateral gland were analyzed. Data are given as average from two animals that showed mammary outgrowths ± range.
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Results |
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ERKO-derived mammary epithelium does not proliferate in a wild-type stromaERKO or wild-type female mice and recombined them with an ER-positive stroma to analyze whether epithelial ER is a prerequisite for mammary gland growth in mature mice. For isolation of mammary cells, donor mice were treated with E2 and progesterone for 3 wk beginning at 4 wk of age to induce epithelial cell proliferation (21). Whole-mount analysis of hormone-treated donor mice at 11 wk of age (data not shown) proved that mammary epithelial growth was induced in ERKO and wild-type mice, similar to published results (21). The 11-wk-old donor ERKO or wild-type mice were killed and mammary cells isolated. Wild-type-derived mammary cells were separated with a Percoll gradient yielding an enriched epithelial cell population (17, 22). A single-cell suspension of the enriched mammary epithelial cells was injected into the left gland-free inguinal mammary fat pad of 3-wk-old immature, virgin wild-type mice. After injection, the host mice were either left untreated or treated by sc pellet implants containing high doses of E2 (0.25 mg E2/mouse) and/or progesterone (50 mg progesterone/mouse). Sixty days after hormone treatment, the 13-wk-old host mice were killed and the mammary fat pad containing the injected epithelial cells and each control contralateral inguinal gland were collected. Injection of wild-type mammary cells into a wild-type fat pad yielded a complete mammary ductal outgrowth similar to the epithelial ductal structure seen in the contralateral gland (Fig. 1
, A vs. B, and Table 1). When treated with E2 and progesterone, we observed extensive side-branching of the wild-type mammary outgrowth, indicating that isolated epithelial cells are fully capable of responding to mammogenic hormones and to regenerate a normal mammary gland structure (Fig. 1, D vs. E, and Table 1). As negative control, we injected culture medium without cells into the cleared fat pad of wild-type mice, which showed no epithelial outgrowth in contrast to the contralateral gland (data not shown).
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ERKO mammary cells into wild-type stroma yielded no outgrowth when the recipient mice were not treated, in contrast to the full mammary development of the contralateral gland (Fig. 1
, A vs. C, and Table 1). This result clearly suggests that epithelial ER is required for epithelial outgrowth in the adult mammary gland. However, complete mammary growth of ER-negative cells occurred when the recipient mice were treated with E2 and progesterone (Fig. 1F). In addition, the observed mammary development was similar to that seen in the wild-type/wild-type recombination (Fig. 1, E vs. F, and Table 1).
Rudimentary epithelial growth does not require ER
The reciprocal tissue recombination experiments were performed using ERKO stroma from 4- to 6-wk-old recipient ERKO mice. The inguinal gland of female ERKO mice was cleared by removing the epithelial rudiment as described above. Mammary cells from female wild-type or ERKO donor mice were injected into the cleared ERKO fat pad, and the recipient mice were treated with progesterone only or E2 and progesterone as described above. Untreated mice did not produce any ductal outgrowth derived from either wild-type or ERKO epithelial cells injected into the ERKO fat pad (Fig. 2, A vs. B and C; Table 1). In contrast, when treated with either progesterone only or E2 and progesterone, both wild-type and ERKO epithelial cells developed a rudimentary epithelium when injected into ERKO fat pads (Fig. 2, E and F, H and I; Table 1). The rudimentary ductal structure was similar to that seen in the contralateral gland of ERKO recipient mice (Fig. 2, D and G). The negative control showed no epithelial outgrowth in contrast to the rudimentary ductal structure of the contralateral gland (data not shown). These results indicate that neither epithelial nor stromal ER was required for the development of the epithelial rudiment in adult mice. It should be noted here that the mature ERKO stroma was capable to stimulate proliferation and ductal morphogenesis of single mammary cells despite the lack of ER in adult mice. These proliferative and morphogenic effects required treatment with progesterone, which is in line with earlier reports that showed stimulation of epithelial proliferation by progesterone in mammary glands of adult mice (23).
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and PR in mammary outgrowths is regulated by E2 and progesterone-negative epithelial cells depends on the ovarian steroid hormones E2 and progesterone. Previous analysis of steroid hormone receptor expression (reviewed in Refs. 2, 24 , and 25) and the description of female ERKO and PRKO mice (10, 11) clearly showed that both ER and PR are required for tertiary side-branching and lobuloalveolar development of the mammary gland. To further understand the responsiveness of the mammary outgrowths to E2 and progesterone, we studied the pattern of ER and PR expression in the mammary outgrowths and the respective contralateral glands by immunohistochemistry. In the wild-type stroma/wild-type epithelium recombinants, ER was expressed predominantly in the luminal epithelium and less so in the stroma (Fig. 3B), whereas the PR was expressed in the luminal epithelium (Fig. 3, H and K). Treatment with E2 and progesterone completely suppressed ER expression in the epithelium of mammary outgrowths (Fig. 3E and Table 1). Luminal staining was present in the contralateral gland but at a reduced level, and stromal staining for ER appeared to be predominant (Fig. 3D and Table 1). In contrast, luminal PR staining was abundant in treated mammary outgrowths and contralateral wild-type glands (Fig. 3, J and K). These observations are in line with previous findings that showed suppression of epithelial ER by E2 and progesterone in mature mice (24, 25). This indicated also that regulation of steroid hormone receptor expression in the mammary outgrowths may be similar to that in normal mammary glands.
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ERKO cells did not regenerate the epithelial duct structure in a wild-type fat pad when the mice were not treated with steroid hormones. In this case, stromal ER expression was detectable, proving that the lack of epithelial growth was not due to lack of stromal ER (Fig. 3C). In contrast, the contralateral wild-type gland exhibited predominantly luminal epithelial staining for ER, which was reduced by E2 and progesterone treatment (Fig. 3, A and D; Table 1). In recipient wild-type mice treated with E2 and progesterone, ER staining of luminal epithelial cells in wild-type- and ERKO-derived mammary outgrowths was absent with some staining in the stromal compartment (Fig. 3, E and F; Table 1). No distinct PR staining in cleared wild-type fat pads without epithelial outgrowths was detectable in the stroma (Fig. 3I). Epithelial staining for PR in mammary outgrowths of ERKO epithelial cells in E2- and progesterone-treated mice appeared to be less when compared with mammary outgrowths of wild-type epithelial cells (Fig. 3, L vs. J, and Table 1; L.I., 39 ± 6% vs. 61 ± 2%). The contralateral wild-type gland showed abundant luminal PR expression when untreated or when treated with E2 and progesterone (Fig. 3, G and J). This further supported the view that in adult tissue luminal PR expression is regulated by both epithelial and stromal ER (24, 25).
An ER-deficient fat pad did support rudimentary mammary outgrowths of wild-type or ERKO mammary cells only when the ERKO mice were treated with progesterone or a combination of E2 and progesterone. In both cases, ER staining was not detectable in the stromal or epithelial compartment (data not shown). PR staining showed low abundance in the contralateral ERKO gland compared with the contralateral wild-type gland (Table 1; Fig. 4, A and D and G vs. Fig. 3, G and J) indicating that epithelial PR expression depends on stromal and epithelial ER expression. When wild-type mammary cells were injected into the ERKO stroma, PR staining was more abundant in outgrowths treated with progesterone (L.I., 4 ± 1%; Table 1) or a combination of E2 and progesterone (L.I., 40 ± 3%; Table 1) compared with the ERKO epithelial outgrowths (L.I., 0 and 18 ± 3%, respectively; Table 1; see also Fig. 4, E and H vs. D, G, F, and I). This would be in line with the view that epithelial ER may be required for induction of epithelial PR expression in the absence of stromal ER. No distinct stromal staining for PR was detectable in untreated, cleared ERKO fat pads injected with either wild-type or ERKO cells (Fig. 4, B and C). Luminal and stromal PR staining was absent in outgrowths of ERKO epithelial cells when treated with progesterone (Fig. 4F), which might be due to suppression of PR expression by progesterone in the absence of ER. However, luminal epithelial PR staining of ERKO mammary outgrowths was present in E2 and progesterone-treated mice (Fig. 4I and Table 1). These data indicate that progesterone and epithelial PR could be involved in mammary growth in an ER-deficient environment. These results are in line with earlier reports that showed that PR signaling is not compromised in the ER-deficient uterus (26) and are also supported by a recent study that showed that PR was expressed in proliferating epithelial cells in the mammary gland of pubertal and mature mice, whereas ER was predominantly expressed in the nonproliferating epithelium (27).
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Discussion |
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ERKO and Balb/c neonatal mice were grafted under a renal capsule in nude mice (15). Under these conditions, epithelial outgrowth was observed only when Balb/c stroma was present, regardless of the genotype of the epithelial tissue. The authors concluded that stromal ER mediated the growth induction of the neonatal mammary epithelium. Our data clearly prove that stromal ER is required for epithelial growth under physiological conditions in mature mice. When recipient wild-type mice were treated with high doses of E2 and progesterone, ERKO epithelial cells were capable to undergo extensive tertiary side-branching of epithelial ducts and formation of alveolar buds. However, ER-deficient mammary cells were unable to develop epithelial ductal structures in an ER-positive stroma, indicating that epithelial ER is also required for mammary growth under physiological conditions in mature mice (Table 2).
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and PR expression and their responsiveness to steroid hormones is highly dependent on the developmental stage of the mammary gland. Although ER is present in the mammary epithelium and stroma of the newborn mouse, ER is not responsive to E2 and epithelial proliferation does not occur (reviewed in Refs. 2, 9, 24 , and 25). The mammary gland phenotype described for female ERKO mice proved that ER is not required for the formation of the epithelial rudiment in the neonatal and immature mouse (10). But, after the onset of puberty, ER expression increases in the murine stroma and epithelium and the mammary gland becomes responsive to E2. Additionally, complete mammary growth including side-branching and formation of alveolar buds occurs only in mature mice when epithelial PR is detectable and epithelial ER expression is increased (11, 24, 25). The mammary gland in adult mice is then responsive to both E2 and progesterone. The neonatal epithelium used by Cunha et al. (15) might therefore lack responsive ER and PR, which would explain that epithelial ER is not required for epithelial growth in neonatal tissues. In the study presented, we have shown that the outgrowths of mature mammary cells are responsive to steroid hormones. It is therefore reasonable to assume that, in the mature tissue, ER and PR are the main mediators of mammary gland growth, whereas in neonatal tissue, stromal growth factors stimulate epithelial growth.
Two observations reported here were most intriguing (Table 2). First, the lack of outgrowth of ER-deficient epithelial cells injected into an ER-positive stroma and, second, the capability of ER-negative stroma to support proliferation and formation of ductal structures of ER-deficient epithelium in the presence of progesterone. The first observation leads to the conclusion that ER in both epithelium and stroma is necessary for mammary gland development in a mature mouse. We provided further evidence that luminal epithelial ER but not PR is suppressed by E2 and progesterone in the murine mammary gland (24, 25). Furthermore, our data indicated that the induction of epithelial PR expression is mediated by both epithelial and stromal ER. These lines of evidence support a model of mammary epithelial growth regulation in which stromal and epithelial ER govern mammary gland development in cooperation with epithelial PR in adult mice. Mammary gland development is also dependent on several growth factor signaling pathways as well as lipids and extracellular matrix molecules that are secreted by the fat pad (reviewed in Refs. 3, 8 , and 12). The second observation (Table 2) was puzzling to us, considering the fact that single ER-negative mammary cells were able to undergo epithelial proliferation and morphogenesis leading to mammary ductal structures in an ER-deficient fat pad. A recent report by this laboratory (21) showed that ovarian steroid hormones stimulated growth of the mature ERKO mammary rudiment, resulting in extensive ductal elongation and formation of alveolar buds. Here, we also observed stimulation of epithelial growth and morphogenesis of a mammary ductal epithelium in ERKO fat pads treated with progesterone only or treated with progesterone and E2. Because treatment with progesterone only induced growth in an ER-deficient environment, we conclude that PR is sufficient for an initial epithelial proliferation and ductal morphogenesis. This conclusion supports earlier studies by Haslam (23), who showed that progesterone in the absence of E2 could stimulate mammary epithelial growth (reviewed in Ref. 9).
Outgrowths of ER-positive and ER-negative epithelium in an ERKO fat pad were comparable, indicating that epithelial ER is not involved in this growth stimulation. Besides the feasible contribution of the PR, the ERKO stroma may have adapted to ER deficiency and allowed development of a mammary rudiment due to elevated or altered growth factor levels in the absence of ER. Several stromal-derived growth factors that induce mammary epithelial proliferation and morphogenesis are known, including fibroblast growth factor (FGF), keratinocyte growth factor (KGF), or hepatocyte growth factor (HGF) (reviewed in Ref. 3). However, a contribution of these growth factors to an ER-independent mammary growth stimulation remains speculative. Another candidate for the regulation of mammary epithelial proliferation is ERß, as its mRNA as well as ERß protein have been detected in the rodent mammary gland (27, 28). We analyzed the outgrowths for ERß expression by immunostaining, and only a few stromal and myoepithelial cells stained positive for ERß (data not shown). In contrast, Saji et al. (28) found high expression of ERß in the mammary epithelium of rats. However, a study by Zeps et al. (27) confirmed our results of low ERß expression levels in the mammary gland of adult female mice. In addition, we found a similar expression pattern of ERß in untreated mice without mammary outgrowths and in the steroid hormone-treated mice that developed mammary ductal structures. Considering the normal mammary gland development in mice lacking ERß (Ref. 29 and reviewed in Ref. 30) and the data presented here, ERß is most likely not involved in epithelial growth regulation in the mammary gland.
Another explanation for epithelial growth induction in the ERKO mammary gland may be the contribution of a splice variant of ER present in ERKO mice (31). mRNA encoding the ER splice variant E1 was detected in the uterus of ERKO mice, in vitro-translated E1 protein is transcriptionally active, and E1 protein was detected in testicular cells from ERKO mice (31, 32). Because E1 has reduced transcriptional activity in vitro when compared with ER (31), the splice variant E1 may mediate a rudimentary epithelial growth in ERKO mice. Regardless of a speculative role for E1, mammary growth of ERKO epithelium may also be mediated by the E2 metabolite catecholestrogen (33, 34). Das et al. (33) showed that catecholestrogen induced PR expression in the uterus of wild-type mice, and this induction could not be inhibited by ICI 182,780, a full antagonist of ER and ERß. These reports suggested that PR could be regulated by pathways not involving ER. This may be relevant for our observations and supports the idea of epithelial growth induction mediated by progesterone and PR in an ER-deficient mammary gland. Tissue recombinations employing the ERKO, ßERKO, and the double knockout for ER and ERß, the ßERKO mouse (35), would help to determine the role of ERß and the ER splice variant E1 in ER-independent mammary growth.
In conclusion, this report provided evidence that stromal and epithelial ER in cooperation with epithelial PR play a pivotal role in epithelial growth regulation and morphogenesis in the mammary gland of sexually mature mice. However, the mammary stromal compartment and epithelial PR can induce epithelial proliferation and ductal morphogenesis in the absence of ER. The report presented here opens an avenue for studies to elucidate the exact contribution of growth factors, the PR, ER, and ERß in mammary gland development. Studies employing an immortalized mammary epithelial ERKO-derived cell line (22) that is capable of developing mammary epithelial ductal structures (our unpublished observation) are under way in this laboratory. Injection of this cell line genetically engineered to express exogenous ER, ERß, and/or PR into a cleared fat pad of wild-type, ERKO, ßERKO, ßERKO, and PRKO mice should help to answer the remaining questions in mammary gland development in mature mice.
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Acknowledgments |
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Footnotes |
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1 * Present address: Merck KGaA, Institute of Toxicology, 64271 Darmstadt, Germany. 
Abbreviations: CMF, Calcium and magnesium free; ERKO, estrogen receptor knockout; L.I., labeling indices; PRKO, progesterone receptor knockout.
Received August 13, 2001.
Accepted for publication February 6, 2002.
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References |
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knockout mice. Endocrinology 141:2982–2994 knock-out and wild-type mice. In Vitro. Cell Dev Biol 36:620–624
knockout mouse uterus. Proc Natl Acad Sci USA 96:3646–3651 and ER ß status. J Histochem Cytochem 47:1323–1330 and ß in the rodent mammary gland. Proc Natl Acad Sci USA 97:337–342 knock-out and wild-type mice expressing functional ER or ERß. J Androl 22:652–664[Abstract]
-deficient mice reveal a distinct estrogen signaling pathway. Proc Natl Acad Sci USA 94:12786–12791 and ß. Science 286:2328–2331This article has been cited by other articles:
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E E Connor, D L Wood, T S Sonstegard, A F da Mota, G L Bennett, J L Williams, and A V Capuco Chromosomal mapping and quantitative analysis of estrogen-related receptor alpha-1, estrogen receptors alpha and beta and progesterone receptor in the bovine mammary gland J. Endocrinol., June 1, 2005; 185(3): 593 - 603. [Abstract] [Full Text] [PDF]
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