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Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Ming Zhang, Ph.D. Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: mzhang{at}bcm.tmc.edu.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The normal mammary gland depends strongly on the hormones and their receptors for its development (1, 2). It is well established that estrogen and its receptor ER play a central role in early ductal development (3, 4). Mice lacking ER
gene (ERKO) did not have normal ductal development. An early transplantation experiment demonstrated that the stromal expressed ER contributed to the early ductal development (5). However, a recent study showed that ER is primarily present in epithelial, and ERß is present in both epithelial and stromal cells. Both ER and ERß are responsible for proliferation through estrogen action (6). Other hormones such as insulin-like hormones, prolactin, and GH also contribute to the ductal development (1, 7). For example, hypophysectomized mice do not develop normal mammary glands (2). In contrast, progesterone and progesterone receptor (PR) are thought to mediate a role in ductal side branching in mature mammary gland and in alveolar development during pregnancy. The conclusion derived from the study using PR knockout mice (PRKO). Mice lacking PR had normal ductal structures when examined at late stage after puberty but had severely defective side branching and alveologenesis in pregnancy (8). When PRKO and wild-type mice were exposed to the combined treatment of estrogen and progesterone, the wild-type breast tissues responded with side branching and lobular development, whereas the mammary glands of PRKO mice remained essentially unchanged (9, 10). However, early reports have focused on the effect of progesterone and PR on this later stage of mammary development. Their effects on early ductal elongation during pubertal stage of mammary development have not been examined carefully.
We have identified another role of progesterone and PR in early ductal development through the study of maspin knockout mice. Maspin is a member of the family of serine protease inhibitor with tumor suppressing function (11). In female mammary gland, maspin gene expression is changed during cycles of mammary development (12, 13), suggesting that its expression may be subject to hormonal regulation. In fact, human maspin promoter contains a hormonal response element (HRE) that acts as a negative regulatory element in vitro (14). To study the role of maspin in normal mammary development and tumor progression, we attempted to generate maspin null mice by gene target deletion. Interestingly, the homozygous maspin knockout mice were embryonic lethal at periimplantation stage (15), which precluded the analysis of mammary phenotype at a later stage. Therefore, we examined the effect of maspin on mammary development in maspin heterozygous mice. Here we report that maspin heterozygous mice have a defect in ductal development at pubertal stages. However, this defect arises from a reduced level of progesterone systemically. We showed that the level of progesterone in maspin heterozygous mice was consistently reduced during puberty. When both maspin heterozygous and wild-type mice were ovariectomized, treatment with progesterone by itself stimulated ductal development. Treatment of wild-type mice with progesterone inhibitor RU486 resulted in decreased ductal growth as was observed in maspin heterozygous mice, and the inhibitory effect was dose dependent. Furthermore, we demonstrated that progesterone action was dependent of the presence of PR. A recent study by Mueller et al. (16) showed that progesterone alone stimulated ductal epithelial cell proliferation in mammary glands of mice with ER deletion (ERKO). These findings indicate that like estrogen, progesterone also plays a role in early ductal elongation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). No difference of ductal development between sibling Mp+/+ and Mp+/– mice was observed at 5 wk of age (Fig. 1, A–D). However, a reduction in ductal elongation and number of side branches was observed in Mp+/– mammary glands at 7 wk of age (Fig. 1). To quantitate the difference, morphometric analysis was carried out using a panel of animals, all paired sisters from Mp+/+ and Mp+/– background. To quantitate the mammary ductal development, we arbitrarily set up two standards. One is the length of ductal elongation that measures the ductal distance from the lymph node to the TEBs; the other is the number of side branches, which counts all of branching points of mammary ducts within the area from the lymph node to the TEBs. As shown in Fig. 1G, a significant difference was found between Mp+/+ and Mp+/– glands for the length of ductal elongation. For example, Mp+/+ mice had a mean value of 5659 µm (±474.9, n = 9) for the ductal length, whereas that of Mp+/– mice was 3673 µm (± 449.6, n = 9, P < 0.001). The difference in ductal side branching points was also highly significant (Mp+/+; 136.8 ± 10.7, Mp+/–; 30.4 ± 4.8, P < 0.001, n = 9) (Fig. 1H). Because the mitotic activity in the TEB represents proliferation status of epithelial cells, we examined the rate of DNA synthesis in the body cells of TEB of Mp+/– and Mp+/+ glands (Fig. 1, E and F). The mitotic index of Mp+/– glands in the body cells of TEB was significantly reduced than that of Mp+/+ glands (Mp+/+, 5.74% ± 1.13, n = 5; Mp+/–, 3.01% ± 0.63, n = 5; P < 0.038).
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Ovarian and pituitary hormones are known to affect mammary development through different signal pathways. We carried out reciprocal ovary transplantation between Mp+/+ and Mp+/– mice at 4 wk of age and collected the mammary glands for whole mount analysis at the end of 7 wk. As shown in Fig. 2, transfer of ovaries from Mp+/– donor to Mp+/+ recipient resulted in a defective ductal elongation and side branching. However, the transfer of ovary from Mp+/+ donor to Mp+/– recipient rescued the defect of ductal development (Fig. 2A). The transfer of ovaries between Mp+/+ mice served as controls. The ductal outgrowths of these ovary-transplanted mice were quantitated (Fig. 2B). The overall ductal lengths in Mp+/+ mice that underwent transplantation with ovaries from Mp+/– mice (termed Mp+/+/ Mp+/–) were significantly reduced when compared with that of Mp+/– mice transferred with Mp+/+ ovaries (Mp+/–/ Mp+/+) (Fig. 2B, P < 0.005). Transfer of ovaries from Mp+/+ to Mp+/– mice (Mp+/–/ Mp+/+) rescued the defective ductal outgrowth in Mp+/– mice. No significant difference was observed for ovary transplantation between the groups of Mp+/–/ Mp+/+ and Mp+/+/ Mp+/+ mice (Fig. 2B). This result suggests that a systemic difference in ovarian hormone(s), rather than factor(s) derived from other organs, contributes directly to the mammary developmental defect observed in Mp+/– mice.
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Progesterone Is Required for Mammary Ductal Development in Pubertal Stage
To determine that the reduced progesterone level resulted in a defect of ductal development in Mp+/– mice, we treated the Mp+/– mice with either progesterone (10 mg/pellet·mouse) or control pellets starting at 4 wk of age for 3 wk. The mammary glands were collected by the end of 7 wk for whole mount analysis. Treatment with progesterone completely rescued the ductal defect in Mp+/– mice. Mp+/+ mice had a mean value of 4182 µm (4182± 426.2, n = 9) for the ductal length, whereas that of Mp+/– mice was 2497 µm (2497 ± 449.6, n = 9; P < 0.001). The difference in ductal side branching points was also highly significant (Mp+/+; 137.3 ± 10.9, Mp+/–; 30.4 ± 4.8, n = 9; P < 0.001).
The reduced progesterone level may lead to an increase of PR, which renders the cells more sensitive to exogenous progesterone stimulation. Another possibility is that the PR level may be changed in Mp+/– epithelial cells. To test these possibilities, we analyzed the expression of PR in Mp+/+ and Mp+/– mammary glands at 7 wk of age. The pattern (mixed PR-positive and -negative cells) and intensity of PR staining were similar between these two groups of mice (Fig. 3A). Quantitative analysis showed that the percentage of PR-positive epithelial cells was not significantly changed between these two groups of mice (Fig. 3B).
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). Such stimulatory effect has been observed by others (19, 20). Therefore, the E + P-treated samples were mainly served as a control. Interestingly, progesterone alone resulted in an extensive ductal development (Fig. 4A, Mp+/+, progesterone; or Mp+/–, progesterone). Quantitative analysis of P treatment showed that the number of ductal elongation and side branching points in Mp+/– mice were comparable with that of the Mp+/+ mice that were not ovariectomized (Fig. 4, B and C).
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, A and B). At a higher dosage (0.5 mg/pellet), the progesterone antagonist completely blocked ductal development (Fig. 5, A and B). When Mp+/+ mice were treated by both RU486 and a high dose of progesterone simultaneously, the inhibitory effect of RU486 was completely reversed (Fig. 5). We also compared the number of side-branching points between control pellet-treated and RU486-treated Mp+/+ mice (Fig. 5). These results indicated that RU486 significantly inhibited the number of ductal side branches in a dose-dependent manner in pubertal mice.
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), suggesting PR is required for progesterone action during this stage of ductal development. When the ovariectomized wild-type mice were treated with progesterone + RU486 (0.5 mg/pellet), ductal growth was blocked by RU486 (0.5 mg/pellet), despite of the presence of high level of progesterone. Because PR level is reduced in the absence of estrogen in ovariectomized mice, these data suggest that the basal level of PR is sufficient for mediating the effect of progesterone in the absence of RU486 antagonist. However, a high dosage of RU486 (0.5 mg/pellet) is now able to compete with progesterone for its interaction with the low level of PR in the ovariectomized mice (Fig. 6A). We have also examined the mammary gland development in PRKO mice with intact ovaries at 7 wk and 10 wk of age. Figure 6B showed that there was a defect of ductal development in PRKO mice at 7 wk of age compared with that of the wild-type controls. Quantitative analysis showed a significant difference in ductal elongation between wild-type and PRKO glands (P < 0.001). However, the defect in mammary ductal growth was rescued after 7 wk of age. By the time the mice were 10 wk old, there was no obvious difference in ductal elongation between PRKO and wild-type mice (data not shown). This suggests that another factor is activated to rescue the mammary developmental defect in PRKO mice during the period from 7–10 wk, which is critical for mammary gland development.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In addition to its role in the normal mammary gland, PR has been found to stimulate proliferation in cell culture and in some mammary tumors. For example, progesterone was shown to stimulate proliferation of virgin and pregnant mammary epithelial cells (31, 32). In combination with estrogen, progesterone has been reported to induce cyclin D1 expression in mammary epithelial cells in vivo (20). Murine mammary gland carcinogenesis has been shown to be critically dependent on PR function (33, 34). In recent clinical trials, progesterone, when administered as a component in hormonal replacement therapy, was reported to increase the risk of breast cancer (35, 36). These reports underscore the importance of progesterone and PR in both normal mammary gland development and tumorigenesis.
Despite these reports, the question whether the progesterone-regulated signal transduction pathway plays a role in early ductal development has not been well studied. Our report provides direct evidence that progesterone and PR play a role in early ductal growth during pubertal stage. This study was initiated by the observation that maspin heterozygous mice display a defect in ductal development. However, this defect was not the result of the loss of one copy of maspin in mammary epithelial cells, but rather from a reduction in the expression of the ovarian hormone, progesterone.
It is well known that E is an important factor for ductal proliferation at puberty. A recent report by Cheng et al. (6) showed that E acts on both ER and ERß to promote the epithelial cell proliferation. Once ER receives the proliferation signal from estrogen, it initiates the DNA synthesis, and is then lost from cells. The subsequent steps in proliferation can proceed in the absence of either ER and ERß. This explains why the proliferating epithelial cells do not have detectable ER expression. In another report, using mammary transplantation Mueller et al. (16) have shown that both stromal and epithelial ER are required for complete mammary gland development. Treatment of mice with antiestrogen ICI182,780 has highlighted the requirement of E and ER in ductal development. Shyamala et al. (37) showed that prepubertal mice (4 wk old) when treated with antiestrogens for 3 wk exhibited decreased ductal development. Tamoxifen treatment causes ERß degradation, thus affecting cell response to estrogen (6). We have treated the wild-type mice with antiprogesterone RU486 during pubertal stage and shown a dose-dependent effect on ductal development. For example, treatment of RU486 at 0.1 mg/pellet in wild-type mice gave rise to a mammary phenotype similar to that of maspin heterozygous mice in which progesterone was consistently reduced (Figs. 1
and 5), whereas at a dosage of 0.5 mg/pellet completely blocked ductal development (Fig. 5). In conclusion, these data support a role of progesterone in regulating early ductal development. Our data show that this action is PR dependent.
The key puzzle for the reduced production of progesterone in Mp+/– mice has been recently solved by our laboratory. We have obtained evidence that maspin heterozygous female mice display a sever defect in ovarian development including defective corpora lutea development and decreased ovulation (Porter, W., and M. Zhang, unpublished data). When primed by pregnant mare serum gonadotropin and human chorionic gonadotropin, the number of ovulated eggs within the oviduct of Mp+/– female mice was significantly reduced as compared with the Mp+/+ mice. Analysis of ovary follicle showed that the Mp+/– mice had smaller size of ovaries with hemmorrhagic cysts. As the result, the fertility rate of Mp+/– mice was significantly reduced. In addition to ovary defect, we examined the mammary glands of Mp+/– mice during mid and late pregnancy. There was a reduction in alveolar development in Mp+/– mice as compared with that of Mp+/+ mice. Serum hormonal analysis showed that the progesterone level was significantly reduced in Mp+/– mice compared with that in Mp+/+ mice at mid and late pregnancy. However, during lactation Mp+/– female mice were able to nurse their pups without noticeable defect (data not shown).
How can we reconcile the conflicting views of progesterone action in ductal development? Clearly, PRKO mice have normal ductal development when they are examined at a late stage. Earlier reports of PRKO phenotypes were based on observation of mammary gland in mice older than 10 wk (8, 27). We confirmed in this study that indeed mammary ducts of PRKO mice were similar to that of wild-type mice at postmaturation stage (data not shown). However, as often observed in many mouse mutants, permanent deletion of a gene may result in a compensatory effect by others. For example, in virgin mice, glucocorticoid receptor –/– outgrowths displayed abnormal ductal morphorgenesis, but this defect was rescued during pregnancy, possibly due to the mineralocorticoid receptor (38). The compensatory factor(s) in PRKO mammary gland is currently unknown. It is noteworthy that prolactin signal axis may serve as one of the candidates of such compensatory factor. Prolactin and its receptor are indispensable for all stages of mammary development (25, 39, 40). Progesterone signal regulates the action of prolactin through prolactin receptor (41). There is also report that progesterone and prolactin may mutually interact to stimulate ductal proliferation (42). In addition, gene expression profiling using mammary tissues from PRKO and wild-type mice at pubertal stage may also help us reveal the identity of other compensatory factor(s).
Our study indicates that both estrogen and progesterone appear to play important roles in ductal development. Although PR is dispensable because the defect from PR deletion can be rescued by other factor(s), the requirement for ER seems to be more stringent because its deletion causes a defect that cannot be rescued by other compensatory mechanism. Hence, one observes the defective phenotype in ERKO mice even at a late stage of mammary development. A recent report by Mueller et al. (16) showed that progesterone alone stimulated epithelial cell proliferation and ductal morphogenesis in mammary glands. Atwood et al. (43) showed that progesterone by itself increased ductal side branching in wild-type mice. These reports support the observation that progesterone, in addition to estrogen is involved in early ductal morphogenesis. We are currently investigating the signal pathway by which maspin affects progesterone production and subsequently how progesterone may affect early ductal proliferation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mammary Transplantation
The left and right inguinal mammary glands of 3-wk-old athymic mice were cleared from epithelial cells according to the procedure by Deome et al. (45). Epithelial cells (tissue fragment, 1-mm3 size) from either Mp+/+ or Mp+/– mammary tissues (12 wk of age) were inoculated to the cleared fat pad. Twenty athymic mice were grouped into two groups for implanting Mp+/+ and Mp+/– epithelial cells. The mammary glands were kept for outgrowth for 8 wk before they were biopsied for whole mount analysis. Eight of 10 Mp+/– epithelia transplantation and seven of 10 Mp+/+ epithelia transplantation developed ductal outgrowth with similar pattern.
Ovary Transplantation
The ovary transfer was carried out based on a procedure provided by Dr. Richard Behringer (M. D. Anderson Cancer Center, Houston, TX). Four-week-old recipient female mice were anesthetized by the injection of avertin. The ovary and uterus were exposed by surgical procedure. We used fine scissors to make a small incision in the bursa on the side opposite the opening of the oviduct. The recipient ovary was removed and a donor ovary was inserted into the bursal sac. The ovary and the uterus were gently return to the body cavity. The body wall was sewed up with suture. The mice were kept for mammary gland biopsy by the end of 7 wk.
Hormone Pellets and Treatment
Bee’s wax pellet containing progesterone, E + P, or RU486 or blank control were prepared and implanted as described previously (9). The dosages are as follows: progesterone (10 mg), E + P (10 mg progesterone, 10 µg estrogen), and RU486 (0.1 or 0.5 mg). All pellets were implanted to mice when they just reached 4 wk of age and were maintained for a period of 11 d. They were exchanged for new pellets for additional 11 d. The mice were killed by the end of 7 wk of age. For ovariectomy, female mice at 3 wk of age were ovariectomized 1 wk before they were treated by hormone pellets as described above.
Whole Mount Analysis
Mammary gland whole mounts were performed by spreading the fourth inguinal gland on a glass slide followed by fixation in 4% paraformaldehyde for 2 h at 4 C. Tissues were rinsed in 70% ethanol and stained by hematoxylin (46). Mammary whole mounts were photographed using a SZX12 dissecting microscope (Olympus, Inc., Melville, NY) and an Olympus Z12 digital camera.
Quantitation of Ductal Outgrowth
The inguinal No. 4 mammary glands were excised from mice at 7 wk of age. The mammary glands were whole mounted on slides and photographed using a digital camera.
Photoimages were analyzed using the National Institutes of Health imaging software (http://rsb.info.nih.gov/nihimage). Ductal length, the number of branch points, and the number of TEBs was determined from the images. To quantitate the mammary ductal development, we arbitrarily set up two standards. One is the length of ductal elongation that measures the ductal distance from the lymph node to the TEBs; the other is the number of side branches that counts all of the branching points of mammary ducts within the area from the lymph node to the TEBs.
Immunostaining for PR
The mammary glands from 7 wk of age were fixed in 4% paraformaldehyde and sectioned at 5 µm for immunostaining as described previously (8). Polyclonal anti-PR antibody (DAKO, Inc., Carpinteria, CA) at 1:500 dilution was incubated with the slides for 1 h at room temperature. Biotinylated secondary antibody and ABC reagent (Vector Laboratories, Inc.) were used according to the manufacturer’s recommendations. Sections were counterstained with hematoxylin. Quantitation of PR-stained cells was done by counting the percentage of PR-positive cells in total epithelial cells. Mammary sections from six Mp+/+ and six Mp+/– mice were used for PR staining. Three 200x fields were counted for each slide. Total 3942 nuclei from Mp+/+ cells and 2472 nuclei from Mp+/– cells were counted (Mp+/– glands had less ductal epithelial cells).
Mitotic Index of Mammary Epithelial Cells in the TEB
The mammary gland from 7 wk of age were fixed in 4% paraformaldehyde and sectioned for the immunostaining with antibody against phosphorylated histone 3, a mitotic marker of DNA synthesis. Polyclonal anti-H3P antibody (Upstate Biotechnology, Inc., at 1:1000 dilution was incubated with the slides for 1 h at room temperature. Biotinylated secondary antibody and ABC reagent (Vector Laboratories, Inc.) were used according to the manufacturer’s recommendations. Because the cap cells do not express PR and the subtending luminal cells are in quiescent state, they were excluded from the counting. Thus, we counted only the body cells within the TEB for H3P-positive cells. Mitotic index is the percentage of H3P-positive cells among the body cells within TEB. Slides from five paired groups of mammary samples, each containing four to five TEB structures, were used in the analysis. A total of 3421 Mp+/+ ductal cells and 2326 Mp+/– ductal cells were counted from five groups of mice. The difference in H3P counting was analyzed by a prism t test statistical program.
Serum Hormone Analysis
Blood was collected from 7-wk-old mice at the end point. Serum samples were assayed for estrogen and progesterone by ELISA immunoassay using the kits from DS Lab, Inc. (Dallas, TX).
Statistical Analysis
The statistical analysis for ductal growth was performed using two-tailed t test. P < 0.05 is considered as significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: E + P, Estrogen + progesterone; ER, estrogen receptor; ERKO, mice lacking ER gene; HRE, hormonal response element; PR, progesterone receptor; PRKO, R-deficient; TEBs, terminal end buds.
Received for publication February 5, 2004. Accepted for publication May 21, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ER ß in proliferation in the rodent mammary gland. Proc Natl Acad Sci USA 101:3739–3746. Endocrinology 143:2357–2365NURSA Molecule Pages Link:
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