Progesterone action in normal mouse mammary gland.

0013-7227/90/1275-2183$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society

Vol. 127, No. 5 Printed in U.S.A.

Progesterone Action in Normal Mouse Mammary Gland* SHIQING WANG, LAURA J. COUNTERMAN, AND SANDRA Z. HASLAM Physiology Department, Michigan State University, East Lansing, Michigan 48824

ABSTRACT. Previously it has been shown that progesterone, as well as estrogen, plays an important role in the growth of the mammary gland. Eighty percent of mammary progesterone receptors (PgR) are estrogen-inducibile and are localized in the epithelium; the remaining 20% of PgR are estrogen-independent and appear to be localized in the mammary stroma. The purpose of the present study was to investigate how progestins promote mammary growth in relation to their interactions with epithelial and stromal components of the gland and to assess the role of estrogen in these interactions. Progestins [progesterone, [17/3methyl-3H]promogestone (R5020), and medroxy progesterone acetate] alone or in combination with estrogen were combined with Elvax 40P and implanted directly into mammary glands. The effect of hormones on cell proliferation was determined by observing changes in mammary gland morphology and by quantitating DNA synthesis in both epithelial and stromal cells by DNA histoautoradiography. The results demonstrate that in mammary epithelial cells the effects of progestins on mammary gland morphology and DNA synthesis are locally mediated such that proliferative changes in the hormone-implanted glands were

S

EVERAL lines of evidence indicate that progesterone (P) plays an important role in stimulating normal mammary gland growth and differentiation in rodents in vivo (1, 2) and in vitro (3, 4), as well as in human mammary tissue in vivo (5, 6). Recently we have reported that in sexually mature mice, P has a potent effect on the stimulation of DNA synthesis of mammary ductal epithelium (7). Estrogen (E) is also required for proliferation of normal mammary epithelium; it is quite likely that one way in which E acts to promote proliferation is through its ability to increase progesterone receptor (PgR) concentration (8-10). E-inducible PgR constitute 80% of the total PgR present in the mammary gland and appear to be localized mainly in mammary epithelial cells (11,12). Constitutive, E-independent PgR are also present in the adult mammary gland, but this class of PgR appears to be localized in the mammary stroma (12). The relative contribution of the two types of progestin binding sites to mammary gland growth and development is not known. Received May 21, 1990. Address all correspondence and requests for reprints to: Dr. Sandra Z. Haslam, Physiology Department, Giltner Hall, Michigan State University, East Lansing, Michigan 48824. * This work was supported by NIH Grant CA-40104.

greater than in contralateral control glands. Dose-response studies with estrogen and R5020 revealed that the extent of progestin activity was only partially dependent upon the R5020 dose with the major determining factor being the dose of estrogen. Analysis of the effect of estrogen on mammary PgR concentration indicates that the degree and pattern of the morphological response of ductal sidebranching and increases in DNA synthesis are largely due to the increase in estrogen-dependent PgR. The antiprogestin, 11(8- (4-dimethylamino-phenyl) 1 -17/3- hydroxy17a-(prop-lynyl)-estra-4,9-diene-3-one (RU486), blocks the proliferation in the epithelium that is mediated through estrogen-dependent PgR. In contrast, in stromal cells progestin activity is not estrogen-dependent, and stimulation of DNA synthesis was not confined to the hormone-implanted glands. Furthermore, RU486 stimulates stromal cell DNA synthesis, and this response is augmented by estrogen. While progestin effects in epithelial cells appear to be mediated by estrogen-dependent PgR, the mechanism operative in stromal cells appears to be different and remains to be elucidated. (Endocrinology 127: 2183-2189, 1990)

The purpose of the present investigation was to elucidate further how progestins mediate mammary growth and interact with the epithelial and stromal components of the gland and to assess the role of E in these interactions. Using Elvax 40P implants it is possible to confine bioactive molecules locally to the mammary gland (13). We have recently been able to distinguish between local vs. systemic effects of E on mammary gland proliferation using this method (14). In order to study the potential local vs. systemically mediated effects of progestins and estrogenic regulation of P effects, Elvax 40P also was used. The effects of E and/or P on mammary gland morphology and DNA synthesis in both epithelial and stromal cells were investigated. Materials and Methods Chemicals [17,9-methyl-3H]promogestone (R5020; SA, 71 Ci/mmol) and radioinert R5020 were purchased from New England Nuclear Corp. (Boston, MA). ll/M4-dimethylamino-phenyl)l-17/?-hydroxy-17a-(prop-lynyl)-estra-4,9-diene-3-one (RU486) was a gift from Roussel Uclaf (Romainville, France). All other hormones were purchased form Sigma Chemical Co. (St. Louis, MO). [Methyl-3H]Thydmidine (50 Ci/mmol) was purchased 2183

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PROGESTERONE EFFECTS IN NORMAL MAMMARY GLAND

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from ICN Radiochemicals (Irvine, CA). All other chemicals were reagent grade. Animals Ten-week-old BALB/c mice from our own colony were ovariectomized 1 week before Elvax implantation. Elvax pellets containing either P, R5020, medroxy progesterone acetate (MPA), or RU486 (0.1 or 1 jtg/pellet) alone or combined with varying amounts of 17/3-estradiol (5, 50, or 500 ng/pellet) were prepared as previously described (13,14). Hormone-containing pellets were implanted into the right inguinal gland, and control pellets were implanted in the contralateral left inguinal gland. DNA histoautoradiography Two or 4 days after implantation, mice received a single ip injection of 3H-thymidine (2 /uCi/g body wt) 1 h before death. The mammary glands were removed and processed for whole mount analysis as well as autoradiographic analysis as previously described (7, 15). Determination of mammary epithelial and stromal cell labeling indices (LI, percent labeled nuclei) was accomplished with the use of a computer-interfaced morphometric digitizing system (7). Steroid hormone binding assay Hormone- or control-implanted mammary glands were removed, homogenized separately, and prepared as cytoplasmic extracts, as previously described (8). Extracts were incubated with 1-20 nM [3H]R5020 with or without a 100-fold excess of radioinert R5020 in the presence of 100-fold excess radioinert dexamethasone (to suppress progestin binding to glucocorticoid binding sites). Specific binding was determined using a dextrancoated charcoal assay procedure (8), and binding data were analyzed according to Scatchard (16). Tissue DNA was quantitated as previously described (17). Statistical analysis All data are expressed as the mean ± SEM, and analysis of variance was applied to all of the data to test for statistical significance.

Results Mammary gland morphology Implantation of Elvax pellets containing either R5020 or E alone had no effect on mammary gland morphology (Fig. 1, A and B). However, when R5020 + E were combined and implanted together, ductal sidebranching similar to that which occurs in early pregnancy was observed. The sidebranching occurred directly adjacent to the implant (Fig. 1C), and no sidebranching was observed in the contralateral control gland (Fig. ID), confirming the localized nature of the response. Mammary gland wholemounts were analyzed 2, 4, and 6 days after implantation with the maximal morphological re-

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sponse observed on day 4 (data not shown). P and MPA, when combined with E, both produced morphological responses similar to R5020; the antiprogestin RU486 either alone or combined with E had no stimulatory effect on mammary gland morphology (data not shown). Dose-response experiments with R5020 and E revealed that the dose of E appears to be the limiting factor determining the extent of mammary gland response to progestins. When a low dose of R5020 (0.1 ^g) was used, increasing doses of E (5, 50, and 500 ng) caused the area and density of ductal sidebranching surrounding the implant to increase (Fig. 2). At 5 or 50 ng E no ductal sidebranching was observed in the contralateral control gland, indicating that the progestin response was localized to the hormone-implanted gland. At 500 ng E, plumping of the duct ends without sidebranching was

observed in the contralateral gland, indicating that at this dose the effect was due to E (Fig. 2), whereas the progestin effect still was confined to the hormone-implanted gland. When a high dose of R5020 (1 /xg) was used, increasing doses of E caused much greater increases in the area of sidebranching in the implanted gland (Fig. 3). Furthermore, the sidebranching now also extended to the contralateral gland, indicating that the R5020 effect was no longer limited to the implanted gland. These results demonstrate that the extent of the effect of R5020 was only partially dose dependent and that a major factor determining the extent of the progestin response was the amount of E. Regulation of PgR concentration One major regulatory effect of E on progestin-mediated effects is through regulation of PgR concentration. To determine if this was responsible for the limiting effect of E observed above, the effect of varying doses of E on PgR concentration in hormone-implanted and contol contralateral glands was analyzed. The results shown in Table 1 demonstrate that at low dose of E (5 ng) only PgR in the implanted gland is increased. However at the 50 and 500 ng doses of E, both contralateral control and E-implanted glands had elevated PgR levels. These results demonstrate that the dose dependent effects of E on the degree and pattern of increased ductal sidebranching induced by R5020 + E at various doses are most likely determined by the amount of E-induced PgR. Mammary gland DNA synthesis The effects of hormone implants in DNA synthesis of mammary epithelial cells are shown in Fig. 4. In initial experiments, DNA synthesis was analyzed at both 2 and 4 days after implantation. The greatest increase in LI was consistently observed at 2 days (data not shown), therefore all subsequent experiments were analyzed at



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FIG. 1. Effect of progestin R5020 and E on mammary gland morphology. Mice were ovariectomized 1 week before implantation with Elvax pellets containing either no hormone (control) (a), 1 ng R5020 (b), or 1 Mg R5020 + 5 ng E (c) in the right inguinal gland and control pellets in the contralateral left inguinal gland (d). Mammary gland wholemounts were analyzed 4 days after implantation. Magnification, x64.

day 2. In contrast to the effect on mammary gland morphology, progestins alone significantly increased DNA synthesis (Fig. 4). R5020 had a greater effect than MPA, and P had the least effect (P < 0.05). This order and magnitude of responses in the mammary gland is compatible with the progestin potentcy of these compounds and relative binding affinity to PgR observed in other target organs (18, 19). When progestins were combined with E, much greater increases in DNA synthesis were observed (Fig. 5). As above, R5020 had a greater effect than MPA, which had a greater effect than P (P < 0.05). The antiprogestin, RU486, alone or in combination with E, did not stimulate DNA synthesis. However, the E + R5020-induced increase in DNA synthesis was blocked in the presence of RU486 (Fig. 6). The inhibitory effect of RU486 indicates that the R5020 effect on DNA synthesis was due to its progestin activity and was mediated via E-induced PgR. In all cases the stimulation of epithelial cell DNA synthesis was much greater in hormone-implanted glands than in the contralateral control gland, implying a local mode of progestin action. The results of progestin effects on stromal cell DNA synthesis are shown in Figs. 7 and 8. Progestins alone increased stromal cell DNA synthesis, and the biological potency of the progestins followed the same pattern as that observed for epithelial cells (Fig. 7). One major difference, however, was that RU486 by itself increased stromal cell DNA synthesis. In contrast to its effect in epithelial cells, E did not increase the effects of R5020, MPA, or P in stromal cells (Fig. 8). However, the effect

of RU486 was increased by estrogen. In all cases DNA synthesis in stromal cells was increased in both hormoneand control-implanted glands. These results further demonstrate that the regulating effect of E on progestin effects observed in mammary epithelium is not operative in stromal cells. Discussion The ability of progestins to promote mammary gland growth and differentiation in vivo was assessed in two ways by their ability to: 1) stimulate DNA synthesis in epithelial and stromal cells; and 2) produce ductal sidebranching that gives rise to lobuloalveoar morphogenesis that occurs during pregnancy. Furthermore, the role of E in these events was also assessed. The results demonstrate that progestins tested herein produce their stimulatory effects on epithelial DNA synthesis and ductal sidebranching by virtue of intrinsic progestin activity. The high activity of R5020 and MPA, which have reduced sensitivity to metabolic inactivation, and the lesser activity of P, which is rapidly metabolized, indicate that most likely the native compounds rather than progestin metabolites are responsible for the activity. It is of interest to note that whereas RU486 inhibited the increase in epithelial cell DNA synthesis resulting from estrogen + R5020 treatment, it did not reduce the DNA synthesis increase observed with R5020 alone. One possible explanation for this observation is that RU486 interacts preferentially with newly synthesized E-in-


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FIG. 2. Effect of low R5020 dose plus E on mammary gland morphology. Mice were ovariectomized 1 week before implantation with Elvax pellets containing 0.1 Mg R5020 + 5 ng E (a), 50 ng E (c), or 500 ng E (e) respectively in the right inguinal gland and control pellets in the contralateral left inguinal gland (b, d, and f). Mammary gland wholemounts were analyzed 4 days after implantation. Magnification, X64.

duced PgR. We have previously reported that PgR, newly synthesized in response to E, appears to be different from preexisting old PgR with regard to molecular size and/or form (20). Thus, these differences may be the basis for the observed differential effect of RU486. Progestins can produce a localized effect on mammary gland morphology and epithelial DNA synthesis, implying direct action of progestins in the mammary gland. E enhances the stimulatory effect of progestins on epithelial cell DNA synthesis. Analysis of the effect of E dose on PgR concentration in conjunction with the extent of and patterns of ductal sidebranching indicate that the limiting factor for P-mediated effects in epithelial cells is E-induced PgR concentration. Although progestins alone increase DNA synthesis, this does not appear to be sufficient to cause sidebranching. It is possible that a higher rate of DNA synthesis is achieved with the addition of estrogen that results in sidebranching. We also

have observed that there is significant heterogeneity in the distribution of epithelial estrogen and progesterone receptors (our unpublished observations). This suggests that topographic differences also may exist with regard to localization of DNA synthesis, and this also may be a determining factor for the occurrence of sidebranching. Thus, these results extend our previous observations and further support the concept that in normal mammary epithelial cells progestins have major mitogenic activity. Furthermore, by using Elvax implants we have been able to demonstrate that the progestin-induced response is the result of direct/local action in the mammary gland and that it is mediated via E-induced PgR. Progestins also stimulate mammary stromal cell DNA synthesis. However, the mechanisms operative in stromal cells appear to be different from those occurring in epithelial cells. Firstly, even at low progestin doses the hormone effects are not confined to the implanted



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FIG. 3. Effect of high R5020 dose plus E on mammary gland morphology. Mice were ovariectomized 1 week before implantation with Elvax pellets containing 1 ng R5020 + 5 ng E (a), 50 ng E (c), or 500 ng E (e) respectively in the right inguinal gland and control pellets in the contralateral left inguinal gland (b, d, and f). Mammary gland wholemounts were analyzed 4 days after implantation. Magnification, X64.

TABLE 1. Effect of varying dose estrogen implants on mammary progesterone receptor concentration

Edose

Specific 3H-R5020 binding (fm/mg DNA) Right

Control 5 ng 50 ng 500 ng

400 ± 600 ± 786 ± 787 ±

35 49° 57 155

Left 408 ± 425 ± 712 ± 735 ±

39 50 39 163

0

P = 0.05 that right gland specific 3H-R5020 binding is greater than that of left gland.

gland but are expressed equally in the control contralateral gland. Secondly, estrogen does not synergize to enhance the proliferative effect of progestins. Thus it can be concluded that progestin effects on stromal cells are not mediated via E-induced PgR. This finding is compatible with our previous observations that stromal cell PgR are E-independent (12). Unexpectedly, DNA syn-

thesis was also stimulated by the antiprogestin, RU486. Whereas estrogen did not synergize with progestins, it did increase the effect of RU486. Others recently have shown that RU486 also can stimulate proliferation of certain breast cancer cell lines in vitro (21, 22); however, the basis for this response has not been determined. RU486 also has potent antiglucocorticoid activity (23, 24), and it remains to be established whether or not the progestin effects in stromal cells are due the progestin or antiglucocorticoid activity of the compounds. Thus, at this time the mechanism(s) underlying progestin effects on stromal cell proliferation remain to be elucidated and are the subject of continuing investigations.

Acknowledgment The authors thank Ms. Esther Brenke for the excellent preparation of this manuscript.



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Endo • 1990 Vol 127 • No 5

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References 1. Nandi S 1958 Endocrine control of mammary gland development and function in the C3H/HeCrgl mouse. J Natl Cancer Inst 21:1039 2. Bresciani F 1968 Topography of DNA synthesis in the mammary gland of the C3H mouse and its control by ovarian hormones. Cell Tissue Kinet 1:51 3. Edery M, McGrath M, Larson L, Nandi S 1984 Correlation between in vitro growth regulation of estrogen and progesterone receptors

control R5020 MPA

Pg

RU486

Hormone Treatment FIG. 7. Effect of progestins on mammary gland stromal cell DNA synthesis. Mice were ovariectomized 1 week before implantation with Elvax pellets containing 1 tig R5020,1 tig MPA, 1 tig P, or 1 tig RU486 (•) in the right inguinal gland and control pellets (M) in the contralateral left inguinal gland. Two days after implantation, LI was determined in stromal cells by DNA histoautoradiography. Each value represents the mean ± SEM of two or three experiments; each experimental group contained three or four animals. *, P < 0.05 that hormoneand control-implanted glands are greater than control group. in rat mammary epithelial cells. Endocrinology 115:1691 4. Imagawa W, Tamooka Y, Hamamoto S, Nandi S 1985 Stimulation of mammary epithelial cell growth in vitro: interaction of epidermal growth factor and mammogenic hormones. Endocrinology 116:1514 5. Masters JRW, Drife JO, Scarisbreck JJ 1977 Cyclic variation of DNA synthesis in human breast epithelium. J Natl Cancer Inst 58:1263 6. Brown JB 1981 Hormone profiles in young women at risk of breast cancer. In: Pick MC, Sitteri PK, Welsch CW (eds) Hormones and Breast Cancer. Banbury Report 8, Cold Spring Harbor Laboratory,


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FlG. 8. Effect of progestins plus E on mammary gland stromal cell DNA synthesis. Mice were ovariectomized 1 week before implantation with Elvax pellets containing either 5 ng E alone or in combination with either 1 ng R5020, 1 ng MPA, 1 ng P, or 1 ng RU486 (•) in the right inguinal gland and control pellets (M) in the contralateral left inguinal gland. Two days after implantation, LI was determined by DNA histoautoradiography. Each value represents the mean ± SEM of two or three experiments; each experimental group contained three or four animals. *, P < 0.05 that hormone-implanted and control-implanted glands in progestin plus E-treated groups are greater than estrogen-alone group. Cold Spring Harbor, p 33 7. Haslam SZ 1988 Progesterone effects on deoxyribonucleic acid synthesis in normal mouse mammary glands. Endocrinology 122:464 8. Haslam SZ, Shyamala G 1979 Effect of oestradiol on progesterone receptors in normal mammary glands and its relationship to lactation. Biochem J 182:127 9. Haslam SZ, Shyamala G 1980 Progesterone receptors in normal mammary gland: receptor modulations in relation to differentiation. J Cell Biol 86:730 10. Haslam SZ 1986 Mammary fibroblast influence on normal mouse

16. 17. 18. 19. 20.

21. 22. 23.

24.

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mammary epithelial cell responses to estrogen in vitro. Cancer Res 46:310 Edery M, Imagawa W, Larson L, Nandi S 1984 Regulation of estrogen and progesterone receptor levels in mouse mammary epithelial cells grown in serum-free collagen gel cultures. Endocrinology 116:105 Haslam SZ, Shyamala G 1981 Relative distribution of estrogen and progesterone receptors among the epithelial, adipose, and connective tissue components of the normal mammary gland. Endocrinology 108:825 Silberstein GB, Daniel CW 1982 Elvax 40P implants: sustained, local release of bioactive molecules influencing mammary ductal development. Dev Biol 93:272 Haslam SZ 1988 Local versus systemically mediated effects of estrogen on normal mammary epithelial cell deoxyribonucleic acid synthesis. Endocrinology 122:860 Banerjee MR, Wood BG, Lin FK, Crump LR 1976 Organ culture of whole mammary gland of the mouse. Tissue Culture Assoc Manual 2:457 Scatchard G 1947 The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660 Ceriotti GA 1952 A microchemical determination of deoxyribonucleic acid. J Biol Chem 198:297 Feil PD, Glasser SR, Toft DO, O'Malley BW 1972 Progesterone binding in the mouse and rat uterus. Endocrinology 91:738 Philbert D, Raynaud JP 1974 Progesterone binding in the immature rabbit and guinea pig uterus. Endocrinology 94:627 Haslam SZ 1987 Role of sex steroid hormones in normal mammary gland function. In: Neville MC, Daniel CW (eds) The Mammary Gland: Development, Regulation and Function. Plenum Press, New York, p 449 Bowden RT, Hissom JR, Moore MR 1989 Growth stimulation of T47D human breast cancer cells by the anti-progestin RU486. Endocrinology 124:2642 Hissom JR, Bowden RT, Moore MR 1989 Effects of progestins, estrogens, and antihormones on growth and lactate dehydrogenase in the human breast cancer cell line T47D. Endocrinology 125:418 Moguilewsky M, Philibert D 1984 RU 38486: potent antiglucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by an impaired activation. J Steroid Biochem 20:271 Philibert D 1984 RU 38486: an original multifaceted antihormone in vivo. In: Agarwal MK (ed) Adrenal Steroid Antagnism. Walter de Gruyter & Co, Berlin, p 77


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