Direct Action of 17/3-Estradiol on Mouse Mammary Ducts Analyzed by Sustained Release Implants and Steroid Autoradiography1

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[CANCER RESEARCH 47, 6052-6057, November 15. 1987] Direct Action of 17/3-Estradiol on Mouse Mammary Ducts Analyzed by Sustained Release Implants and Steroid Autoradiography1

Charles W. Daniel,2 Gary B. Silberstein, and Phyllis Strickland

Department of Biology, Thimann Laboratories, University of California, Santa Cruz, California 95064

ABSTRACT association of stroma, and indeed the stroma might be the site of initial action by the hormone. 17,M .stradini is a recognized mammary mitogen, but uncertainty exists A different approach to the problem of estradiol action has as to whether its normal action is mediated exclusively through the been the direct application of steroid to the gland in vivo. By pituitary or whether in addition direct effects of estradiol on mammary applying estrone to the skin in the nipple area of male monkeys tissue may play a role in mammary growth and development. To further investigate the action of estradiol on the developing mammary ductal (10,11), rabbits (12), and guinea pigs ( 13) lobuloalveolar growth system of young mice, implants of biocompatible ethylene vinyl acetate was stimulated in the treated compared with the untreated copolymer, which deliver small amounts of steroid locally to the target glands. Implantation of estrogens in cholesterol pellets into the tissue, were implanted into the mammary glands of castrated females in mammary glands of ovariectomized virgin mice did not produce which the ductal system was static and end buds had regressed. Within local glandular growth (14). 3 days end buds appeared in the vicinity of the implants but not elsewhere An improved technique for local administration of hormones in the gland and not in other glands of the animal, indicating direct and growth factors has recently become available in which small stimulation. The new end buds were histologically normal, displaying a plastic EVAc implants capable of the slow release of nondena- visible cap (stem) cell layer with high levels of DNA synthesis. The tured bioactive molecules are placed directly into the gland (15). antiestrogen keoxifene, which competes with estrogen for its receptors, inhibited end bud formation in the estradiol-implanted gland but failed EVAc pellets may be implanted to any area of interest with to inhibit growth when implanted into the glands of intact, 5-week-old local effects of released molecules readily distinguishable from females. Time course and dose-response studies of estradiol stimulation systemic influences. This technique allows both qualitative and were carried out in ovariectomized animals and were consistent with a quantitative assessments of hormone effects. direct action for estrogen. Steroid autoradiography revealed estrogen In this communication we report that implanted estrogen can receptors in the lumenal cells of the end bud, in ductal epithelium, and directly stimulate mammary ductal growth in castrated female in stroma adjacent to ducts, but none was detected in the rapidly prolif mice without causing systemic stimulation and that this effect erating cap cells. We conclude that estrogen, perhaps acting on nonepi- can be blocked by a competitive inhibitor of estradiol binding. thelial target cells and probably in conjunction with extramammary Steroid autography revealed a gradient of estradiol receptors in factors, directly stimulates mammary ductal growth. the mammary ducts and the presence of receptors in the stroma of the developing gland. INTRODUCTION In vivo, estradiol3 is known to play a critical role in stimulat MATERIALS AND METHODS ing growth of the mouse mammary gland during the ductal Materials. EVAc (Elvax 40P) was a gift of DuPont Chemical Com phase of development (1). Administered systemically to cas pany, Universal City, CA. Steroid hormones and tatnoxifen were ob trated female mice, it has the specific effect of restoring the tained from Sigma Chemical Company, St. Louis, MO. The antiestro mammary end buds, the growth points for ductal elongation. gen keoxifene (LY 156758) was a gift of Eli Lilly Company, Indian These restored end buds display normally high levels of DNA apolis, IN (16). synthesis (2). Because estradiol is ineffective in restoring mam Animals and Surgical Procedures. Virgin C57/BL/crl mice were mary growth in ovariectomized-hypophysectomized animals, castrated at 5 wk of age and were used for implant experiments 6 wk its action on the gland is considered to be indirect,4 through or more after surgery. Preparation of Implants. EVAc implants were prepared as described stimulation of pituitary prolactin secretion (3, 4). Further evi by Rhine et al. (17). EVAc was washed for 1 wk prior to use in several dence supporting an indirect role for the steroid is found in changes of 95% ethanol with continuous stirring and dissolved in several in vitro studies in which estradiol added to culture mÃ©thylÃ¨nechlorideto give a 20% w/v solution. Steroids were dissolved medium failed to stimulate proliferation in primary cultures of directly in the EVAc solution, and the solution was quick frozen in an mouse mammary epithelial cells (5-7). acetone-dry ice bath for 10 min. The frozen pellet was stored for 2 days An additional, more direct role for estrogen in mammary at -20Â°C,after which it was placed under a mild vacuum for another 2 growth and development is indicated by recent in vitro studies days at room temperature. The dried pellet was cut into approximately 1.0-mm2 pieces and weighed. of mixed cultures of mammary epithelial and mammary stromal cells, in which epithelial proliferation was enhanced by the Release of Estradiol from EVAc. The release kinetics of estrogen was studied in vitro. Pellets prepared with 86 ng/mg EVAc. and containing addition of estradiol to the culture medium (8, 9). These exper 9 x 10" cpm/mg EVAc 17/3-[3H]estradiol (NEN), were cut into implant- iments suggest that estradiol action may require the close sized pieces, weighed, and sealed in porous nylon mesh bags (Nitex) which were then placed in 1.5 ml normal saline at 37Â°Con a rotator (1 Received 5/18/87: revised 8/11/87; accepted 8/18/87. The costs of publication of this article were defrayed in part by the payment rpm). At 24-h intervals, the bags were removed and 0.5 ml of the saline of page charges. This article must therefore be hereby marked advertisement in was counted in Biofluor (NEN). Bags were then rinsed and replaced in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by NSF Grant PCM83-0814. fresh saline. 2To whom requests for reprints should be addressed. Implantation of EVAc. Recipient mice were anesthetized with Nem- 3The abbreviations used are: estradiol. 170-estradiol; EVAc, ethylene vinyl butal (60 Â¿ig/gbodyweight) and the third and fourth mammary glands acetate copolymer (Elvax 40P; DuPont Chemical Company). 4Our use of the terms "direct" and "local" throughout this paper do not imply were exposed by reflecting the abdominal skin. A small pocket was made in the mammary fat pad using Dumont forceps which were then a particular mode of action at the cellular level; estradiol could act either as a primary mitogen or. as suggested by these and other results, as a means of used to insert the implant. In the case of double implants, both were sensitizing the gland to systemic factors. inserted into the same pocket. The abdominal skin was closed with 6052

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. DIRECT STIMULATION OF MAMMARY DUCTS BY ESTRADIOL wound clips and the animals were allowed to recover in an atmosphere virgin mice at 5 wk of age is arborated and fills approximately of95%O2-5%CO2. 50% of the available fatty stroma. In hormonal h intact animals Steroid Autoradiography. Virgin female C57/BL mice were ovariec- the tips of most branches display large end buds (19). Ovariec tomized at 5 wk of age, 2 days prior to treatment, to reduce the levels tomy resulted in an approximately linear decline in end bud of endogenous estrogen. Each of three experimental animals received an injection of 200 //C ['HJestradiol in 40 ^1 100% ethanol with 0.2 ml number, end bud size, and ductal caliber. After 2 wk ducts were balanced salt solution and 2 UKBSA. Control animals also received a thin, no end buds were seen (Figs. 1 and 2), and there was no second injection containing 200-fold excess estradiol or testosterone DNA synthesis in the regressed gland (Fig. ID). propionate in the same vehicle, and animals in the experimental group The time course of the estrogen response was investigated. received a second injection consisting of vehicle only. Mice, ovariectomized for at least 6 wk, were implanted with a Animals were sacrificed by cervical dislocation 2 h after injection. single EVAc pellet containing 25 ng estradiol, a dose which Strips of mammary' glands were taken in the end bud region and near produced a maximal local response. Whole mount preparations the nipple. Control tissues included uterus, bladder, and mammary fat of glands were scored for numbers of end buds after which pad from an area of the gland not occupied by mammary ducts. Tissue representative areas were excised and processed for [3H]thymi- was flattened on strips of cardboard, flash frozen in liquid propane, and stored at -70Â°C. Sections of 5 pm were cut on a Tissue Tek II dine autoradiography from which labeling indices were deter cryostat at â€”30Â°C.Inthe dark, frozen sections were transferred to mined. Fig. 3 shows that end buds appeared on Day 3, were slides predipped in Kodak NTB-2 photographic emulsion diluted 1:1 most numerous on Day 4, and decreased in number and size with water by the method of Stumpf and Sar (18). Slides were stored through Day 10. Significant DNA synthesis began in ductal in the dark at 4Â°Cfor 3-5 mo, developed in Kodak D-19 developer tips on Day 2, before the appearance of end buds, peaked on diluted 1:1 with water at 15Â°Cfor 3 min, rinsed for 30 s in water, and Days 3 and 4, and declined to negligible levels by Day 8. Because fixed for 5 min. Slides were lightly stained in hematoxylin/eosin for the decline in DNA synthesis was more rapid than the disap examination. pearance of end buds, during Days 6-8 small end buds were Tissue Preparation for Microscopic Studies. Glands were fixed for 2 present which were no longer actively growing. or more h in Tellyesniczky's fluid and whole mounts were prepared as The kinetics of estrogen release from EVAc was determined described (15). In all cases a sample from an unimplanted mammary in vitro and was essentially linear after the first day. In the first gland was taken in order to evaluate systemic effects of treatment. For 24 h, approximately 4% of the total estrogen contained in the histology, pieces of whole mount preparations were embedded in par affin for sectioning. In cases in which DN A autoradiography was carried plastic was released; this declined to 1.5% by Day 10 (Fig. 3). out, animals were injected with 100 ^Ci ['Hjthymidine (1CN, Irvine, Dose Response of Estradiol Stimulation. Ovariectomized CA) 30 min prior to anesthesia. Paraffin sections of this material were hosts were implanted in the third mammary fat pad with pellets dipped in Kodak NTB-2 emulsion, exposed for 12 days, and developed containing 6-48 ng estradiol. Fig. 4 shows that glands in in Kodak D-19 diluted 1:1 with water. Cells displaying three or more ovariectomized animals responded to increasing amounts of silver grains/nucleus were scored as positive in both DNA and steroid estradiol up to 25 ng with increased numbers of end buds local autoradiographic experiments. to the implant. At higher doses the response became systemic with end buds first appearing in the nipple region of both the implanted and nonimplanted glands. At still higher doses, RESULTS stimulation progressed to the middle of the gland, finally in Local Stimulation of Growth and Morphogenesis by Estradiol. volving the entire ductal network (data not shown). As a control EVAc pellets containing 25 ng estradiol were implanted near for specificity of the 170-estradiol effect, pellets of 17Â«-estradiol the tips of regressed ducts in the thoracic thirol mammary were implanted in a second set of mice and only at doses of glands of 3-mo-oId mice which had been ovariectomized at 5 1,200 ng/pellet was a modest, nonsystemic effect observed. wk of age. Whole mount preparations of glands taken after 4 Estradiol Inhibitor Studies. Two nonsteroidal antiestrogens, days of treatment showed the reappearance of large end buds keoxifene and tamoxifen, were investigated for their ability to at the ductal tips. Regions of the same gland but beyond the inhibit estradiol stimulation. Ovariectomized hosts received immediate influence of the implant were unaffected. Contralat- simultaneous implants in the third mammary fat pad of 32 ng eral and ipsilateral glands also showed neither end buds nor estradiol and the antiestrogen keoxifene in the same glandular autoradiographic evidence of DNA synthesis (Fig. 1). The location. Separate control animals were implanted with equiv branching pattern and ductal caliber of treated glands were alent doses of estradiol and BSA. Fig. 5 shows that after 4 days, normal in all respects and in no case were hyperplasias or other 0.35-mg keoxifene treatment resulted in 92% inhibition of the structural abnormalities observed. local stimulation but did not affect the systemic response. The newly formed end buds displayed typical histology, which Keoxifene was also tested for its ability to inhibit the growth included the restoration of the cap cell layer and an epithelial of mammary ducts in intact, 5-wk female mice by placing stem cell population located between the lumenal cells and the inhibitor-containing EVAc implants close to the growing end stroma (Fig. 1C). Autoradiographs of glands pulsed with buds. No significant reduction in end bud numbers or labeling ['H]thymidine revealed extensive epithelial DNA synthesis, par index was observed (data not reported). ticularly in cap cells (Fig. 1C), at levels that were comparable Tamoxifen, an antiestrogen with estrogenic properties (20), to those in intact, 5-wk animals (data not shown). Thus, local was not inhibitory under any experimental conditions and in ized estradiol-induced cell proliferation was coupled with ap fact stimulated moderate numbers of end buds when implanted parently normal ductal morphogenesis. Glands implanted with into ovariectomized animals (data not shown). 40 ng BSA in EVAc showed no stimulation (Fig. \B) and Estradiol Receptor Distribution in the Mammary Tree. Freeze- thymidine autoradiography revealed no DNA synthesis (Fig. thaw steroid autoradiography (18) was used to determine the ID). distribution of estradiol receptors in the mammary ductal sys Time Course Studies. The time course of end bud regression tem. Cells displaying three or more silver grains over their after ovariectomy was investigated in order to be certain that nuclei were found in many but not all regions of the duct (Fig. the effects seen with implanted estrogen occurred against a 6, C and D). The basal layer of undifferentiated stem cells, the clearly growth-quiescent background. The ductal system in cap cells, were unlabeled in all preparations examined. Myo- 6053

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Fig. 1. Local stimulation of ductal growth in ovariectomized hosts treated with estradiol. A, appearance of local end buds (EB) after 4 days exposure to 25 ng estradiol. B, contralateral gland exposed 4 days to blank EVAc only. A and B, x 10.2. Implant is at the top of the photograph. C, autoradiograph of longitudinal section of an end bud from gland pictured in A. D, autoradiograph of terminal duct (TD) from gland pictured in B. The pattern of DNA synthesis in estradiol-treated glands is identical to that seen in glands from hormonally intact animals (not pictured); no labeled cells were seen in the contralateral control gland. C and D, x 350. CC. cap cells.

about 7 silver grains/labeled cell. The ducts immediately sub tending end buds displayed labeling of similar intensity to that 24 30 seen in the luminal epithelium of the end bud. Ducts in the nipple region displayed even greater receptor activity, with an 25 -Ã¯- 20 average of 14 silver grains/labeled cells. These results indicate 16 20 that there is a gradient of estrogen receptor levels in the mam mary duct, with no receptors detected in the end bud cap cells or in myoepithelial cells, and maximum numbers in ducts near the nipple area (Figs. 6 and 1). B TO Â£ In stroma surrounding the mammary ducts patchy distribu 8 tion of labeled cells was observed, with clusters of receptor- positive fibroblastic cells seen in fibrous regions near the mam 4567 U 16 18 mary ducts (Figs. 6 and 1). Distant from mammary tissue, few Doysafter ovariectomy labeled cells were observed and no receptor activity was deter Fig. 2. Gland regression following ovariectomy measured by enumerating end mined in adipocytes. buds (EB) at the growing front and determining their maximum diameter. The addition of excess, cold estradiol to the injection mixture Mammary ducts are maintained in the absence of ovarian steroids but shrink in caliber (not plotted). End bud number is plotted as the mean Â±SE per gland; n greatly reduced labeling (Fig. 6) while testosterone propionate = 4 glands. A representative experiment is plotted. End bud diameter is plotted had no effect (data not shown), thus demonstrating that the as the mean Â±SE. The range of n was 43-11 end buds for Days 1-8. observed binding was specific for estradiol. Uterine myome- trium, an estrogen target tissue, was included as a positive epithelial cells, which occur along the differentiated duct and control for estrogen receptors while bladder tissue, which has are derived from cap cells (Ref. 19; Fig. 6D) were also unlabeled. no estrogen receptors, served as a negative control. When In contrast, cells of the multilayered epithelial tissue bordering incubated with labeled estradiol, the uterine tissue was heavily the end bud lumen were frequently labeled, with an average of labeled while the bladder was not (data not shown). 6054

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4567 17a 20 Days after implant Fig. 3. Time course of estradici stimulation measured by changes in DNA synthesis and end bud numbers local to slow-release implant. Ovariectomized hosts were implanted with a pellet containing 25 ng estradici, a dose which gives 10 20 30 40 50 1200 2000 a maximum local response. Animals were sacrificed every' 24 h for 10 days. Representative sections of glands were cut out and used for thymidine autora- Estradiol (ng) diography. Results are plotted as the mean number of end buds Â±SE: end bud number was 3-8 for Days 3-10. % labeled cells/EB, number of end buds evaluated Fig. 4. Estradiol dose-response study. In the dose range to the left of the ranged from 3-5 for Days 1-10. Insel, in vitro release of [JH]estradiol from EVAc. dashed line, end buds were found almost exclusively in the region of the implant An EVAc pellet containing 86 ng/mg unlabeled estradiol with 9 x 10*cpm/mg and no DNA synthesis was seen in contralateral or ipsilateral control glands. At triliated estradiol, was cut into implant-sized pieces, which were incubated in higher doses end buds appeared Tirsi in the nipple region of the implanted gland saline as described in "Materials and Methods." Results are the average of two and then in nonimplanted glands. 17i>-Eslradiol had a mammogenic effect only trials in which 12 and 15 1-mg pieces were incubated. The percentage of the total at very high doses. Independent values derived from several experiments are counts released after 14 days was 33%; release of unlabeled estradiol was calcu plotted. lated on the basis of percentage of label released. Mean no. EBs local to implant/gland DISCUSSION CO 4- M o 00 The local ductal mammogenic effects of implanted estrogen described herein are unambiguous in demonstrating a direct action of estradiol on mammary tissue. Estradiol, at doses lower than 25 ng, acted only in the implanted gland to restore ductal growth in castrated female mice (Figs. 1 and 4) while leaving unaffected other regions of the treated gland as well as the untreated glands in the same animal. Stimulation was specific for the biologically active (17-/Ã•)form of the hormone and thus could not be attributed to nonspecific steroid action (Fig. 4). Nor could stimulation have been due to lingering systemic effects of ovarian steroids. Postcastration ductal regression was complete after 2 wk (Fig. 2) and estrogen was never implanted earlier than 6 wk postovariectomy, thus stimulation occurred against a growth-quiescent background. As in the case of injected estradiol (2), the restored end buds demonstrated high levels of DNA synthesis and appeared to be Fig. 5. Inhibition of estradiol action. An EVAc pellet containing 32 ng participating in normal branching morphogenesis. It is also estradiol was placed in the same pocket as a pellet containing 0.35 mg keoxifene and end buds were counted after 4 and 5 days (D); controls received co-implants significant that the newly formed end buds displayed a regen of estradiol and BSA (O). Results are plotted as the mean number of end buds erated population of cap cells, the putative stem cell population local to an implant Â±SE; n (number of glands evaluated) = 5 (Day 4) and 4 (Day of the mammary ducts which is absent from the regressed ductal 5). tips of estradiol-deprived mice (19). In a variety of other systems the binding of estrogen to its (Fig. 5). Local stimulation must therefore be due, at least Â¡n receptor and the steroid effects that follow can be inhibited by part, to direct effects of the steroid. competitive blockade with nonestrogenic, nonsteroidal inhibi While keoxifene effectively inhibited estradiol action in cas tors such as keoxifene (16). In our mammary gland experi trated mice, it was ineffective in reducing proliferation when ments, co-implantation of estrogen and keoxifene resulted in implanted into endocrine-intact, 5-wk-old females in which the up to 90% inhibition of the action of the steroid, demonstrating gland was vigorously growing (data not shown). This could be that the end bud growth that was correlated with estrogen due to the "open" character of the system, in which estrogen is implantation could be blocked by interfering with estrogen continuously supplied through the circulation while the concen binding to its receptors in the immediate vicinity of the implant tration of inhibitor progressively declines, making it impossible 6055

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Fig. 6. Autoradiographs of tissues exposed in n'ra to |3H]estradiol. A, longitudinal section of end bud, outlined by dashed line. Multilayered lumenal epithelial cells of the end bud body demonstrate moderate labeling while the cap cells (CC) are not labeled. B, an end bud from an animal treated with 200-fold excess cold eslradiol is without significant label. C longitudinal section of duct close to its end bud. Labeling is similar to that seen in end bud body cells. D, duct near the nipple region of the gland in which the cells are more heavily labeled. Note labeled stromal cells (black arrows) and unlabeled myoepithelial cells (clear arrows). All autoradiographs were exposed for 22 wk before being developed, x 630. to maintain an inhibitor concentration sufficient to depress estradiol receptors reflects a secondary function of the steroid, estrogen stimulation. Alternatively, growth factors not imme perhaps induction of progesterone receptors which are known diately dependent on estrogen may be able to sustain ductal to appear in mammary epithelial cells as a result of direct growth for a time, despite a keoxifene blockade. estrogen stimulation (23). The lack of identifiable estradiol The time course of the estrogen response was consistent with receptors in both the cap and myoepithelial cells is consistent a primary effect of the steroid on glandular targets. Epithelial with the observation that myoepithelium in benign breast le DNA synthesis was stimulated after 48 h, followed in 24 h by sions of the human (24) and in normal lactating glands of the the appearance of end buds (Fig. 3). This timing was identical mouse (25) are also negative for estrogen receptors when ex to that observed for stimulation by implanted cholera toxin (21, amined by thaw-mount autoradiography. 22) and epidermal growth factor (unpublished), both of which Estrogen receptors were also detected in the stroma, with have been shown to act directly and nonsystemically on the most of the labeled cells appearing in fibrous tissue in the gland. vicinity of end buds and ducts (Figs. 6 and 7). Although the cell Steroid receptor distribution was investigated to determine types could not be identified with certainty, adipocytes could possible sites of estradici action. No receptors were detected in be excluded and the labeled cells tentatively identified as mes the cap cell layer, the zone of most active DNA synthesis (Figs. enchymal or fibroblastic. The detection of stromal estrogen 6 and 7). In contrast, lumenal cells in the end bud and imme receptors is consistent with other studies in which estradiol diately subtending duct, which also actively synthesize DNA, receptors were identified in homogenates of both normal gland displayed estrogen receptors. Epithelia in the nipple region, and in epithelia-free fat pads (26) and suggests that the mito- where little or no DNA synthesis takes place, had about twice genie action of estrogen may require stromal intermediaries. the receptor number per cell compared to the more peripheral The possibility that the action of estrogen is mediated by cells (Fig. 7). This distribution is the inverse of what would be stromal elements is not surprising, for the role of the stroma as predicted if estrogen were acting directly on the epithelium as a primary, sex-steroid target in developing tissues is well doc a primary mitogen and suggests that the presence of epithelial umented in other systems (27). In our investigation the distri- 6056

6. Yang, J., Richards, J., Guzman, R., Imagawa, W., and Nandi, S. Sustained growth in primary culture of normal mammary epithelial cells embedded in collagen gels. Proc. Nati. Acad. Sci. USA, 77: 2088-2092, 1980. 7. Imagawa, W., Tomooka, Y., and Nandi, S. Serum-free growth of normal and tumor mouse mammary epithelial cells in primary culture. Proc. Nati. Acad. Sci. USA, 79:4074-4077, 1982. 8. McGrath, C. M. Augmentation of the response of normal mammary epithe lial cells to estradiol by mammary stroma. Cancer Res.. 43: 1355-1360, N 1983. 9. Haslam, S. Z., and Levely, M. L. Estradiol responsiveness of normal mouse mammary cells in primary cell culture: association of mammary fibroblasts with estradiol regulation of progesterone receptors. Endocrinology, 116: 1835-1844, 1985. 10. Speert, H. Mode of action of estrogens on the mammary gland. Science (Wash. DC), 92: 461-462, 1940. 11. Chamberlin, T. L., Gardner, W. V., and Allen, E. Local responses of the sexual skin and mammary glands of monkeys to cutaneous applications of estrogen. Endocrinology, 28: 753-757, 1941. 14.0+0.09 9.7Â±2.1 6.8Â±0.87.411.10.0 12. Lyons, W. R., and Sako, Y. Direct action of estrone on the mammary gland. Recent Prog. Horm. Res., 14: 398-401, 1940. 13. Nelson, W. Growth of the mammary gland following local application of estrogenic hormone. Am. J. Physiol., 133: 397-398, 1941. 14. Nagasawa, H., and Yanai, R. Increased mammary gland response to pituitary Fig. 7. Diagrammatic representation of estradiol receptor distribution in the mammotropic hormones by estrogen in rats. Endocrino!. Jpn., 18: 53-56, mammary tree. The number of silver grains Â±SEper labeled cell is indicated in 1971. various regions. Cells with 3 or more silver grains were considered receptor 15. Silberstein, G. B., and Daniel, C. W. EVAc 40P implants: sustained, local positive, and eight or more structures from each area were counted. Labeled release of bioactive molecules influencing mammary ductal development. stromal cells were much more widely distributed along the ducts than is indicated. Dev. Bio!., 95:272-278, 1982. L, lumen; CC, cap cells; N, nipple; S, stroma. 16. Clemens, J. A., Bennett, D. R., Black, L. J., and Jones, C. D. Effects of a new antiestrogen, keoxifene (LY156758), on growth of carcinogen-induced mammary tumors and on LH and prolactin levels. Life Sci., 32: 2869-2875, 1983. bution of epithelial receptors does not support a role for estro 17. Rhine, W. D., Dean, S. T., Hsieh, D. S. T., and Langer, R. L. 1980. Polymers gen as a direct epithelial mitogen while the dose-response for sustained macromolecule release: procedures to fabricate reproducible delivery systems and control release kinetics. J. Pharmacol. Sci., 69: 265- results suggest a complex, possibly indirect mechanism of stim 270, 1980. 18. Stumpf, W. E., and Sar, M. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. Direct Action of 17β-Estradiol on Mouse Mammary Ducts Analyzed by Sustained Release Implants and Steroid Autoradiography

Charles W. Daniel, Gary B. Silberstein and Phyllis Strickland

Cancer Res 1987;47:6052-6057.

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