Progesterone Stimulates Mammary Gland Ductal Morphogenesis by Synergizing with and Enhancing Insulin-Like Growth Factor-I Action
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内分泌学杂志 2005年第3期
Neuroendocrine Unit, Departments of Medicine (W.R., D.L.K.) and Physiology (M.E.M.), New York University School of Medicine and Department of Veterans Affairs Medical Center, New York, New York 10016
Address all correspondence and requests for reprints to: David L. Kleinberg, Department of Medicine, New York University School of Medicine, 550 First Avenue, New York, New York 10016. E-mail: david.kleinberg@med.nyu.edu.
Abstract
Progestins have been implicated in breast cancer development, yet a role for progesterone (Pg) in ductal morphogenesis (DM) has not been established. To determine whether Pg could cause DM, we compared relative effects of Pg, estradiol (E2) and IGF-I on anatomical and molecular biological parameters of IGF-I-related DM in oophorectomized female IGF-I(–/–) mice. Pg had little independent effect on mammary development, but together with IGF-I, in the absence of E2, Pg stimulated an extensive network of branching ducts, occupying 92% of the gland vs. 28.3% with IGF-I alone, resembling pubertal development (P < 0.002). Its major effect was on enhancing duct length and branching (P < 0.002). Additionally, Pg enhanced phosphorylation of IRS-1, increased cell division, and increased the antiapoptotic effect of IGF-I. Pg action was inhibited by RU486 (P < 0.01). E2 also stimulated DM by enhancing IGF-I action but had a greater effect on terminal end bud formation and side branching (P < 0.002). In contrast to previous findings, long-term exposure to E2 alone, without IGF-I, caused formation of ducts and side branches, a novel finding. Both IGF-I and E2 were found necessary for Pg-induced alveolar development. In conclusion, Pg, through Pg receptor can enhance IGF-I action in DM, and E2 acts through a similar mechanism; E2 alone caused formation of ducts and side branches; there were differences in the actions of Pg and E2, the former largely affecting duct formation and extension, and the latter side branching; and both IGF-I and E2 were necessary for Pg to form mature alveoli.
Introduction
RECENT REPORTS SUGGEST that progestins increase breast cancer incidence (1, 2, 3, 4, 5). A better understanding of the role of progesterone (Pg) in normal mammary development is needed as a baseline to better understand the clinical dilemma of Pg or progestins as mammary carcinogens. It is well established that Pg is important for formation of mammary gland alveolar structures (6, 7). Based on the observation that Pg receptor (PgR)-deficient animals have normal mammary development during puberty, Pg is not necessary for and may or may not play a role in ductal morphogenesis (DM) (8, 9). To further investigate whether Pg has a role in this process and also to extend knowledge on the relative roles of estradiol (E2) and IGF-I in DM, we studied the individual and combined effects of IGF-I, E2, and Pg on mammary development in an experimental mouse model in which mammary development is impaired but can be induced when requisite hormones are administered. The results are presented here.
Materials and Methods
Animals
IGF-I(–/–) null mice were bred in our laboratory, as previously described (10). Breeding pairs were a gift from Lynn Powell-Braxton (Genentech, Inc., South San Francisco, CA). IGF-I(+/–) males were mated with IGF-I(+/–) females. Although 25% of offspring were expected to be IGF-I(–/–) animals, only 2% of all mice born alive were IGF-I(–/–) knockouts. Fifty-six percent were males, and 44% were females. Wild-type female litter mates weighed a mean of 28 g compared with IGF-I(–/–) animals weighing a mean of 6.2 g at 56 d of age. Animals were housed in sterile cages. Sterile technique was used for handling in our Association for Assessment and Accreditation of Laboratory Animal Care-approved facility (VA Medical Center, New York, NY). Absence of the IGF-I gene was confirmed by PCR on DNA extracted from mouse tails as previously described.
Animals were oophorectomized at 8 wk old. Anesthesia was induced with a 1.25% solution of tribromoethanol-amylene hydrate (0.2 ml/10 g body weight ip). A small midline dorsal skin incision was made approximately halfway between the middle of the back (the hump) and the base of the tail. The peritoneal cavity on the left side was accessed by blunt dissection of the connective tissue and by making a small cut with pointed scissors into the muscle. The left ovary was removed through the muscle incision by grasping the periovarian fat. The junction between the Fallopian tube and the uterine horn, together with all accompanying blood vessels and fat, was severed with a single cut, and the horn was returned into the abdominal cavity. The right ovary was removed in the same way. The skin incision was closed using a 6-0 single suture. Two weeks after oophorectomy, animals were treated with various hormone combinations. Pumps and pellets were administered under anesthesia.
We also employed castrated female Ames dwarf animals to determine whether Pg was acting through the PgR. We turned to this model in which mammary development is also impaired (deficient in GH, prolactin, and TSH) because of declining yields of IGF-I(–/–) female animals due to various breeding problems (11). Dr. A. Bartke (Southern Illinois University School of Medicine, Carbondale, IL) supplied breeding pairs. Heterozygous males were mated with heterozygous females. Of litters of six to 10, 12.5% were female Ames homozygous. At 8 wk of age, animals were oophorectomized, as above, and pellets containing 0.25 mg thyroxine (IRA, Sarasota, FL) were implanted sc. After 2 wk, animals were treated with IGF-I, with or without pellets containing Pg, and with or without RU486.
Hormones and delivery
Des(1-3)IGF-I (IGF-I) (a gift of Genentech, Inc.) was administered by Alzet miniosmotic pumps (Alza Corp., Palo Alto, CA) to IGF-I(–/–) animals. For the 5-d studies, pumps (model 1007) containing 20 μg were implanted sc on the upper back of the mice and delivered 0.25 μl/h. Experiments were carried out for either 5 or 28 d [model 1002 pumps containing 40 μg des(1-3) IGF-I changed at 14 d]. E2 (Calbiochem, La Jolla, CA) was administered via slow-release SILASTIC brand capsules (Dow Corning, Midland, MI) inserted in the subscapular region, as previously described (12). Pg (Sigma Chemical Co., St. Louis, MO) was given in a SILASTIC brand capsule of exactly the same dimensions as that for E2. Each capsule contained 4 mg Pg. Separate capsules were used for each steroid. RU486 (5 mg/kg) (Sigma) was administered sc in sesame oil daily for 5 d. Those not receiving RU486 received vehicle only.
Experimental design
To examine effects of IGF-I, E2, and Pg on DM, hormones were given either singly or in combination with IGF-I(–/–) female mice that had been oophorectomized at 56 d of age. In one experiment, we employed oophorectomized female Ames dwarf animals. The IGF-I(–/–) animals were then treated for either 5 or 28 d with E2 alone, Pg alone, IGF-I alone, IGF-I + E2, IGF-I + Pg, or placebo (control). E2 + Pg and IGF-I + E2 + Pg were administered for 28 d only. The animals were 75 (5-d treatment) or 98 (28-d treatment) d old by the time the entire experimental process was completed. Ames dwarfs were treated for only 5 d and were 75 d old at the completion of those experiments. Animals were anesthetized to remove the lumbar mammary glands and then killed. The mammary glands were fixed in 10% formalin solution and stained in whole mount with iron hematoxylin (12).
Stained whole mounts were stored in 50-ml jars (Nalge Company, Zaventum Zuid, Belgium) containing methyl salicylate solution. Aspects of mammary development and differentiation were assessed quantitatively by examining the whole mount under a dissecting microscope at magnification x15 (Nikon SMZ-U, Nikon, Melville, NY). Terminal end buds (TEBs), alveolar structures, branch points (13), and side branches (14) were counted. Photographs of whole mounts (x24) were examined using computer imaging analysis system (Image-Pro Plus, Media Cybernetics, Baltimore, MD). The area of the mammary fat pad that is occupied by glandular elements was measured and expressed as a percentage of the entire fat pad two dimensionally. TEBs were identified because they appeared like club-shaped structures on whole mount. When evaluated histologically, they were multilayered structures containing epithelial body cells and cap cells surrounding the structure. When cell division within these structures was measured by Ki67 immunostaining, there was significantly more division in TEBs than in the glandular ducts in mammary glands treated with IGF-I alone (P < 0.02), IGF-I with Pg (P < 0.01), or IGF-I + E2 (P < 0.01). We were unable to do these Ki67 measurements in the 28-d E2-treated animals and have, therefore, called these TEB-like in this case. To evaluate length of ducts, we identified ducts and manually outlined the length of each duct using the trace feature of the computer system. Once identified, the computer automatically calculates the mean length of ducts. The number of branch points was determined by enlarging photomicrographs and identifying each of the branch points as they form at acute angles and then counted manually. Side branches were identified and counted in whole mounts.
Statistical results were assessed in each of two to three animals per hormone combination so that mean effects could be analyzed.
The contralateral mammary gland was used for histological analysis in a subset. Those were routinely processed and paraffin embedded, each in the same orientation so that like sagittal sections could be made on a microtome. Histological analysis was carried out on 4-μm sections. Sections to be studied were identified by staining with hematoxylin eosin. Three sections from each gland were taken to make a single observation. There were generally 20–30 sections in each gland. If one sort of staining was done on sections 5, 10, and 15, another was done on sections 6, 11, and 16, and so on. In this study, tissues were stained for phosphorylated IRS-1, which is important in mammary development (15) and Ki67 antigen. The sections were deparaffinized with xylene, rehydrated through descending alcohols to water, and rinsed in PBS (pH 7.6). For nonenzymatic antigen retrieval, sections were heated in 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven (700 W) for 15 min and allowed to cool for 20 min at room temperature. To inactivate endogenous peroxidase activity, slides were then rinsed with PBS and incubated in 3% H2O2 for 10 min and blocking with 10% normal goat serum for 1 h at room temperature, and incubated overnight at 4 C or 1 h at room temperature with primary antibodies. The primary antibodies used were rabbit anti-IRS-I pY896 (1:100 dilution, Biosource International, Inc., Camarillo, CA) and rabbit Ki67 antibody (1:1000, Novocastra, Newcastle, UK). Slides were then incubated for 1 h at room temperature with goat antirabbit Igs and horseradish peroxidase (1:100 dilution, Dako, Carpinteria, CA). Detection was achieved by incubation for 5 min with a diaminobenzidine tetrahydrochloride solution containing 5 mg diaminobenzidine tetrahydrochloride (Sigma) and 1 ml 1% H2O2 in 100 ml Tris buffer (pH 7.6). The slides then counterstained with hematoxylin or 0.5% methyl green (pH 4), mounted with Permont. Negative controls for IRS-1 identification were incubated with primary antibody preabsorbed with 200-fold molar excess of the IRS-1 (pY896) phospho peptide. This treatment blocked staining for pY IRS-1 in an MCF-7 cell tumor and in sections from a mammary gland from a 10-d lactating rat (Fig. 1). Omission of the primary antibody and replacement with nonimmune IgG was used as a negative control for Ki67 staining. Apoptotic cells were detected by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) staining with ApopTag peroxidase in situ apoptosis detection kit, according to the manufacturer’s instruction (Chemicon International Inc., Temecula, CA). Mammary gland sections from three different levels of each gland were examined by taking photographs using a Nikon E400 microscope at x200 magnification attached to a Nikon digital camera DXM1200 running software Nikon ACT-1 on a personal computer system. Morphometry was carried out by identifying positive brown staining cells in the sections. They were then submitted to analysis by a semiautomatic computer system (Image-Pro Plus, version 4.5, Media Cybernetics). All stained epithelial cells were counted for pY IRS-1 antibody. Ki67 and TUNEL staining were expressed as the percentage of labeled cells by total number of epithelial cells. At least 2000 cells/section of a mammary gland were counted. In the sections from control or E2- or Pg-treated animals, there was less epithelium because of the impaired development of epithelial elements but more than 1000 cells/three sections were counted. Antibody specificity for pY IRS-1 was determined by Western blotting of MCF-7 proteins. Protein samples from control and IGF-I-treated cells were analyzed by SDS-PAGE on a 10–15% gradient gel. We found that there was an increase in signal of IRS-1 in protein extracted for IGF-I treated cells.
FIG. 1. Paraffin sections of mammary gland from a rat lactating for 10 d (A and B) and from an MCF-7 cell tumor grown in a nude mouse (C and D) stained for IRS-1 pY 896 (A and C) or IRS-1 pY896 with blocking peptide (B and D). Note that IRS-1 blocking peptide blocked brown color staining in cytoplasm of epithelial cells.
Statistics were done employing groups of three (or in some cases two) IGF-I(–/–) animals each for each hormone or hormone combination. The unpaired two-tailed Student’s t test was employed for statistical analysis.
The institutional committee on animal care approved these studies, and animals were maintained in accordance with the National Institutes of Health guidelines for the Care and Use of Laboratory Animals.
Results
IGF-I and E2 in DM (Fig. 2; Table 1)
We had previously shown that the first 14 d of partial mammary development was caused by IGF-I and E2 (12, 16). The present studies, which were designed to compare effects of E2, Pg, and IGF-I, confirm those observations and extend them to 28 d at which time pubertal DM was probably complete. After 5 d of treatment with IGF-I and E2, a mean of 32% of the mammary gland was occupied by ducts and TEBs (Table 1). After 28 d, almost the entire fat pad was filled with TEBs, and ducts formed by branching and also side branching. There were also some immature alveolar structures (vide infra) (Fig. 2, E and F). Both ducts and TEBs were hyperplastic histologically. There were also significant increases over control in the number of ducts, number of branch points, and length of ducts at 5 d, and further increases in all parameters of development at 28 d. Although the number of TEBs was similar at both time points, there was a relative reduction in TEBs at 28 d when expressed as TEBs/area of fat pad occupied by ducts (P < 0.002) (Fig. 3). At both time points, the combined effects of IGF-I and E2 were significantly greater than the effect of either IGF-I or E2 alone (P < 0.002; Table 1). IGF-I alone stimulated formation and branching and elongation of ducts at 5 and further at 28 d (P < 0.02 compared with control) and increased the number of TEBs at both time periods, but as with IGF-I + E2, there was a falloff in TEBs relative to the area occupied by ducts. Although E2 alone had no effect at 5 d, it stimulated formation of branching ducts and side branches at 28 d (P < 0.002) and a minimal but significant effect on gland area (Fig. 2, B and C). This effect of E2 on TEB formation only occurred after 28 d of exposure.
FIG. 2. Whole mounts of lumbar mammary glands from 98-d-old oophorectomized IGF-I(–/–) female mice treated for 28 d with various hormones or nothing. Each variable is presented at a magnification of x12 to be able to evaluate them in whole mount, and some are also presented at x24 to show glandular detail. A, Control at x12. Mammary development is very impaired, and only a small area of immature ducts is found. B, E2 alone. There were more sides branches, and the area occupied by ducts was increased (x12). C, Higher power of B to better illustrate TEBs (x24). The small structures at the end of ducts are not typical TEBs. D, IGF-I alone at x12. Note extension of glandular elements led by TEBs, an increase in branch points, and side branches. E, IGF-I + E2 at x12. The entire mammary gland is filled with branching ducts, side branches and TEBs, and immature alveolar structures, particularly in the area of the gland pointed to by the arrow. F, High power (x24) of the leading edge of the previous developing gland (E) under the influence of E2 and IGF-I. There was an increase in TEBs (arrowheads), branched ducts, and lateral branches and even a few alveolar structures (arrow). G, Pg alone at x12. The area of fat pad occupied by ducts is slightly but significantly increased compared with control. H, IGF-I + Pg, the entire fat pad is filled with a network of narrow elongated branching ducts and side branches. I, IGF-I + Pg at x24. Note the relatively simple branching thin ducts and the difference between this effect and that of IGF-I +E2. J, The combination of Pg- and E2-stimulated development of side branches and gland area, as E2 did (x12). We do not show a high power of this section, but if we did, it would appear almost identical with the effect of E2 alone as in C. K, When IGF-I was added to the combination of Pg + E2, there was a profusion of formation of alveoli as one sees during midpregnancy (x12). L, High power of K (x24).
TABLE 1. Effects of IGF-I, Pg, and E2 alone and in combination on mammary development in oophorectomized IGF-I(–/–)
FIG. 3. Data from Table 1 and Fig. 2, E and H, are plotted in two panels, one for the effects of IGF-I + E2 (upper panel) and the other for IGFD-I + Pg (lower panel). Note the relative fall-off in TEB formation while ducts continue to branch and extend into the mammary fat pad. Also note that alveolar structures were formed over time with the E2 IGF-I combination but not with the other.
IGF-I and Pg in DM
To determine whether Pg had an effect on experimental DM, we carried out studies similar to those described above. We administered IGF-I alone, Pg alone, or a combination of Pg and IGF-I for periods of 5 and 28 d to oophorectomized IGF-I(–/–) null female animals. When IGF-I was given together with Pg, a significant increase in the area of the fat pad occupied by a network of ducts was observed at 5 and 28 d. At 5 d, this hormone combination was more effective than IGF-I + E2 on area occupied by ducts (P < 0.04). By 28 d, the duct network occupied almost the entire mammary gland (Fig. 2, H and I; Table 1) and resembled a gland from a fully pubertal nulliparous animal. There were significant increases in branching duct and length of ducts. In fact, duct length in response to IGF-I + Pg was significantly greater at 5 d than with IGF-I + E2. In contrast, Pg had little effect on TEB formation. At both 5 and 28 d, the combined effect of IGF-I and Pg on TEB number was not greater than with IGF-I alone. Pg alone had no effect on TEB formation (Fig. 2G) but did cause a slight but significant increase in the area of the fat pad occupied by ducts (P < 0.04; Table 1).
Does the PgR mediate the action of Pg in DM?
To determine whether Pg was working via PgR in stimulating DM, we employed another animal model, the oophorectomized female Ames dwarf. RU486 was used to block Pg action through PgR. RU486 significantly inhibited the action of Pg + IGF-I in DM. Specifically, there was a significant reduction in TEB formation, duct number, area occupied by ducts, and branch points (Fig. 4).
FIG. 4. Effect of RU486 on the effect of IGF-I + Pg on mammary development in oophorectomized Ames dwarf female mice. A, Upper panel, representative gland having been treated with IGF-I + Pg for 5 d. Middle panel, Gland from an animal treated with those hormones + RU486. Note that development is impaired. B, Graph (lower panel) showing parameters of mammary development in groups of three animals treated with different hormone combinations.
Enhancement of other actions of IGF-I by Pg and E2
We tested the effect of the various hormone combinations on molecular biological actions of IGF-I. This was done after exposure to various hormone combinations for a period of 5 d as seen in whole mount (Fig. 5). These included expression of phosphorylated IRS-1 (Fig. 6), cell division (Fig. 7), and apoptosis (Fig. 8). Both Pg and E2 were found to amplify the actions of IGF-I about equally. Both Pg and E2, each together with IGF-I, significantly increased pY IRS-1 expression over and above that of IGF-I alone (Fig. 6). Pg and E2 also enhanced the stimulatory effect of IGF-I on cell division (staining for Ki67; Fig. 7) and enhanced the inhibitory effect of IGF-I on apoptosis (TUNEL; Fig. 8).
FIG. 5. Whole mounts of lumbar mammary glands removed from oophorectomized IGF-I(–/–) female mice treated with various hormones for 5 d. A, Control; B, E2 alone; C, Pg alone; D, IGF-I alone; E, IGF-I + E2; F, IGF-I + Pg. Details of development can be found in Table 1. (Magnification, x12).
FIG. 6. A, Histological sections from representative lumbar mammary glands treated for 5 d with: A, control; B, E2; C, Pg; D, IGF-I; E, IGF-I + E2; or F, IGF-I + Pg. Magnification, x40. These sections were immunostained for pY IRS-1. Note that there was little or no staining for pY IRS-1 in the control or E2- or Pg-treated sections (A–C), but there was staining in cytoplasm of TEBs and ducts in sections from animals treated with IGF-I (D), IGF-I + E2 (E), and IGF-I + Pg (F). Arrows point to TEBs and arrowheads to ducts. B, This figure in the lower panel shows combined data on all animals and indicates that the Py IRS-1 stimulated by IGF-I was significantly enhanced by the addition of either E2 or Pg.
FIG. 7. A, Histological sections from representative lumbar mammary glands treated for 5 d with: A, control; B, E2; C, Pg; D, IGF-I; E, IGF-I + E2; or F, IGF-I + Pg. Cell division was determined by staining for Ki67. Again, cell division was increased over control or E2 or Pg when IGF-I was given. Both E2 and Pg enhanced cell division. Arrows identify Ki67-positive cells (magnification, x400). B, The lower panel presents these data on groups of animals. Results are expressed as percentage of total number of epithelial cells stained for Ki67.
FIG. 8. A, Histological sections from representative lumbar mammary glands treated for 5 d with: A, control; B, E2; C, Pg; D, IGF-I; E, IGF-I + E2; or F, IGF-I + Pg. Apoptosis was assessed by TUNEL assay. Apoptosis was found to occur in mammary glands in controls, E2 -treated, or Pg treated animals. Apoptotic cells are identified by brown staining in the nuclei identified by arrows. Apoptotic cells were largely eliminated in the mammary glands from animals treated with IGF-I alone or with E2 or Pg. B, Data for this parameter are presented in this graph in the lower panel. Results are expressed as total number of labeled cells/total number of epithelial cells.
Formation of alveolar structures requires IGF-I, Pg, and E2
Full mammary development resembling that seen in midpregnancy was observed only when IGF-I was given in combination with Pg and E2 (Table 1; Fig. 2, K and L). Only the three hormones together caused the development of a profusion of single- or sometimes double-layered acinar structures that were too numerous to count. They filled almost the entire mammary fat pad. That IGF-I is essential for true alveolar development is supported by the fact that the effect of Pg + E2 was virtually identical with that of E2 alone (Fig. 2J), where the percent fat pad area occupied by glandular structures was greater and there were increased side branches (P < 0.002). After 28 d of exposure, the combination of IGF-I and E2 stimulated some development of structures more mature than TEBs, which we have called alveolar structures (Fig. 2, E and F), suggesting that IGF-I + E2 can start the process of developing secretory structures but also indicating that Pg is essential for full formation of secretory structures that will eventually produce milk.
Discussion
At puberty, a surge of estrogen stimulates development of the mammary gland as long as the pituitary gland is intact (17, 18). Before that, the mammary gland is composed of a fat pad with a few simple branched ducts, the glandular anlagen (6, 19, 20, 21). In response to IGF-I and estrogen, actively dividing multilayered club-shaped structures, called TEBs, form. They extend into the substance of the fat pad, leaving in their wake a network of branched ducts. When the glandular tree nears the outer borders of the fat pad, TEBs are reduced in number, and the tree-like glandular component becomes and remains relatively quiescent until pregnancy. Lyons (22) first showed that GH was the pituitary hormone responsible for pubertal development. We then demonstrated that the earliest phases of experimental DM in several animal models were under the control of GH, through the action of IGF-I, together with E2 (9, 13, 16, 23, 24, 25). Studies in IGF-I(–/–) female mice revealed that the full effect of GH was mediated by IGF-I. We also studied mammary development for up to 14 d and showed that IGF-I plus E2 caused further partial DM (25).
The present study was designed to determine whether the entire process of DM past the 14-d point could be continued under the influence of IGF-I plus E2 and to determine whether Pg could also affect DM. For this purpose, we oophorectomized female IGF-I(–/–) mice and then administered the three hormones singly and in combination for 5 and 28 d. Our results show that, indeed, IGF-I together with E2 can stimulate partial DM at 5 d and a mammary gland almost completely filled by branching ducts at 28 d. This was due to an increase in TEBs, area of the fat pad occupied by glands, duct number, duct length, and duct branching. In fact, the mammary glands appeared more developed than expected for a completed picture of DM (6, 19). Ordinarily, when the mammary tree reaches the limits of the fat pad, there is regression of end buds, active pubertal development comes to an end (26), and the mammary tree appears quiescent. In this case, there were areas of hyperplasia (confirmed histologically) not ordinarily seen at this stage of development. This might have been due to either enhancement of IGF-I action by E2 or stimulation of IGF-I production. Our data suggest that the effect of E2 is at least partially caused by enhancement of IGF-I action because IGF-I alone stimulated one third or less the degree of mammary development of that of IGF-I plus E2. We also found that each known molecular biological action of IGF-I in mammary development that we tested (increased cell division, increased IRS-1 production, and inhibition of apoptosis) (27, 28, 29, 30, 31, 32) was significantly enhanced by the addition of E2. We have not determined whether other actions of IGF-I would have been enhanced by E2 or Pg, but it is not unreasonable to assume that they would. These include an increase in phosphorylation of IGF-IR and IRS-2 proteins (33, 34, 35) and enhancement of cell cycle progression (36, 37). The synergy might also have been caused by an increase in IGF-I itself. Previous observations have shown that E2 can enhance IGF-I mRNA production (12).
Regarding the mammary gland hyperplasia stimulated by the combination of IGF-I and E2, these studies now show that E2 can have some independent effect on mammary development; side branching and duct formation occur, but normal TEB development does not, whether given for long enough periods of time (28 d). In previous studies, E2 had no independent effect on mammary development in the absence of IGF-I given for as long as 14 d. Although overexpression of IGF-I can cause mammary hyperplasia and increase mammary tumor incidence (38), these data show that the addition of E2 can also cause hyperplasia. If enhancement of IGF-I action was the sole cause of hyperplasia, one would have expected hyperplasia when the same dose of IGF-I was given together with Pg, which also stimulated IGF-I action except where TEB formation was concerned. Thus, although enhancement of IGF-I action is likely one cause of improved mammary development, there are probably other pathways of activity, as well. An independent effect of E2 not related to IGF-I, or one that might circumvent the IGF-IR (29) is also a possibility. Because E2 did not have an earlier independent effect, one might speculate that IGF-II could have. In fact, the pathway by which E2 acts without IGF-I might be the one that caused the hyperplastic changes noted above. If IGF-I action was participating in formation of the structures that appeared with long-term estrogen, we would have expected formation of at least one true TEB because IGF-I is the common denominator for TEB formation.
The role of Pg in DM is less well worked out than that of E2. Established is that Pg is intricately and essentially involved in the formation of secretory lobuloalveolar structures (39). However, data are meager regarding a physiological role for Pg in DM (40, 41). We know that Pg is not essential for pubertal mammary development because DM takes place in PgR knockout mice (7, 8, 42). However, a very recent report indicated that maspin heterozygous mice had impaired mammary development that could be overcome by administration of Pg (43). Our study supports and extends those observations. They clearly demonstrate that Pg is capable of enhancing the effect of IGF-I in DM in the absence of estrogen. Gross anatomical appearance of mammary gland development caused by IGF-I + Pg shows that the ductal tree looks like normal fully developed pubertal gland. To determine whether the action of Pg was via PgR, we assessed the effect of RU486 on Pg- and IGF-I-induced mammary development and found that it significantly inhibited development. Like the interaction between E2 and IGF-I, Pg also acts by enhancing the action of IGF-I. However, there are differences between the two hormone combinations because Pg was mostly responsible for duct growth and branching. Unlike E2, Pg does not enhance the action of IGF-I on TEB formation or cause ductal decorations. Therefore, there are differences in actions of E2 and Pg on mammary development. Pg has more of an effect on duct formation, extension, and branching, whereas E2 has more of an effect on TEB and slightly more mature alveolar structures. These observations were unexpected because we consider the action of Pg one of enhancement of alveolar formation.
It was previously thought that Pg could not act in the absence of estrogen (44). Because PgR is present in the mammary gland at puberty (39) whether ovaries are present or not (45), and some of the receptors are estrogen dependent and are others not (42), the machinery for Pg action is there. With the caveat that the mice employed in these studies may have seen ovarian estrogen before oophorectomy at 21 d of age, our data support the concept that Pg can cause mammary development in the absence of estrogen. Assuming that our animal model in which the combination of IGF-I, E2, and Pg caused highly active formation of alveolar structure over 28 d, the action of Pg on formation of alveoli does require estrogen and IGF-I. In no case did Pg cause alveolar formation unless the other two hormones were present. Thus, Pg and presumably progestins (46) stimulate alveolar formation during preparation for lactation but have a more important effect on duct extension and branching during experimental pubertal development. This aspect of mammary development requires the action of prolactin, but this important activity was outside the purview of this manuscript and, therefore, not discussed (47, 48).
Overexpression of IGF-I is associated with an increase in mouse mammary gland tumor formation, and it also inhibits mammary gland involution due to a decrease in apoptosis (38, 49, 50). In view of our findings that Pg can enhance the action of IGF-I in duct formation, cell division, and production of phosphorylated IRS-1 and also increase IGF-I’s effect on inhibition of apoptosis, this provides an understanding of how Pg can affect the mammary gland. It also may provide insight into the observations that exposure to Pg or progestins can increase breast cancer incidence. Overall, if progestins or Pg are carcinogenic, consideration should be given to employing PgR antagonists in the treatment of breast cancer (51). That both Pg and E2 work on mammary gland via their effects on enhancing IGF-I action, consideration should also be given to blocking the effects of both Pg and E2 by antagonists of IGF-I (52).
Acknowledgments
We are grateful to Fabio Rotundo and Kalmon Kovacs for help and advice in immunostaining methodology, Marie Monaco for running Western blots and reviewing the manuscript, and Inn Ling Eng for expert animal breeding and making histological sections.
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Address all correspondence and requests for reprints to: David L. Kleinberg, Department of Medicine, New York University School of Medicine, 550 First Avenue, New York, New York 10016. E-mail: david.kleinberg@med.nyu.edu.
Abstract
Progestins have been implicated in breast cancer development, yet a role for progesterone (Pg) in ductal morphogenesis (DM) has not been established. To determine whether Pg could cause DM, we compared relative effects of Pg, estradiol (E2) and IGF-I on anatomical and molecular biological parameters of IGF-I-related DM in oophorectomized female IGF-I(–/–) mice. Pg had little independent effect on mammary development, but together with IGF-I, in the absence of E2, Pg stimulated an extensive network of branching ducts, occupying 92% of the gland vs. 28.3% with IGF-I alone, resembling pubertal development (P < 0.002). Its major effect was on enhancing duct length and branching (P < 0.002). Additionally, Pg enhanced phosphorylation of IRS-1, increased cell division, and increased the antiapoptotic effect of IGF-I. Pg action was inhibited by RU486 (P < 0.01). E2 also stimulated DM by enhancing IGF-I action but had a greater effect on terminal end bud formation and side branching (P < 0.002). In contrast to previous findings, long-term exposure to E2 alone, without IGF-I, caused formation of ducts and side branches, a novel finding. Both IGF-I and E2 were found necessary for Pg-induced alveolar development. In conclusion, Pg, through Pg receptor can enhance IGF-I action in DM, and E2 acts through a similar mechanism; E2 alone caused formation of ducts and side branches; there were differences in the actions of Pg and E2, the former largely affecting duct formation and extension, and the latter side branching; and both IGF-I and E2 were necessary for Pg to form mature alveoli.
Introduction
RECENT REPORTS SUGGEST that progestins increase breast cancer incidence (1, 2, 3, 4, 5). A better understanding of the role of progesterone (Pg) in normal mammary development is needed as a baseline to better understand the clinical dilemma of Pg or progestins as mammary carcinogens. It is well established that Pg is important for formation of mammary gland alveolar structures (6, 7). Based on the observation that Pg receptor (PgR)-deficient animals have normal mammary development during puberty, Pg is not necessary for and may or may not play a role in ductal morphogenesis (DM) (8, 9). To further investigate whether Pg has a role in this process and also to extend knowledge on the relative roles of estradiol (E2) and IGF-I in DM, we studied the individual and combined effects of IGF-I, E2, and Pg on mammary development in an experimental mouse model in which mammary development is impaired but can be induced when requisite hormones are administered. The results are presented here.
Materials and Methods
Animals
IGF-I(–/–) null mice were bred in our laboratory, as previously described (10). Breeding pairs were a gift from Lynn Powell-Braxton (Genentech, Inc., South San Francisco, CA). IGF-I(+/–) males were mated with IGF-I(+/–) females. Although 25% of offspring were expected to be IGF-I(–/–) animals, only 2% of all mice born alive were IGF-I(–/–) knockouts. Fifty-six percent were males, and 44% were females. Wild-type female litter mates weighed a mean of 28 g compared with IGF-I(–/–) animals weighing a mean of 6.2 g at 56 d of age. Animals were housed in sterile cages. Sterile technique was used for handling in our Association for Assessment and Accreditation of Laboratory Animal Care-approved facility (VA Medical Center, New York, NY). Absence of the IGF-I gene was confirmed by PCR on DNA extracted from mouse tails as previously described.
Animals were oophorectomized at 8 wk old. Anesthesia was induced with a 1.25% solution of tribromoethanol-amylene hydrate (0.2 ml/10 g body weight ip). A small midline dorsal skin incision was made approximately halfway between the middle of the back (the hump) and the base of the tail. The peritoneal cavity on the left side was accessed by blunt dissection of the connective tissue and by making a small cut with pointed scissors into the muscle. The left ovary was removed through the muscle incision by grasping the periovarian fat. The junction between the Fallopian tube and the uterine horn, together with all accompanying blood vessels and fat, was severed with a single cut, and the horn was returned into the abdominal cavity. The right ovary was removed in the same way. The skin incision was closed using a 6-0 single suture. Two weeks after oophorectomy, animals were treated with various hormone combinations. Pumps and pellets were administered under anesthesia.
We also employed castrated female Ames dwarf animals to determine whether Pg was acting through the PgR. We turned to this model in which mammary development is also impaired (deficient in GH, prolactin, and TSH) because of declining yields of IGF-I(–/–) female animals due to various breeding problems (11). Dr. A. Bartke (Southern Illinois University School of Medicine, Carbondale, IL) supplied breeding pairs. Heterozygous males were mated with heterozygous females. Of litters of six to 10, 12.5% were female Ames homozygous. At 8 wk of age, animals were oophorectomized, as above, and pellets containing 0.25 mg thyroxine (IRA, Sarasota, FL) were implanted sc. After 2 wk, animals were treated with IGF-I, with or without pellets containing Pg, and with or without RU486.
Hormones and delivery
Des(1-3)IGF-I (IGF-I) (a gift of Genentech, Inc.) was administered by Alzet miniosmotic pumps (Alza Corp., Palo Alto, CA) to IGF-I(–/–) animals. For the 5-d studies, pumps (model 1007) containing 20 μg were implanted sc on the upper back of the mice and delivered 0.25 μl/h. Experiments were carried out for either 5 or 28 d [model 1002 pumps containing 40 μg des(1-3) IGF-I changed at 14 d]. E2 (Calbiochem, La Jolla, CA) was administered via slow-release SILASTIC brand capsules (Dow Corning, Midland, MI) inserted in the subscapular region, as previously described (12). Pg (Sigma Chemical Co., St. Louis, MO) was given in a SILASTIC brand capsule of exactly the same dimensions as that for E2. Each capsule contained 4 mg Pg. Separate capsules were used for each steroid. RU486 (5 mg/kg) (Sigma) was administered sc in sesame oil daily for 5 d. Those not receiving RU486 received vehicle only.
Experimental design
To examine effects of IGF-I, E2, and Pg on DM, hormones were given either singly or in combination with IGF-I(–/–) female mice that had been oophorectomized at 56 d of age. In one experiment, we employed oophorectomized female Ames dwarf animals. The IGF-I(–/–) animals were then treated for either 5 or 28 d with E2 alone, Pg alone, IGF-I alone, IGF-I + E2, IGF-I + Pg, or placebo (control). E2 + Pg and IGF-I + E2 + Pg were administered for 28 d only. The animals were 75 (5-d treatment) or 98 (28-d treatment) d old by the time the entire experimental process was completed. Ames dwarfs were treated for only 5 d and were 75 d old at the completion of those experiments. Animals were anesthetized to remove the lumbar mammary glands and then killed. The mammary glands were fixed in 10% formalin solution and stained in whole mount with iron hematoxylin (12).
Stained whole mounts were stored in 50-ml jars (Nalge Company, Zaventum Zuid, Belgium) containing methyl salicylate solution. Aspects of mammary development and differentiation were assessed quantitatively by examining the whole mount under a dissecting microscope at magnification x15 (Nikon SMZ-U, Nikon, Melville, NY). Terminal end buds (TEBs), alveolar structures, branch points (13), and side branches (14) were counted. Photographs of whole mounts (x24) were examined using computer imaging analysis system (Image-Pro Plus, Media Cybernetics, Baltimore, MD). The area of the mammary fat pad that is occupied by glandular elements was measured and expressed as a percentage of the entire fat pad two dimensionally. TEBs were identified because they appeared like club-shaped structures on whole mount. When evaluated histologically, they were multilayered structures containing epithelial body cells and cap cells surrounding the structure. When cell division within these structures was measured by Ki67 immunostaining, there was significantly more division in TEBs than in the glandular ducts in mammary glands treated with IGF-I alone (P < 0.02), IGF-I with Pg (P < 0.01), or IGF-I + E2 (P < 0.01). We were unable to do these Ki67 measurements in the 28-d E2-treated animals and have, therefore, called these TEB-like in this case. To evaluate length of ducts, we identified ducts and manually outlined the length of each duct using the trace feature of the computer system. Once identified, the computer automatically calculates the mean length of ducts. The number of branch points was determined by enlarging photomicrographs and identifying each of the branch points as they form at acute angles and then counted manually. Side branches were identified and counted in whole mounts.
Statistical results were assessed in each of two to three animals per hormone combination so that mean effects could be analyzed.
The contralateral mammary gland was used for histological analysis in a subset. Those were routinely processed and paraffin embedded, each in the same orientation so that like sagittal sections could be made on a microtome. Histological analysis was carried out on 4-μm sections. Sections to be studied were identified by staining with hematoxylin eosin. Three sections from each gland were taken to make a single observation. There were generally 20–30 sections in each gland. If one sort of staining was done on sections 5, 10, and 15, another was done on sections 6, 11, and 16, and so on. In this study, tissues were stained for phosphorylated IRS-1, which is important in mammary development (15) and Ki67 antigen. The sections were deparaffinized with xylene, rehydrated through descending alcohols to water, and rinsed in PBS (pH 7.6). For nonenzymatic antigen retrieval, sections were heated in 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven (700 W) for 15 min and allowed to cool for 20 min at room temperature. To inactivate endogenous peroxidase activity, slides were then rinsed with PBS and incubated in 3% H2O2 for 10 min and blocking with 10% normal goat serum for 1 h at room temperature, and incubated overnight at 4 C or 1 h at room temperature with primary antibodies. The primary antibodies used were rabbit anti-IRS-I pY896 (1:100 dilution, Biosource International, Inc., Camarillo, CA) and rabbit Ki67 antibody (1:1000, Novocastra, Newcastle, UK). Slides were then incubated for 1 h at room temperature with goat antirabbit Igs and horseradish peroxidase (1:100 dilution, Dako, Carpinteria, CA). Detection was achieved by incubation for 5 min with a diaminobenzidine tetrahydrochloride solution containing 5 mg diaminobenzidine tetrahydrochloride (Sigma) and 1 ml 1% H2O2 in 100 ml Tris buffer (pH 7.6). The slides then counterstained with hematoxylin or 0.5% methyl green (pH 4), mounted with Permont. Negative controls for IRS-1 identification were incubated with primary antibody preabsorbed with 200-fold molar excess of the IRS-1 (pY896) phospho peptide. This treatment blocked staining for pY IRS-1 in an MCF-7 cell tumor and in sections from a mammary gland from a 10-d lactating rat (Fig. 1). Omission of the primary antibody and replacement with nonimmune IgG was used as a negative control for Ki67 staining. Apoptotic cells were detected by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) staining with ApopTag peroxidase in situ apoptosis detection kit, according to the manufacturer’s instruction (Chemicon International Inc., Temecula, CA). Mammary gland sections from three different levels of each gland were examined by taking photographs using a Nikon E400 microscope at x200 magnification attached to a Nikon digital camera DXM1200 running software Nikon ACT-1 on a personal computer system. Morphometry was carried out by identifying positive brown staining cells in the sections. They were then submitted to analysis by a semiautomatic computer system (Image-Pro Plus, version 4.5, Media Cybernetics). All stained epithelial cells were counted for pY IRS-1 antibody. Ki67 and TUNEL staining were expressed as the percentage of labeled cells by total number of epithelial cells. At least 2000 cells/section of a mammary gland were counted. In the sections from control or E2- or Pg-treated animals, there was less epithelium because of the impaired development of epithelial elements but more than 1000 cells/three sections were counted. Antibody specificity for pY IRS-1 was determined by Western blotting of MCF-7 proteins. Protein samples from control and IGF-I-treated cells were analyzed by SDS-PAGE on a 10–15% gradient gel. We found that there was an increase in signal of IRS-1 in protein extracted for IGF-I treated cells.
FIG. 1. Paraffin sections of mammary gland from a rat lactating for 10 d (A and B) and from an MCF-7 cell tumor grown in a nude mouse (C and D) stained for IRS-1 pY 896 (A and C) or IRS-1 pY896 with blocking peptide (B and D). Note that IRS-1 blocking peptide blocked brown color staining in cytoplasm of epithelial cells.
Statistics were done employing groups of three (or in some cases two) IGF-I(–/–) animals each for each hormone or hormone combination. The unpaired two-tailed Student’s t test was employed for statistical analysis.
The institutional committee on animal care approved these studies, and animals were maintained in accordance with the National Institutes of Health guidelines for the Care and Use of Laboratory Animals.
Results
IGF-I and E2 in DM (Fig. 2; Table 1)
We had previously shown that the first 14 d of partial mammary development was caused by IGF-I and E2 (12, 16). The present studies, which were designed to compare effects of E2, Pg, and IGF-I, confirm those observations and extend them to 28 d at which time pubertal DM was probably complete. After 5 d of treatment with IGF-I and E2, a mean of 32% of the mammary gland was occupied by ducts and TEBs (Table 1). After 28 d, almost the entire fat pad was filled with TEBs, and ducts formed by branching and also side branching. There were also some immature alveolar structures (vide infra) (Fig. 2, E and F). Both ducts and TEBs were hyperplastic histologically. There were also significant increases over control in the number of ducts, number of branch points, and length of ducts at 5 d, and further increases in all parameters of development at 28 d. Although the number of TEBs was similar at both time points, there was a relative reduction in TEBs at 28 d when expressed as TEBs/area of fat pad occupied by ducts (P < 0.002) (Fig. 3). At both time points, the combined effects of IGF-I and E2 were significantly greater than the effect of either IGF-I or E2 alone (P < 0.002; Table 1). IGF-I alone stimulated formation and branching and elongation of ducts at 5 and further at 28 d (P < 0.02 compared with control) and increased the number of TEBs at both time periods, but as with IGF-I + E2, there was a falloff in TEBs relative to the area occupied by ducts. Although E2 alone had no effect at 5 d, it stimulated formation of branching ducts and side branches at 28 d (P < 0.002) and a minimal but significant effect on gland area (Fig. 2, B and C). This effect of E2 on TEB formation only occurred after 28 d of exposure.
FIG. 2. Whole mounts of lumbar mammary glands from 98-d-old oophorectomized IGF-I(–/–) female mice treated for 28 d with various hormones or nothing. Each variable is presented at a magnification of x12 to be able to evaluate them in whole mount, and some are also presented at x24 to show glandular detail. A, Control at x12. Mammary development is very impaired, and only a small area of immature ducts is found. B, E2 alone. There were more sides branches, and the area occupied by ducts was increased (x12). C, Higher power of B to better illustrate TEBs (x24). The small structures at the end of ducts are not typical TEBs. D, IGF-I alone at x12. Note extension of glandular elements led by TEBs, an increase in branch points, and side branches. E, IGF-I + E2 at x12. The entire mammary gland is filled with branching ducts, side branches and TEBs, and immature alveolar structures, particularly in the area of the gland pointed to by the arrow. F, High power (x24) of the leading edge of the previous developing gland (E) under the influence of E2 and IGF-I. There was an increase in TEBs (arrowheads), branched ducts, and lateral branches and even a few alveolar structures (arrow). G, Pg alone at x12. The area of fat pad occupied by ducts is slightly but significantly increased compared with control. H, IGF-I + Pg, the entire fat pad is filled with a network of narrow elongated branching ducts and side branches. I, IGF-I + Pg at x24. Note the relatively simple branching thin ducts and the difference between this effect and that of IGF-I +E2. J, The combination of Pg- and E2-stimulated development of side branches and gland area, as E2 did (x12). We do not show a high power of this section, but if we did, it would appear almost identical with the effect of E2 alone as in C. K, When IGF-I was added to the combination of Pg + E2, there was a profusion of formation of alveoli as one sees during midpregnancy (x12). L, High power of K (x24).
TABLE 1. Effects of IGF-I, Pg, and E2 alone and in combination on mammary development in oophorectomized IGF-I(–/–)
FIG. 3. Data from Table 1 and Fig. 2, E and H, are plotted in two panels, one for the effects of IGF-I + E2 (upper panel) and the other for IGFD-I + Pg (lower panel). Note the relative fall-off in TEB formation while ducts continue to branch and extend into the mammary fat pad. Also note that alveolar structures were formed over time with the E2 IGF-I combination but not with the other.
IGF-I and Pg in DM
To determine whether Pg had an effect on experimental DM, we carried out studies similar to those described above. We administered IGF-I alone, Pg alone, or a combination of Pg and IGF-I for periods of 5 and 28 d to oophorectomized IGF-I(–/–) null female animals. When IGF-I was given together with Pg, a significant increase in the area of the fat pad occupied by a network of ducts was observed at 5 and 28 d. At 5 d, this hormone combination was more effective than IGF-I + E2 on area occupied by ducts (P < 0.04). By 28 d, the duct network occupied almost the entire mammary gland (Fig. 2, H and I; Table 1) and resembled a gland from a fully pubertal nulliparous animal. There were significant increases in branching duct and length of ducts. In fact, duct length in response to IGF-I + Pg was significantly greater at 5 d than with IGF-I + E2. In contrast, Pg had little effect on TEB formation. At both 5 and 28 d, the combined effect of IGF-I and Pg on TEB number was not greater than with IGF-I alone. Pg alone had no effect on TEB formation (Fig. 2G) but did cause a slight but significant increase in the area of the fat pad occupied by ducts (P < 0.04; Table 1).
Does the PgR mediate the action of Pg in DM?
To determine whether Pg was working via PgR in stimulating DM, we employed another animal model, the oophorectomized female Ames dwarf. RU486 was used to block Pg action through PgR. RU486 significantly inhibited the action of Pg + IGF-I in DM. Specifically, there was a significant reduction in TEB formation, duct number, area occupied by ducts, and branch points (Fig. 4).
FIG. 4. Effect of RU486 on the effect of IGF-I + Pg on mammary development in oophorectomized Ames dwarf female mice. A, Upper panel, representative gland having been treated with IGF-I + Pg for 5 d. Middle panel, Gland from an animal treated with those hormones + RU486. Note that development is impaired. B, Graph (lower panel) showing parameters of mammary development in groups of three animals treated with different hormone combinations.
Enhancement of other actions of IGF-I by Pg and E2
We tested the effect of the various hormone combinations on molecular biological actions of IGF-I. This was done after exposure to various hormone combinations for a period of 5 d as seen in whole mount (Fig. 5). These included expression of phosphorylated IRS-1 (Fig. 6), cell division (Fig. 7), and apoptosis (Fig. 8). Both Pg and E2 were found to amplify the actions of IGF-I about equally. Both Pg and E2, each together with IGF-I, significantly increased pY IRS-1 expression over and above that of IGF-I alone (Fig. 6). Pg and E2 also enhanced the stimulatory effect of IGF-I on cell division (staining for Ki67; Fig. 7) and enhanced the inhibitory effect of IGF-I on apoptosis (TUNEL; Fig. 8).
FIG. 5. Whole mounts of lumbar mammary glands removed from oophorectomized IGF-I(–/–) female mice treated with various hormones for 5 d. A, Control; B, E2 alone; C, Pg alone; D, IGF-I alone; E, IGF-I + E2; F, IGF-I + Pg. Details of development can be found in Table 1. (Magnification, x12).
FIG. 6. A, Histological sections from representative lumbar mammary glands treated for 5 d with: A, control; B, E2; C, Pg; D, IGF-I; E, IGF-I + E2; or F, IGF-I + Pg. Magnification, x40. These sections were immunostained for pY IRS-1. Note that there was little or no staining for pY IRS-1 in the control or E2- or Pg-treated sections (A–C), but there was staining in cytoplasm of TEBs and ducts in sections from animals treated with IGF-I (D), IGF-I + E2 (E), and IGF-I + Pg (F). Arrows point to TEBs and arrowheads to ducts. B, This figure in the lower panel shows combined data on all animals and indicates that the Py IRS-1 stimulated by IGF-I was significantly enhanced by the addition of either E2 or Pg.
FIG. 7. A, Histological sections from representative lumbar mammary glands treated for 5 d with: A, control; B, E2; C, Pg; D, IGF-I; E, IGF-I + E2; or F, IGF-I + Pg. Cell division was determined by staining for Ki67. Again, cell division was increased over control or E2 or Pg when IGF-I was given. Both E2 and Pg enhanced cell division. Arrows identify Ki67-positive cells (magnification, x400). B, The lower panel presents these data on groups of animals. Results are expressed as percentage of total number of epithelial cells stained for Ki67.
FIG. 8. A, Histological sections from representative lumbar mammary glands treated for 5 d with: A, control; B, E2; C, Pg; D, IGF-I; E, IGF-I + E2; or F, IGF-I + Pg. Apoptosis was assessed by TUNEL assay. Apoptosis was found to occur in mammary glands in controls, E2 -treated, or Pg treated animals. Apoptotic cells are identified by brown staining in the nuclei identified by arrows. Apoptotic cells were largely eliminated in the mammary glands from animals treated with IGF-I alone or with E2 or Pg. B, Data for this parameter are presented in this graph in the lower panel. Results are expressed as total number of labeled cells/total number of epithelial cells.
Formation of alveolar structures requires IGF-I, Pg, and E2
Full mammary development resembling that seen in midpregnancy was observed only when IGF-I was given in combination with Pg and E2 (Table 1; Fig. 2, K and L). Only the three hormones together caused the development of a profusion of single- or sometimes double-layered acinar structures that were too numerous to count. They filled almost the entire mammary fat pad. That IGF-I is essential for true alveolar development is supported by the fact that the effect of Pg + E2 was virtually identical with that of E2 alone (Fig. 2J), where the percent fat pad area occupied by glandular structures was greater and there were increased side branches (P < 0.002). After 28 d of exposure, the combination of IGF-I and E2 stimulated some development of structures more mature than TEBs, which we have called alveolar structures (Fig. 2, E and F), suggesting that IGF-I + E2 can start the process of developing secretory structures but also indicating that Pg is essential for full formation of secretory structures that will eventually produce milk.
Discussion
At puberty, a surge of estrogen stimulates development of the mammary gland as long as the pituitary gland is intact (17, 18). Before that, the mammary gland is composed of a fat pad with a few simple branched ducts, the glandular anlagen (6, 19, 20, 21). In response to IGF-I and estrogen, actively dividing multilayered club-shaped structures, called TEBs, form. They extend into the substance of the fat pad, leaving in their wake a network of branched ducts. When the glandular tree nears the outer borders of the fat pad, TEBs are reduced in number, and the tree-like glandular component becomes and remains relatively quiescent until pregnancy. Lyons (22) first showed that GH was the pituitary hormone responsible for pubertal development. We then demonstrated that the earliest phases of experimental DM in several animal models were under the control of GH, through the action of IGF-I, together with E2 (9, 13, 16, 23, 24, 25). Studies in IGF-I(–/–) female mice revealed that the full effect of GH was mediated by IGF-I. We also studied mammary development for up to 14 d and showed that IGF-I plus E2 caused further partial DM (25).
The present study was designed to determine whether the entire process of DM past the 14-d point could be continued under the influence of IGF-I plus E2 and to determine whether Pg could also affect DM. For this purpose, we oophorectomized female IGF-I(–/–) mice and then administered the three hormones singly and in combination for 5 and 28 d. Our results show that, indeed, IGF-I together with E2 can stimulate partial DM at 5 d and a mammary gland almost completely filled by branching ducts at 28 d. This was due to an increase in TEBs, area of the fat pad occupied by glands, duct number, duct length, and duct branching. In fact, the mammary glands appeared more developed than expected for a completed picture of DM (6, 19). Ordinarily, when the mammary tree reaches the limits of the fat pad, there is regression of end buds, active pubertal development comes to an end (26), and the mammary tree appears quiescent. In this case, there were areas of hyperplasia (confirmed histologically) not ordinarily seen at this stage of development. This might have been due to either enhancement of IGF-I action by E2 or stimulation of IGF-I production. Our data suggest that the effect of E2 is at least partially caused by enhancement of IGF-I action because IGF-I alone stimulated one third or less the degree of mammary development of that of IGF-I plus E2. We also found that each known molecular biological action of IGF-I in mammary development that we tested (increased cell division, increased IRS-1 production, and inhibition of apoptosis) (27, 28, 29, 30, 31, 32) was significantly enhanced by the addition of E2. We have not determined whether other actions of IGF-I would have been enhanced by E2 or Pg, but it is not unreasonable to assume that they would. These include an increase in phosphorylation of IGF-IR and IRS-2 proteins (33, 34, 35) and enhancement of cell cycle progression (36, 37). The synergy might also have been caused by an increase in IGF-I itself. Previous observations have shown that E2 can enhance IGF-I mRNA production (12).
Regarding the mammary gland hyperplasia stimulated by the combination of IGF-I and E2, these studies now show that E2 can have some independent effect on mammary development; side branching and duct formation occur, but normal TEB development does not, whether given for long enough periods of time (28 d). In previous studies, E2 had no independent effect on mammary development in the absence of IGF-I given for as long as 14 d. Although overexpression of IGF-I can cause mammary hyperplasia and increase mammary tumor incidence (38), these data show that the addition of E2 can also cause hyperplasia. If enhancement of IGF-I action was the sole cause of hyperplasia, one would have expected hyperplasia when the same dose of IGF-I was given together with Pg, which also stimulated IGF-I action except where TEB formation was concerned. Thus, although enhancement of IGF-I action is likely one cause of improved mammary development, there are probably other pathways of activity, as well. An independent effect of E2 not related to IGF-I, or one that might circumvent the IGF-IR (29) is also a possibility. Because E2 did not have an earlier independent effect, one might speculate that IGF-II could have. In fact, the pathway by which E2 acts without IGF-I might be the one that caused the hyperplastic changes noted above. If IGF-I action was participating in formation of the structures that appeared with long-term estrogen, we would have expected formation of at least one true TEB because IGF-I is the common denominator for TEB formation.
The role of Pg in DM is less well worked out than that of E2. Established is that Pg is intricately and essentially involved in the formation of secretory lobuloalveolar structures (39). However, data are meager regarding a physiological role for Pg in DM (40, 41). We know that Pg is not essential for pubertal mammary development because DM takes place in PgR knockout mice (7, 8, 42). However, a very recent report indicated that maspin heterozygous mice had impaired mammary development that could be overcome by administration of Pg (43). Our study supports and extends those observations. They clearly demonstrate that Pg is capable of enhancing the effect of IGF-I in DM in the absence of estrogen. Gross anatomical appearance of mammary gland development caused by IGF-I + Pg shows that the ductal tree looks like normal fully developed pubertal gland. To determine whether the action of Pg was via PgR, we assessed the effect of RU486 on Pg- and IGF-I-induced mammary development and found that it significantly inhibited development. Like the interaction between E2 and IGF-I, Pg also acts by enhancing the action of IGF-I. However, there are differences between the two hormone combinations because Pg was mostly responsible for duct growth and branching. Unlike E2, Pg does not enhance the action of IGF-I on TEB formation or cause ductal decorations. Therefore, there are differences in actions of E2 and Pg on mammary development. Pg has more of an effect on duct formation, extension, and branching, whereas E2 has more of an effect on TEB and slightly more mature alveolar structures. These observations were unexpected because we consider the action of Pg one of enhancement of alveolar formation.
It was previously thought that Pg could not act in the absence of estrogen (44). Because PgR is present in the mammary gland at puberty (39) whether ovaries are present or not (45), and some of the receptors are estrogen dependent and are others not (42), the machinery for Pg action is there. With the caveat that the mice employed in these studies may have seen ovarian estrogen before oophorectomy at 21 d of age, our data support the concept that Pg can cause mammary development in the absence of estrogen. Assuming that our animal model in which the combination of IGF-I, E2, and Pg caused highly active formation of alveolar structure over 28 d, the action of Pg on formation of alveoli does require estrogen and IGF-I. In no case did Pg cause alveolar formation unless the other two hormones were present. Thus, Pg and presumably progestins (46) stimulate alveolar formation during preparation for lactation but have a more important effect on duct extension and branching during experimental pubertal development. This aspect of mammary development requires the action of prolactin, but this important activity was outside the purview of this manuscript and, therefore, not discussed (47, 48).
Overexpression of IGF-I is associated with an increase in mouse mammary gland tumor formation, and it also inhibits mammary gland involution due to a decrease in apoptosis (38, 49, 50). In view of our findings that Pg can enhance the action of IGF-I in duct formation, cell division, and production of phosphorylated IRS-1 and also increase IGF-I’s effect on inhibition of apoptosis, this provides an understanding of how Pg can affect the mammary gland. It also may provide insight into the observations that exposure to Pg or progestins can increase breast cancer incidence. Overall, if progestins or Pg are carcinogenic, consideration should be given to employing PgR antagonists in the treatment of breast cancer (51). That both Pg and E2 work on mammary gland via their effects on enhancing IGF-I action, consideration should also be given to blocking the effects of both Pg and E2 by antagonists of IGF-I (52).
Acknowledgments
We are grateful to Fabio Rotundo and Kalmon Kovacs for help and advice in immunostaining methodology, Marie Monaco for running Western blots and reviewing the manuscript, and Inn Ling Eng for expert animal breeding and making histological sections.
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