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Department of Physiology, Michigan State University, East Lansing, Michigan 48824
Address all correspondence and requests for reprints to: Sandra Z. Haslam, Ph.D., Department of Physiology, 108 Giltner Hall, Michigan State University, East Lansing, Michigan 48824. E-mail: shaslam{at}pilot.msu.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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Animals, surgical procedures, and hormone administration
Ten-week-old or 9- to 12-month-old BALB/c female mice from our
own colony were bilaterally OVX under Nembutal anesthesia 1 or 5 weeks
before E treatment. Varying doses of E in 0.85% NaCl were administered
by ip or sc injection; in pilot studies results were not significantly
different for the two injection routes. Injection was chosen as the
method of systemic administration to optimize the precision and
reproducibility of hormone dosage. Serum levels of E were determined
after OVX and E injection by RIA using a commercial kit
(Diagnostic Products, Los Angeles, CA) according to the
supplier’s instructions. Time-course studies revealed that after E
injection, maximal DNA synthesis and morphological responses were
observed at 72 and 96 h, respectively. Therefore, in all
experiments except time-course studies, mammary glands were analyzed at
72 h after E treatment for DNA histoautoradiography and at 96
h after E treatment for morphological analysis.
DNA histoautoradiography
At various times after E injection, animals received a single ip
injection of [3H]thymidine in a dosage of 2 µCi/g BW
1 h before death. The mammary glands were removed, fixed, and
processed as wholemounts, followed by embedding and sectioning for
histoautoradiographic analysis as described previously (15).
Histological sections were examined at x400 magnification to determine
the DNA labeling indexes (LIs) of epithelial and stromal cells. In view
of the stimulatory effect of E on duct ends, epithelial cell LIs were
also quantitated separately in ducts vs. duct ends. In
parallel, stromal cell LIs were determined for areas adjacent to and
distal from duct ends. Adjacent areas were defined to be within 400
µm of the duct end (i.e. in the same microscope field at
x400 magnification), and the distal areas were defined to be more than
400 µm away from the duct end. A minimum of 10 individual structures
were evaluated for each of 5–9 mice/experimental group. Determination
of LI was facilitated by the use of a computer-interfaced morphometric
digitizing system as described previously (15).
Steroid hormone binding assay
To determine the progesterone receptor (PR) concentration,
cytoplasmic extracts were prepared separately for mammary gland and
uteri as described previously (16). Mammary gland and uterine
cytoplasmic extracts were incubated with a single saturating
concentration of 20 nM [3H]R5020 with a
100-fold excess of radioinert dexamethasone (for suppression of
progestin binding to glucocorticoid receptors) or with a 100-fold
excess of radioinert R5020. Specific binding was determined using a
dextran-coated charcoal assay procedure as previously described (16).
Tissue DNA was quantitated as previously described (17).
Estrogen receptor (ER) immunohistochemistry
The immunohistochemical procedure was carried out on frozen
sections of mammary tissue using anti-ER monoclonal antibody, H222 (10
µg/ml; gift from Abbott Laboratories, Abbott Park, IL),
and was used as described previously (18). To quantitate the number of
ER-positive cells by light microscopy, 3,3'-diaminobenzidine
tetrahydrochloride chromogen substrate was used, and quantitation of
the percentage of ER-positive cells was facilitated by the use of a
computer-interfaced morphometric digitizing system (18). To quantify
the amount of ER per cell, the same immunohistochemical procedure was
carried out as described above, except that ER antibody was visualized
in tissue sections after incubation with a fluorescein-conjugated, goat
antirat (1:36 dilution; 45 min at 20 C) secondary antibody (Cappel
Research Products, Durham, NC). Fluorescently labeled sections were
mounted onto slides with Slow Fade (Molecular Probes, Inc., Eugene, OR) and analyzed under an Odyssey Laser Scanning
Confocal Microscope (Noran Instruments, Inc., Madison, WI). The
confocal microscope was equipped with Image-1 Software (Universal
Imaging Corp., Media, PA), which allows the image storage and
fluorescence intensity measurements as described previously (19). For
fluorescein, the excitation and primary barrier filters were 488 and
515 nm, respectively. A x40 objective was used for capturing images,
and the binding of anti-ER antibody was quantified using the function
Brightness Measurement: Area Brightness of the Image-1. The amount of
ER in antibody-treated sections was determined by quantitating the
intensity of fluorescence staining. To determine the intensity of
fluorescence staining, individual cells were outlined using the Area
Brightness function, and the average pixel brightness within the
circumscribed areas that was due to anti-ER antibody staining was
calculated by the Image-1 program. For anti-ER staining, the background
level of fluorescence obtained with normal serum in parallel control
sections was subtracted from fluorescence intensity obtained with
anti-ER antibody. In all cases images were captured and stored by
24 h after antibody treatment to reduce variability due to
fluorescence fading. There were no significant differences in
fluorescence measurements between sections from the same treatment
groups.
Statistical analysis
All data are expressed as the mean ± SEM and
were analyzed for significance using Student’s t test or
ANOVA as appropriate. P 
0.05 was chosen for
significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows
that the percentage of mammary glands exhibiting a morphological
response after E treatment increased with increasing length of time
after OVX, with 100% of the glands exhibiting stimulation of duct ends
in animals at 5 weeks post-OVX. In control injected mice 1 and 5 weeks
post-OVX, mammary gland morphology was identical and was characterized
by simple ducts lacking significant side-branching and alveolar buds
(Fig. 2, c and d). E treatment at 1 week
post-OVX had no effect on morphology (Fig. 2a). However, E treatment at
5 weeks post-OVX produced enlarged duct ends (Fig. 2b). Histological
analysis of the enlarged duct ends revealed the presence of multiple
layers of epithelial cells (Fig. 3a).
These enlarged duct ends were similar in appearance to end buds growing
in response to endogenous E in mammary glands of ovary-intact, immature
pubertal mice (Fig. 3c). End buds are the epithelial structures that
exhibit the highest degree of proliferation and are major growth points
leading to ductal elongation during puberty. Estrogen withdrawal by OVX
in both immature and adult mice produced quiescent duct ends
characterized by a single layer of epithelial cells and a reduced
number of DNA synthetic cells (Fig. 3, b and d).
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, the extent of the stimulatory response
to E as a function of time after OVX was identical to that obtained in
10-week-old mice. Furthermore, the morphological response was also
identical (data not shown). Because length of time after OVX rather
than age was the major determining factor of the extent of the E
response, in the interest of reducing the time and cost required for
these studies, all further experiments were carried out using
10-week-old mice. Mice OVX 1 or 5 weeks before E treatment were chosen
to represent early and late postmenopausal states, respectively, in
this study. Sex steroid hormones are also produced by the adrenal glands. One possible explanation for the enhanced response to E observed with increasing time after OVX was a compensatory increase in circulating E derived from adrenal gland biosynthesis. This did not appear to be a likely explanation based upon the persistent attenuated ductal morphology observed at 5 weeks post-OVX. However, to address the question of adrenal hormone contribution directly, the study was repeated in mice that were OVX as well as adrenalectomized. The morphological and DNA synthesis results obtained were identical to those obtained with OVX alone (data not shown), ruling out a significant adrenal contribution to the E response observed at 5 weeks post-OVX.
Time course and dose response of E-induced cell
proliferation
A time-course study revealed that the maximal proliferative
response in mammary epithelial cells and stromal cells occurred at
72 h after E injection (Fig. 4). To
determine whether the enhanced proliferative response to E at 5 weeks
post-OVX was due to an increase in E potency or to an increase in E
efficacy, a dose-response study was carried out (Fig. 5). The maximum proliferative response
observed at 5 weeks post-OVX was 5.6- and 2.5-fold greater than that at
1 week post-OVX for epithelial cells and stromal cells, respectively.
The ED50 values at 1 and 5 weeks post-OVX were identical,
indicating that the potency of E was the same in the two groups. By
contrast, the response to E was significantly greater at 5 weeks
post-OVX than at 1 week post-OVX. Thus, these results indicate that the
increase in E-induced proliferation observed at 5 weeks post-OVX was
due to an increase in E efficacy rather than potency. At 1 and 5 weeks
post-OVX basal serum levels of E were 5.4 ± 0.7 (n = 7) and
5.4 ± 0.5 (n = 8) pg/ml, respectively. Injection of 1 µg E
at 5 weeks post-OVX resulted in a serum E level of 31 ± 7.8 pg/ml
(n = 4) at 24 h after injection. This was similar to that in
ovary-intact mice (25 pg/ml on day 1 of the estrous cycle) (20). Thus,
in the interest of using a physiological dose of E, all subsequent
studies were conducted using 1 µg E.
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), there was a 6.5-fold higher E-induced
stimulation of DNA synthesis in duct ends at 5 weeks post-OVX than at 1
week post-OVX. Furthermore, little or no DNA synthesis was observed in
ducts. Analysis of stromal cell LIs (Fig. 7) revealed a 4-fold higher stimulation
of DNA synthesis in the stromal cells immediately surrounding the
enlarged duct ends at 5 weeks post-OVX vs. that in stroma
adjacent to the quiescent duct ends at 1 week post-OVX. These results
indicate that there is a paracrine interaction between proliferating
duct end epithelium and nearby stroma that occurs only over a short
distance.
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shows that there was no difference in
the percentage of ER-positive epithelial cells in control mice at 1 and
5 weeks post-OVX. Similarly, there was no significant difference in the
percentage of ER-positive stromal cells in control mice at 1 and 5
weeks post-OVX (Fig. 8b). However, the percentage of ER-positive
stromal cells was only about 50% that of ER-positive epithelial cells.
Because of the proposed role of stromal cells to mediate E-induced
epithelial proliferation, we also quantitated the percentages of
ER-positive stromal cells, either adjacent to or distant from duct
ends, at 1 and 5 weeks post-OVX. ER-positive stromal cells were evenly
distributed throughout the mammary gland, and no significant
differences were observed between the 1 and 5 weeks post-OVX groups in
either control or E-injected mice (data not shown). At 24 h after
E injection, the percentages of ER-positive epithelial and stromal
cells were significantly decreased at both 1 and 5 weeks post-OVX,
indicative of ER down-regulation by E.
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, a and b). Decreased cellular ER
content was also observed in both epithelial and stromal cells after E
treatment.
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). In contrast, no significant
increase in PR binding was observed at 5 weeks post-OVX. To determine
whether the lack of increase in PR was due an altered time course of PR
induction at 5 weeks post-OVX, PR levels were measured at 12, 24, and
36 h after E injection (Fig. 11a).
No increase in PR was observed at 5 weeks post-OVX over the entire
period tested. The lack of PR induction in mammary glands was not due
to an overall lack of E responsiveness at 5 weeks post-OVX, as an
8-fold increase in PR was observed in the uteri of the same mice
24 h after injection (Fig. 11b).
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, the
maximal proliferative response in the epithelial cells at 5 weeks
post-OVX was observed after 3 days of E injection and was 2.6-fold
higher than that at 1 week post-OVX. The epithelial cell proliferative
response decreased to a steady state level of proliferation but
remained 2.3-fold higher than that started at 1 week post-OVX. After 3
days of E injection at 5 weeks post-OVX, the mammary gland contained
enlarged duct ends similar to those observed at 96 h after a
single E injection. As a steady state LI was reached at 11–14 days,
the duct ends remained enlarged, but contained fewer
[3H]thymidine-labeled cells (data not shown). A similar
pattern of increased proliferative response to E was seen in mammary
stromal cells at 5 weeks post-OVX. The increased steady state
proliferative response to E in both mammary epithelial and stromal
cells when continuous, long term E treatment was started at 5 weeks
post-OVX indicates that long term OVX confers a permanent alteration in
mammary gland responsiveness to E.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The time points 1 and 5 weeks post-OVX appear to be appropriate choices
to represent early and late postmenopausal states, respectively. Our
results show that 1 week post-OVX is the shortest period of time
required by the adult mammary gland to achieve a baseline against which
a response to exogenously administered E can be measured. Although
serum levels are reduced to baseline in adult mice 2–3 days after OVX
(20), our data in Fig. 4 show that after a single injection of 1 µg
E, cell proliferation was not reduced to a baseline level by 4 days.
However, by 1 week post-OVX, basal levels of proliferation were reached
for both mammary epithelium and stroma and were not further decreased
with increasing time up to 5 weeks post-OVX (Fig. 4). Furthermore,
morphological and histological evidence demonstrate that the mammary
glands of 1 week OVX mice were regressed compared with those in the
ovary-intact state. Thus, the period of 1 week post-OVX is a reasonable
time point to choose for the early postmenopausal state. We have since
carried out studies in mice at 10 and 15 weeks post-OVX and have
observed estrogenic stimulation of duct ends at these time points
(Raafat, A. M., and S. Z. Haslam, unpublished observations).
Thus, 5 weeks post-OVX appears to be an appropriate time point for the
late postmenopausal state and exhibits the responses to E that are also
observed at much longer times post-OVX.
The enlarged duct ends obtained in response to E in long term OVX mice were similar in histological appearance to end buds of immature pubertal mice. End buds are the epithelial structures that exhibit the highest degree of proliferation and are major growth points leading to ductal morphogenesis during puberty. Analysis of E regulation of PR revealed that there is also a lack of PR inducibility at 5 weeks post-OVX. The presence of enlarged duct ends in the form of end buds, and the lack of PR inducibility in response to E treatment are characteristic features of the mammary glands of immature, pubertal mice. The present results indicate that long term OVX can produce these characteristics of the immature gland in adult mice. This observation is of particular interest with regard to mammary gland susceptibility to carcinogenesis, as it is well established that the mammary glands of immature, pubertal mice and rats are the most susceptible to carcinogen-induced mammary tumorigenesis (24). Thus, it will be of great interest to determine the influence of long vs. short term OVX and E replacement on susceptibility to mammary tumorigenesis.
The results of dose-response studies lead us to conclude that enhanced E responsiveness may be at least in part due to an increased efficacy of E. Recent studies using mammary tissue recombinants in ER knockout mice have provided compelling evidence in the mouse that the proliferative effect of E in epithelial cells is indirect and requires the presence of ER in mammary stroma (22). In light of these findings, it was important to determine the number of ER-positive cells and cellular ER content separately for the epithelium and stroma. The results of immunohistochemical analysis of mammary gland ER content revealed that the percentages of ER-positive cells and ER cellular content were not significantly different in short vs. long term OVX mice for either epithelial or stromal cells. Thus, increased efficacy of E at 5 weeks post-OVX does not appear to be due to increased ER content in either the epithelial or stromal cell compartments, and this suggests that amplification of the E response occurs at a postreceptor-ligand binding step(s).
In addition to the enhanced mitogenic effect of E on mammary epithelial cell proliferation, the 3-fold increased proliferative response in mammary stromal cells is noteworthy. Interestingly, proliferation of stromal cells occurred mainly in cells that were close to the proliferating epithelium of duct ends. We have previously shown that the local stromal environment has the capacity to influence mammary epithelial cell behavior and, in particular, can enhance epithelial cell response to E, possibly through the production of growth factors (23). Furthermore, in vitro, under coculture conditions, mammary epithelial cells have been shown to promote E-dependent stromal cell proliferation (25). Thus, the enhanced sensitivity of mouse mammary gland to E after long term OVX is probably mediated through estrogenic effects in the stroma. Furthermore, our results demonstrate that epithelial-stromal cell interactions may be bidirectional, and epithelial cells may influence the proliferative response of nearby stromal cells.
A dissociation between E stimulation of cell proliferation and PR regulation in the mouse mammary gland has previously been reported (26). Although E appears to mediate the proliferation of epithelial cells indirectly via stroma, E acts directly in epithelial cells to increase PR levels (27). Thus, our observations of increased responsiveness to the proliferative effect but decreased responsiveness to the PR regulatory effect of E indicate that mammary epithelial and stromal cells are affected differently by long term OVX. To confirm this observation, we have also used immunohistochemical analysis to determine the percentage of PR-positive cells and PR concentration per cell using a fluorescein-conjugated secondary antibody detection system similar to the ER immunofluorescence assay herein. In concordance with the ligand binding assay results we found that the PR concentration per cell increased 2-fold in 1 week post-OVX E-treated mammary glands, but there was no significant increase in either the number of PR-positive cells or the amount of PR per cell in the 5 week post-OVX E-treated glands (Raafat, A. M., and S. Z. Haslam, unpublished observations). Future studies are planned to determine the mechanistic basis of these different responses to OVX. A similar dissociation of estrogenic regulation of proliferation and PR level has recently been reported for normal human breast (28). It has been proposed that E acts indirectly to stimulate the proliferation of normal human breast epithelium, as ER and markers of cell proliferation do not colocalize in the same epithelial cells. In contrast, as ER and PR do colocalize in the same cells, E appears to act directly to regulate PR in breast epithelial cells. These additional similarities between the human and mouse mammary glands increase the attractiveness of the mouse model to study the hormonal responsiveness of the postmenopausal breast.
Heightened sensitivity to the mitogenic effects of estrogen after withdrawal from estrogen has been previously reported to occur in the MCF-7 human breast carcinoma cell line (29). A shift in the dose for maximal proliferative response to estradiol from 10-10 to 10-14 M was observed in cell culture when MCF-7 cells were deprived of estrogen for 1- 6 months. Although there was a 4-fold increase in the ER concentration, the researchers concluded that this alone could not account for the increased sensitivity to E; no differences in receptor ligand binding affinity were observed in these studies. The enhanced sensitivity to the mitogenic effect of E was also observed in vivo. Nude mice implanted with the E-deprived cells demonstrated an earlier appearance of palpable tumors in response to E than animals bearing wild-type cells. Although no specific mechanism(s) has been identified to explain the increased E sensitivity in E-deprived MCF-7 cells, it does not appear to be the same as the enhanced proliferative response obtained herein after long term OVX. In the case of MCF-7 cells, a shift in the dose-response curve was observed that rendered the E-deprived cells maximally responsive to lower doses of E. In contrast, in the present study, a maximal proliferative response was obtained at the same E dose in long and short term OVX mice. However, the extent of the response was higher in the duct ends of long term OVX mice. Furthermore, no significant difference in ER content between the two groups was observed herein.
We have also examined the proliferative response to E in our model after implanting small doses of E into the mammary gland to assess the direct effect of E on the mammary gland vs. E-induced systemic factors, such as hormones or growth factors/growth inhibitors produced elsewhere in the body (30, 31, 32, 33, 34). Interestingly, the results obtained with systemically administered E herein, were virtually identical to those obtained with E implants with regard to the enhanced proliferation, the histological and morphological characteristics of the response, and the lack of PR inducibility (Raafat, A. M., and S. Z. Haslam, unpublished observations). This suggests that the major effect of estrogen on mammary cells is direct and not due to systemically induced factors.
To our knowledge, only one other in vivo model, the cynomolgus macaque, has been developed to study the effect of HRT on the postmenopausal mammary gland (35). In the cynomolgus macaque, a postmenopausal state was surgically induced by long term OVX (2 yr). Long term treatment of postmenopausal monkeys with estrogen for 36 months resulted in increased thickness and percentage of epithelial tissue and increased epithelial cell proliferation (35). Thus, the results obtained after E treatment in the monkey after long term OVX agree with our findings of increased E-induced epithelial cell proliferation after long term OVX in the mouse.
In women, with the cessation of ovarian function after menopause there is a significant reduction in the mitotic activity of mammary tissue (36). In a recent report, very low levels of mammary epithelial cell proliferation were reported in breast biopsies obtained from postmenopausal women receiving estrogen HRT (37). Although not specified, it is likely that these samples were obtained from women who initiated estrogen HRT in the early postmenopausal period, as that is the time when the majority of women start HRT. In this regard, the low level of E-induced mammary cell proliferation in short term OVX mice is compatible with the finding of low estrogen-induced breast cell proliferation in women when HRT is initiated in the early postmenopausal period. In the same study, the women receiving estrogen HRT also had elevated PR levels in their breast tissue. Thus, another similarity between the short term OVX, early postmenopausal mouse and the early postmenopausal human breast is increased PR after estrogen treatment.
The increased incidence of breast cancer in postmenopausal women may be simply coincidental with advanced age. Alternatively or in addition, it is possible that there is something specific about the postmenopausal breast that enhances the development of mammary cancer. A significant percentage of postmenopausal breast cancers are hormone responsive and regress in response to antiestrogen therapy (38). It seems paradoxical that breast cancers that are quite responsive to ovarian hormones arise at a time when ovarian function has ceased, and the influence of these hormones appears to be minimal. One interpretation is that breast cancers may arise from and/or be comprised of cells that are supersensitive to the growth-promoting effects of estrogen. Our present studies in mice suggest that altered hormonal milieu and long term deprivation of ovarian hormones, rather than advanced age may be the major contributing factor to the enhanced sensitivity to E observed in mice and raise the question of whether enhanced sensitivity to estrogen occurs after menopause in the human breast. The current results obtained in mice considered in conjunction with the results of estrogen HRT obtained in the monkey model and in postmenopausal women lead us to propose that the timing of initiation of HRT with estrogen in early postmenopausal vs. late postmenopausal women could have different consequences for the proliferative response of mammary cells to estrogen. These results have added significance because of a potential new pattern of timing of HRT in women. Previously, women started HRT in the early postmenopausal period to alleviate menopausal symptoms. However, with reports that HRT has beneficial effects on bone density and cardiovascular health in older women, late postmenopausal women who never received HRT in early postmenopause are now being prescribed HRT (11, 12, 13, 14). Our findings of enhanced proliferative response to E with increasing length of time after OVX suggest that the proliferative response of older, late postmenopausal women receiving HRT for the first time may be greater than that of early postmenopausal women. Furthermore, if increased breast cancer risk associated with estrogen HRT in women is due to the mitogenic effect of estrogen in breast tissue, then the timing of HRT could also play a crucial role.
Clearly, hormonal responsiveness of the postmenopausal human breast needs to be investigated more thoroughly to better understand and assess the potential consequences of HRT. An important approach to this problem is the development of suitable experimental animal model systems. We propose that the induction of short vs. long term OVX in mice is one such model system and may provide important information about potential alterations in hormonal responsiveness of the early and late postmenopausal human breast.
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Footnotes |
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Received September 16, 1998.
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