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Monash Institute of Reproduction and Development, Monash University, Monash Medical Center, Clayton, Victoria 3168, Australia; Department of Anatomy, University of California School of Medicine K.A.T., G.R.C.), San Francisco, California 94143; and Department of Urology, University of Illinois College of Medicine (G.S.P.), Chicago, Illinois 60612
Address all correspondence and requests for reprints to: Dr. G. P. Risbridger, Monash Institute of Reproduction and Development, Monash Medical Center, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: gail.risbridger{at}med.monash.edu.au
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Abstract |
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-actin. When cultured in the
presence of 17ß-estradiol or diethylstilbestrol in addition to
testosterone, androgen-induced prostatic growth was reduced, and
differentiation was altered. Although estrogen-treated explants were
smaller than controls, quantification of epithelial, stromal, and
luminal volumes using unbiased stereology revealed significant changes;
the proportion of epithelial cells and lumen decreased, and the
proportion of stroma increased compared with control values. Concurrent
with this reduced growth rate, we observed a disturbance in the
branching pattern and a reduction in ductal canalization. Specifically,
stromal differentiation and organization were disrupted, so that a
discontinuous smooth muscle layer was observed around the epithelial
ducts, and epithelial differentiation was altered. The effects of
estrogens were not accompanied by a decrease in androgen response via
the androgen receptor, because immunolocalization of this receptor
remained constant. These data demonstrate that high doses of estrogens
are growth inhibitory and have direct effects on prostate development
in vitro, which may occur in vivo in
addition to indirect effects via suppression of the
hypothalamic-pituitary-gonadal axis. |
Introduction |
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The developing rodent prostate is also sensitive to other hormones, including estrogens. Exposure to low doses of estrogen during gestation in the mouse have been reported to increase adult prostate weight and androgen receptor levels as well as significantly increase prostatic glandular budding (3, 4, 5). However, the effects of exogenous treatment with low doses of estrogen are still controversial and have not been reproduced in all experiments (6).
In contrast, several studies in which neonatal rats (7, 8, 9, 10, 11) and mice (12, 13) have been exposed to higher doses of estrogen resulted in a permanent suppression of prostate growth, a reduced response to androgens, and an induction of epithelial hyperplasia in adulthood. Prins and colleagues (11) have shown that the reduced responsiveness to androgens is related to a decrease in androgen receptor (AR) expression and may be associated with lower serum androgen levels. However, administration of exogenous androgens only partially restored prostate growth and AR expression (11). These data suggested that the effects of estrogens were mediated not only by changes in androgen levels via suppression of the hypothalamic-pituitary-gonadal axis, but also by additional direct effects on prostate growth.
The majority of studies examining the effect of high doses of estrogens on prostate growth have been carried out in vivo, where exogenous estrogen administration down-regulates the hypothalamic-pituitary-gonadal axis (14). In these studies, altered androgen levels confuse the analysis of direct effects of estrogens on prostate. To address this issue, we used a defined serum-free organ culture method (15, 16) that permits androgen-induced growth and differentiation of the neonatal rat ventral prostate (VP) in vitro and mimics the events that occur in vivo, as documented previously (17, 18, 19, 20). The aim of this study was to determine whether exogenous estrogens have a direct effect on prostate development in the presence of androgens in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Organ culture
Organ culture was carried out as previously described (15).
Briefly, microdissected VPs were cultured on Millicell CM filters
(Millipore Corp., Bedford, MA) floating on 500 µl
nutrient medium in a four-well plate at 37 C in a humidified 5%
CO2 incubator. A basal medium of DMEM/Ham’s F-12
(1:1, vol/vol), supplemented with insulin (10 µg/ml) and transferrin
(10 µg/ml) was used in all experimental groups, and medium was
replenished every 48 h. The medium was supplemented with 10
nM testosterone (T), and treatment groups were cultured
with high doses of 17ß-estradiol (E2; 20
µM) or diethylstilbestrol (DES; 5 µM)
together with 10 nM T. In some experiments, a range of
doses (10 nM to 100 µM) of
E2 and DES was used.
The organs were harvested after 6 days of culture. Explants were photographed, fixed in Bouin’s fixative for 2 h at room temperature, and then processed to paraffin for histological and stereological analysis.
Stereology
An unbiased estimate of prostate volume on day 0 and after 6
days of organ culture was obtained using the Cavalieri principle (21).
Images of paraffin serial sections were captured using a Dage-MTI
digital camera interfaced with a Power Macintosh G3 computer using
Scion Image software (Scion Corp., Frederick, MD) and printed with an
incorporated grid. Section area was estimated using traditional point
counting on a minimum of 10 sections uniformly spaced throughout the
explant. The estimate of volume was based on the following formula:
Vprostate = 1/n x
p x T x a(p), where
n was the fraction of sections analyzed (starting with a
random section), p was the sum of points, T
was the section thickness (5 µm), and a(p) was the area
associated with each point on the grid.
Statistical analysis
All data are expressed as the mean ± SEM.
Comparisons between control and treatment groups were made using a
two-tailed paired t test. All analyses were conducted using
Prism 2.01 software (GraphPad Software, Inc., San Diego,
CA).
Immunohistochemistry
Smooth muscle -actin, high mol wt cytokeratins, cytokeratin
10, and proliferating cell nuclear antigen. Immunohistochemistry
was used to assess smooth muscle cell differentiation with an antibody
to smooth muscle -actin (Sigma, St. Louis, MO; 6.9
µg/ml). Epithelial differentiation was assessed by
immunohistochemistry with antibodies to high mol wt cytokeratins
(HMWCKs; DAKO Corp., Carpinteria, CA; 4.4 µg/ml) and
cytokeratin 10 (CK10; DAKO Corp.; 4.4 µg/ml).
Proliferating cells were detected in tissue sections by
immunohistochemistry with an antibody to proliferating cell nuclear
antigen (PCNA; DAKO Corp.; 3.85 µg/ml).
Immunohistochemistry was performed as previously described (22) with the following modifications. Sections stained for PCNA, HMWCKs, and CK10 were subjected to microwave antigen retrieval in 0.01 M citrate buffer (pH 6.0). In addition, sections stained for HMWCKs were incubated with 0.1% trypsin with 0.2% CaCl2 at 37 C for 10 min. All sections were then treated with 3% (vol/vol) hydrogen peroxide in methanol for 30 min and blocked with CAS block (Zymed Laboratories, Inc., San Francisco, CA).
Sections were then incubated with primary antibodies or concentration-matched mouse IgG (DAKO Corp.; negative control sections) for 1 h at room temperature. Antibodies were detected by incubation with peroxidase-labeled polymer (Envision System, DAKO Corp.), which is conjugated to antimouse and antirabbit Igs, for 30 min at room temperature and then color reacted with 3,3'-diaminobenzidine tetrahydrochloride (liquid substrate kit, Zymed Laboratories, Inc.). The reactions were stopped in water, and sections were counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted.
Androgen receptor. Affinity-purified polyclonal antibody PG-21–39, purification batch 31 (2 µg/ml), was used to localize AR. Sections were incubated with primary antibodies for 1 h at room temperature, and antibodies were detected as previously described (22). Antibodies were preabsorbed with AR21 (AR peptide) and AR462 (unrelated AR peptide) at a 10x molar excess overnight at 4 C before incubation on tissue sections as negative and positive controls.
Double immunohistochemistry for smooth muscle -actin and
HMWCK. Sections were stained for HMWCKs as described above. Before
counterstaining, sections were incubated with double stain enhancer
(Zymed Laboratories, Inc.) for 20 min. After rinsing with
PBS, sections were incubated with CAS (Zymed Laboratories, Inc.) followed by primary antibody for smooth muscle -actin
and developed with peroxidase-labeled polymer (DAKO Corp.
Envision System) as described above. After color reaction with a
peroxidase substrate kit (Vector VIP, Vector Laboratories, Inc., Burlingame, CA), sections were counterstained with
Mayer’s hematoxylin, dehydrated, cleared, and mounted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B), and mesenchyme containing
fibroblasts, smooth muscle cells (Fig. 1, C and D) and other stromal
cells. At birth the smooth muscle cells were organized in a continuous
layer around the epithelial ducts in the proximal region near the
urethra (Fig. 1, C and D).
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). These explants were cultured in the
presence of T (10 nM), which induced extensive branching
over 6 days of culture (Fig. 1F) and a significant increase in the size
of the organ. Unbiased stereology demonstrated a 5-fold increase in
explant volumes from 0.040 mm3 on day 0 to 0.206
mm3 after 6 days of culture in the presence of T
(Fig. 2). Regional variation in the
differentiation of epithelium and mesenchyme was observed after culture
as differentiation occurs in a proximal to distal fashion (Fig. 3, A–C), similar to that previously
described in vivo. Smooth muscle cells formed continuous
layers surrounding the epithelial ducts, particularly in the proximal
region (Fig. 3, A and B). In the distal region, the ducts were solid
undifferentiated cords of epithelium (Fig. 3, A and B). In the proximal
region, ductal canalization occurred and the epithelium underwent
differentiation; basal cells immunopositive for HMWCKs were
interspersed between a single layer of tall columnar luminal epithelial
cells that lined the lumen and were immunonegative for HMWCKs (Fig. 3C).
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, A and B) or in the presence of
E2 plus T (Fig. 4C) or DES plus T (Fig. 4D). Both
estrogens caused a reduction in androgen-induced growth. Although
explants in all treatment groups increased in size over the 6 days of
culture, a significant decrease in the final explant volume on day 6
was observed after culture with E2 (Fig. 5A) and DES (Fig. 5B) compared with
controls treated with T alone. Although estrogen-treated explants were
smaller than controls, quantitation of cellular compartment volumes
showed a significant increase in the proportion of stroma (percentage
of total explant volume) and a significant decrease in the proportion
of epithelium and lumen for both the E2 plus T
(Fig. 5C) and DES plus T (Fig. 5D) groups compared with VP explants
cultured with T alone. Concurrent with the reduced growth rate after
culture in the presence of E2 plus T, normal
budding and branching were interrupted, resulting in fewer epithelial
ducts compared with explants cultured with T alone, as seen
histologically (Fig. 6, A and B).
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). However, culture with
E2 plus T disrupted the differentiation and
organization of smooth muscle, so that discontinuous layers of smooth
muscle actin-containing cells were interspersed between unstained
stromal cells, particularly around proximal ducts (Fig. 6D). This
resulted in an apparent increase in non-smooth muscle stromal cells,
particularly in the proximal region (Fig. 6D), compared with controls.
In addition to disruption of ductal branching and mesenchymal
differentiation, culture in the presence of E2
plus T initiated the process of squamous metaplasia of the epithelium,
which was never observed in explants cultured in the presence of T
alone (Fig. 6, E and F). This was characterized by multilayering and
cornification of epithelial cells, which was confirmed by the
expression of CK10 (Fig. 6F). In the presence of T alone, none of these
metaplastic changes was observed; undifferentiated epithelial cords
gave rise to canalized ducts lined by epithelial cells that were
immunonegative for CK10 (Fig. 6E). The estrogen-induced changes to
prostatic growth were not due to changes in AR localization. In the
presence of T alone, AR was immunolocalized to mesenchymal and
epithelial cells throughout the explant (Fig. 6, G and H). This
expression pattern was not altered after culture in the presence of
E2 plus T (Fig. 6, I and J), and there was no
apparent difference in the staining intensity for AR.
Immunolocalization of PCNA was similar after T treatment or
E2 plus T (Fig. 6, K–N), with a greater degree
of proliferation in the growing distal tips compared with the
differentiating proximal region. Identical results were observed with
DES plus T treatment (data not shown) as described above for
E2 plus T.
The effect of E2 plus T on prostatic growth was
dose responsive. The effects of E2 over a range
of concentrations from 10 nM to 20 µM (Fig. 7, A–F), in the presence of a constant
10-nM T concentration, showed that high doses of
E2 (micromolar concentrations) reduced prostatic
growth rate, whereas decreasing the E2
concentration reduced this inhibitory effect on growth rate such that
these organ cultures (nanomolar concentrations) were not different from
control explants cultured in the presence of T alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Development of the VP in the presence of androgens in vitro proceeds with the appropriate events that are temporally and spatially coordinated as described in vivo (17, 18, 19, 20). Estrogen caused changes to both epithelial and stromal in vitro development. In the epithelium, both E2 and DES induced squamous metaplasia, which was characterized by multilayering of squamous epithelial cells and the onset of CK10 expression. These in vitro epithelial changes are similar to those observed in vivo after neonatal estrogenization (7, 11, 13), although the metaplastic changes described in those studies were prolonged through to adulthood. Concurrent with these changes in epithelial differentiation, we observed reduced ductal canalization; this was confirmed by stereology, which demonstrated a significant decrease in luminal volume.
In addition to these epithelial changes, stromal development was also altered by estrogen exposure. An overall increase in the proportion of stromal volume was measured using stereological techniques after culture with estrogens plus androgens. A similar increase in stromal tissue was reported by Prins and colleagues (20, 23), who described increased proliferation of periductal fibroblasts of neonatally estrogenized adult rat VPs. We have shown that the stromal changes were much more pronounced in the proximal region of the explant; however, as most of the cells in the organ were labeled with PCNA after 6 days of culture, we were unable to discern specific changes in proliferation of these stromal cells. Alternatively, increased production of extracellular matrix or an increase in the size of the stromal cells may occur in addition to cellular proliferation. Concurrent with changes in stromal volume, we observed significant changes to the organization of the smooth muscle cells in the periductal area, where treatment with estrogen resulted in a discontinuous layer of smooth muscle, associated with other stromal cells. This disruption to the smooth muscle layer may be due to fibroblast proliferation or other changes described above.
To date, many studies have attempted to delineate the effects of estrogens due to a removal of androgens from those due to a direct estrogen action. Exogenous administration of estrogens in vivo results in a decrease in androgen levels after suppression of the hypothalamic-pituitary-gonadal axis (14). Studies in canines (24) and rats (25) have attempted to alleviate this complication by administering estrogens in combination with a constant level of androgens. In those studies the observed metaplastic changes were believed to be a direct effect of estrogens, rather than the result of androgen withdrawal (24, 25).
Previous studies have examined the long-term effects of estrogens after short-term neonatal estrogenization. In such experiments, estrogen treatment resulted in a decrease in AR expression and reduced sensitivity to androgens; this decrease was observed as early as postnatal day 10 (after estrogenization on postnatal days 1–5) (11). A decrease in AR expression in vivo cannot be attributed to a reduction in T, as loss of T by neonatal castration or neonatal flutamide exposure does not affect prostatic AR levels by postnatal day 10 (26). This suggests that the decrease in AR observed after neonatal estrogenization is independent of T levels and is regulated through an alternative mechanism. In the present study we did not observe a decrease in AR localization, although this short-term in vitro system investigated the acute effects of estrogenization, and long-term in vitro effects could not be determined.
Alternatively, the effects of high dose estrogens may be mediated
directly through one of the estrogen receptor (ER) subtypes, ER or
ERß. The developing mouse urogenital sinus binds
E2 as early as day 16 of gestation (27), and ER
protein and/or messenger RNA (mRNA) have been detected in the neonatal
(postnatal days 1–5) rat VP. ER was localized in mesenchymal cells
of the proximal region, but not in the distal region (28), and low
levels of ERß mRNA were expressed predominantly in epithelial cells
throughout the gland (29). Neonatal estrogenization significantly
up-regulated expression of ER in the stroma, although ERß mRNA
expression remained constant (29). Localization of ERs in the
epithelium and stroma of the developing prostate is consistent with a
direct action of estrogens, and this concept is supported by the
studies described in this manuscript.
To establish whether estrogen action is mediated via either of the ER
subtypes, it might be possible to inhibit the response using an
antiestrogenic compound, such as tamoxifen. However, tamoxifen can also
act as a potent agonist, as shown in studies on mouse uterus and vagina
(30, 31). Pure estrogen antagonists such as ICI 182,780 are available
(32); however, high concentrations (10- to 100-fold the micromolar
doses of estrogens used) are needed, and these doses of ICI antagonist
are toxic in these organ cultures. Future studies will aim to delineate
the specific role of each ER subtype using specific ER and ERß
antagonists.
This study has been restricted to high doses of E2 and DES and has clearly demonstrated a direct inhibitory effect on prostate growth in the presence of constant androgen levels. Previous studies in mice have suggested that low doses of estrogens, such as DES and bisphenol A can have an opposite effect and stimulate prostate growth (4, 5), although this effect could be mouse strain dependent (6). The rat organ culture model provides researchers with an independent system to study the effects of a range of doses of estrogens or other estrogenic compounds, such as phytoestrogens or environmental estrogens.
In summary, we have demonstrated that high doses of estrogens have a direct inhibitory effect on prostate growth rates in vitro. Therefore, future evaluation of the actions of estrogens in vivo must consider direct actions in addition to indirect effects, which may be mediated through altered androgen levels.
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Footnotes |
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Received May 9, 2000.
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