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Receptors, and Estrogen β Receptors in Experimentally Induced Canine Prostatic Hyperplasia
From the * Facultat de Veterinària,
Universitat Autònoma de Barcelona, Bellaterra, Spain; the
Departament de Patología, Universitat
Pompeu Fabra, Barcelona, Spain; and the Unitat
de Recerca Biomèdica i Servei d'Urologia, Hospital Universitari Vall
d'Hebron Spain, Barcelona, Spain.
| Correspondence to: Dr Teresa Mogas, Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Edifici V, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain (e-mail: teresa.mogas{at}uab.es). |
| Received for publication September 3, 2008; accepted for publication January 5, 2009. |
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Abstract |
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(ER), and estrogen
receptor β (ERβ) in the different cell types of the prostate gland
in an experimental model. Five male beagle dogs were castrated and treated
with 25 mg of 5-androstane-3 and 17β-diol and 0.25 mg
17β-estradiol for 30 weeks. Prostate specimens were surgically obtained
every 45 days (experimental stages M0 to M6: 0, 12, 18, 24, 30, and 36 weeks
from the beginning of the hormonal treatment). The control group consisted of
3 noncastrated dogs treated with a vehicle, from which specimens were only
taken at the time points M0, M1, M4, and M6. Immunohistochemical data revealed
high AR and ER expression in the epithelial and stromal cell nuclei of
all the experimental and control specimens. Weak staining of the cytoplasm was
observed only in epithelial cells. The suspension of hormone treatment led to
a significant reduction in the expression of both receptors. On the contrary,
ERβ was expressed only in epithelial cell nuclei, with no significant
differences in the percentages of stained nuclei between control and
hormonally treated or atrophic prostates. Results indicate that AR, ER,
and ERβ are differently expressed in canine prostate tissue and that they
show specific expression patterns in response to the hormonal induction of
BPH.
Key words: Animal model, benign prostatic hyperplasia, dog, prostate, steroids, androstanediol
Androgens play a central role in regulating the growth of the mammalian prostate gland through the androgen receptor (AR). The secretory epithelial cells of the prostate express AR, requiring continuous stimulation for their survival and functional integrity. The essential role of the AR/androgen signaling pathway in the etiology of BPH remains unclear (McConnell, 1991), but AR localization has been detected in both healthy and hyperplastic prostates (human, Sar et al, 1990; Leav et al, 2001a; dog, Murakoshi et al, 2000a,b).
Estrogens are believed to play a critical role in the pathogenesis of BPH
(Leav et al, 2001b). It is
also known that aromatase activity, and thus estrogen production, correlates
with age. The effects of estrogens on target tissues are now known to be
mediated by 2 ligand-specific transcription factor receptor proteins termed
estrogen receptors and β (ER and ERβ). The presence
of ER is confined to stromal cell nuclei in both healthy (human,
Leav et al, 2001a; rat,
Sar and Welsch, 2000; dog,
Schulze and Barrack, 1987) and
hyperplastic (human, Hiramatsu et al,
1996; Leav et al,
2001a) prostate glands.
To improve our understanding of the role of androgens and estrogens during
the course of induced BPH in dogs, the exact site(s) of action of these sex
steroids needs to be established. This study was designed to identify the cell
types expressing AR, ER, and ERβ in canine prostate tissue. We
undertook an immunohistochemical study to locate these receptors during the
course of experimentally induced BPH in dogs.
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Materials and Methods |
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Hormone Treatment
Animals in G I were castrated and intramuscularly injected with 25 mg of
5-androstane-3 17β-diol (androstanediol) and 0.25 mg of
17β-estradiol (Steraloids Inc, Newport, Rhode Island). The 3 G II dogs
received only 1 mL of triolein vehicle. Treatment was administered 3 times
weekly for 30 weeks. Surgical biopsies of the prostate were obtained first at
baseline before castration and treatment (M0), and then at 6-week intervals
for 36 weeks (M1 to M6: 6, 12, 18, 24, 30, and 36 weeks from the beginning of
the hormonal treatment). As for M0, specimens obtained at the last stage (M6)
lacked hormone treatment. Specimens from control animals were only obtained at
stages M0, M1, M4, and M6.
Surgical Procedures
The dogs fasted for 24 hours before surgery and then were premedicated with
0.4 mg/kg morphine sulfate and 0.05 mg/kg acepromazine maleate. A single dose
of 4 mg/kg of propofol was given intravenously for anesthesia. Tracheal
intubation was achieved with a 9–10-mm endotracheal tube, and anesthesia
was maintained with 1.5%–2% isoflurane in 100% oxygen administered
through a semiclosed circular anesthetic system. All animals were allowed to
breathe spontaneously. Epidural anesthesia-analgesia was achieved by
introducing 0.2 mL/kg lidocaine 2% and 0.1 mg/kg morphine in the lumbosacral
space.
Surgical biopsies were excised from alternating quadrants of the prostate at each experimental interval, using a biopsy punch (0.6 cm). Small samples of tissue were immediately fixed in 10% phosphate-buffered formalin and processed for paraffin embedding.
Postoperative care involved treatment with 15 mg/kg amoxicillin every 48 hours for 15 days. Postoperative analgesia was maintained using fentanyl patches (50 µg/kg/h) placed the day before surgery. The sutures were removed 7–10 days postoperatively and the healing of the prostate was monitored by ultrasonography to detect complications.
Reagents
The immunohistochemical detection of AR was performed using a polyclonal
rabbit antibody (NCL-Arp; Novocastra, Newcastle upon Tyne, United Kingdom) at
a 1:20 dilution for 30 minutes. The antibody against the ER was a
monoclonal mouse antibody (NCL-ER-LH2; Novocastra) used at a 1:50 dilution for
30 minutes. For ERβ, a polyclonal rabbit antibody (H-150: sc-8974; Santa
Cruz Biotech, Santa Cruz, California) was used at a 1:200 dilution for 1
hour.
Immunohistochemical Staining Procedure
Labeling of the 3 different antibodies was performed using an indirect
immunoperoxidase staining procedure in which 4-µm sections were
deparaffinized, rehydrated through a graded alcohol series, and subsequently
incubated in H2O2 (0.3%) to block endogenous peroxidase.
To overcome cross-linking by formalin fixation, paraffin-embedded tissue
sections were pretreated by high-temperature antigen retrieval (autoclave).
Antigen retrieval was conducted in a solution of 10 mM citrate buffer, pH 7.4,
for 3 minutes at 121°C in an autoclave to expose antigenic sites. After
pre-treatment, sections were allowed to cool for 20 minutes at room
temperature (RT).
Antibody labeling was detected by the immunohistochemical DAKO EnVision+ method (DakoCytomation, Glostrup, Denmark). The bound primary antibodies were detected by 30 minutes incubation with a horseradish peroxidase–labeled polymer that is conjugated to a secondary antibody. Incubation with 3,3'-diaminobenzidine tetrahydrochloride for 10 minutes led to stable brown precipitate at labeled sites. The tissues were counterstained with Mayer hematoxylin at RT for 2 minutes. After this, sections were mounted and examined under an Olympus (Barcelona, Spain) B40 light microscope.
Positive and negative controls were included in each labeling procedure. In all immunostained batches, the omission of the primary antibody and substitution with the diluting solution alone served as a negative control. Both cryostat sections of normal canine prostate gland and paraffin sections of human hyperplastic prostate were used as positive controls. For ERs, ovary and uterus tissue were also used as controls.
Scoring of Immunohistochemical Results
Receptor expression in each tissue section was examined at a magnification
of x400 by 2 independent observers. An intensity score and a
proportionality score were obtained in each case. The former reflected the
intensity of the positive reaction in the cell nuclei or cytoplasm (0, no
labeling; 1+, weak labeling; 2+, moderate labeling; 3+, intense labeling; 4+,
very intense labeling), whereas the proportionality score indicated the
percentage of positively labeled cell nuclei in the different cell aggregates.
These 2 scores were obtained in areas containing similar amounts of glandular
epithelium and stromal cells. At least 100 parenchymal and stromal cells were
examined per tissue sample.
Statistical Analysis
All statistical analyses were conducted by analysis of variance implemented
in the MIXED SAS procedure (version 9.1; SAS Institute Inc, New York, New
York). Experimental stage was the repeated measure subjected to the
experimental unit or animal under a given treatment. The animal was considered
a random effect, and treatment and interaction treatment x experimental
stage were the fixed effects. Because sample numbers were not the same for the
2 treatment groups, samples lacking at any given period were considered as
missing values and analyzed using an unbalanced design with the autoregressive
order 1 as covariance structure. Means were computed by least square means
procedures and differences between means were separated by pairwise t
tests. All variables expressed as percentages were square root arcsine
transformed before analysis and their means and standard errors were
backtransformed for the presentation of data. The level of significance was
set at P < .05.
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Results |
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Estrogen Receptor—
Nuclear immunohistochemical staining for ER was observed in the
prostate of the male dog during the hormonal induction and regression of BPH
(Figures 2 and
3). Specific ER antibody
staining was observed in the nuclei of epithelial cells, smooth muscle cells,
and fibroblasts. Weak specific staining was also evident in the cytoplasm of
epithelial cells.
When we analyzed the specimens obtained from control animals, significantly
lower intensity scores were obtained for stage M6 (2+) compared with M0 (4+),
M1 (3.6+), or M4 (3.6+). Similarly, ER positivity was detected in
higher percentages of epithelial and stromal cells at stages M0, M1, and M4
compared with M6, although these differences were not significant.
In the experimental dogs, percentages of positive epithelial cells were
significantly lower 6 weeks after the end of hormone treatment (M6, 50%) when
compared with the remaining specimens (
98%)
(Figure 1F). However, in
stromal cells, both proportionality and mean intensity scores were
significantly higher for M6 (70% and 3.4+, respectively;
Figure 1G) when compared with
the sample obtained just before hormone treatment suspension (M5, 40% and
2.2+, respectively; Figure 1H).
Mean intensity and proportionality scores for experimental M6 specimens (3.2+
and 70%) were significantly higher than those recorded for the control M6
specimens (2+ and 21%).
Estrogen β Receptor— Glandular epithelial cells showed uniformly intense immunostaining for nuclear ERβ. Immunoreactivity was evident in both control and experimental canine prostate specimens and no significant differences were observed among the different samples (M0–M6) or between control and experimental dogs. No nuclear staining was detected in smooth muscle cells or fibroblasts (Figures 1I, 2, and 3).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In contrast to reports by other authors, we observed positive staining for AR in the cytoplasm of prostate epithelial cells. There were no indications that this cytoplasmic staining was nonspecific, because the stromal cell cytoplasm or negative control sections showed no staining for the receptor. One explanation would be a consequence of the high hormone doses used in our experiment, which would induce high levels of AR synthesis in the rough endoplasmic reticulum, thus giving rise to the "ectopic" cytoplasmic localization of these receptors in the cytoplasm. In addition, previous studies in which nuclear and cytosol extracts were separately analyzed have shown both nuclear and cytosol AR distribution in normal, spontaneous hyperplastic, and hormonally induced hyperplastic canine prostate (Trachtenberg et al, 1980), and this could also be in the basis of our observed results.
In the present study, intense nuclear immunohistochemical staining for AR was observed in prostate tissues from both control animals and those with hormonally induced prostatic hyperplasia, with no significant differences observed among specimens obtained during the course of BPH in terms of positive epithelial and stromal cell proportions or staining intensities. When Trachtenberg et al (1980) analyzed cytosol and nuclear prostatic AR contents in experimentally induced canine BPH, results showed that nuclear, but not cytosolic, prostatic AR levels were significantly higher compared with levels in the prostates of age-matched control dogs. Moreover, prostatic cytosolic and nuclear AR contents remained low in untreated castrated dogs. In our study, withdrawal of hormone treatment led to a significant reduction in epithelial and stromal AR signaling, similar to that observed as a result of castration or treatment with a gonadotropin-releasing hormone agonist (Forti et al, 1989), or antiandrogenic agents such as cyproterone acetate (Huang et al, 1985) or chlormadinone acetate (Murakoshi et al, 2000b).
Although prostate tissue is androgen dependent, estrogens influence both
normal functions and pathological changes in this gland. In the present study,
immunolabeling for ER was detected in the nuclei of both glandular
epithelial and stromal cells, as reported in a previous study
(Gallardo et al, 2007). Schulze
and Barrack (1987) reported ER
labeling confined to the nuclei of the canine prostatic stroma and the
prostatic duct epithelium, but, contrary to our findings, they detected no
specific labeling in the acinar epithelium. Similarly, other authors have
reported no ER reaction in epithelial cells, and the presence of
ER was confined to connective tissue nuclei in both healthy (human,
Leav et al, 2001a;
Sar and Welsch, 2000) and
hyperplastic (Hiramatsu et al,
1996; Leav et al,
2001a) prostate tissue. Pelletier et al
(2000) were unable to detect
ER expression in normal rat prostate tissue by immunohistochemistry or
in situ hybridization. These differences in the location of ER with
respect to the observations made in our study could be related to limited
specificity of the antibody used, ineffective methods of antigenic retrieval,
differences in sample processing, or differences in the sensitivity of the
technique employed (Pasquali et al,
2001).
During the hormonal induction of BPH, we observed uniformly intense
immunostaining of epithelial cells for ER, and the proportions of
stained cells decreased significantly after hormone withdrawal. This behavior
was also observed for AR and is probably responsible for the involution of the
gland. In contrast, positive stromal cell staining percentages and mean
intensity scores increased significantly 6 weeks after the end of hormone
treatment when compared with the tissue samples obtained just before the
hormone withdrawal. Similar behavior has been described by Smith et al
(2002), who observed that
ER were up-regulated in cultured prostatic stromal cells after
culturing them in a high concentration of estradiol followed by passage and
growth in the absence of sex hormones. However, another possible explanation
for the increased ER expression detected in stromal cells at stage M6
could be related to the intense inflammatory process associated with several
consecutive surgical procedures (Stygar et
al, 2006). Using a polyclonal antibody that identifies amino acids
1–150 of ERβ, here we immunolocalized the ERβ in epithelial
cell nuclei but not in stromal cells. There are no data for comparison in the
literature because, to our knowledge, our group is the first to address
ERβ expression by immunohistochemistry in the healthy and hyperplastic
canine prostate glands (Gallardo et al,
2007). Studies performed in different species have identified
ERβ in the epithelial cell nuclei of healthy rat prostates
(Pelletier et al, 2000;
Sar and Welsch, 2000) as well
as in epithelial and, to a lesser degree, stromal cell nuclei in human healthy
and hyperplastic prostate tissue (Pasquali
et al, 2001; Fixemer et al,
2003). Intense nuclear staining of ERβ was observed during
the hormonal induction and regression of BPH, and no significant differences
were observed in percentages of positive epithelial and basal cells or in
staining intensities. Moreover, no significant differences were identified
with respect to the normal prostates of age-matched dogs.
The epithelium of the prostate is maintained by 2 functional compartments. The secretory epithelium constitutes the differentiation compartment, which is androgen dependent but has a limited proliferative capacity. In contrast, the basal cell layer consists of generally undifferentiated and androgen-independent basal cells that proliferate under estrogen stimulation and show a low apoptotic index (Bonkhoff et al, 1994; Bonkhoff and Remberger, 1996). These basal cells may express nuclear estrogen and progesterone receptors (Wernert et al, 1988; Isaacs and Coffey, 1989). Following hormone inhibition or castration, studies in animal models have suggested that basal cells are resistant to castration-induced apoptosis and are still able to proliferate following androgen repletion (Evans and Chandler, 1987). In the present study, we noted a persistently high percentage of ERβ-positive cells after the withdrawal of hormone treatment, contrary to what was observed for the other receptors. Hence, we could speculate that ERβ labeling might be attributed to the presence of basal cells that are still active despite the withdrawal of hormone treatment. This hypothesis, however, requires experimental confirmation.
In summary, the canine prostate tissue examined in this experimental model
showed uniformly intense nuclear and cytoplasmic staining for AR and ER
in glandular epithelial cells. Both receptors were also localized in the
nuclei of fibromuscular stromal cells. Although AR and ER levels
remained elevated in both control and hormone-treated dogs, cell positivity
decreased after treatment. ERβ were localized only in epithelial cell
nuclei and, regardless of the hormone treatment, high positivity percentages
were observed; these percentages were similar to those recorded in age-matched
dogs. These observations indicate that AR, ER, and ERβ are
differentially expressed in prostate tissue and that they respond differently
during the hormonal induction of BPH. The experimental model used here is a
potentially valuable tool for investigating the respective roles of epithelial
and stromal hormone receptors and for its applicability in the study of the
genesis of human BPH. These hormone receptors are critical for understanding
the mechanisms through which hormones regulate prostatic epithelial growth,
differentiation, and function, and in turn for identifying the paracrine
mediators involved in these regulatory cell-to-cell interactions.
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Footnotes |
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