ARTICLE |
Correspondence to: G. Pelletier, Oncology and Molecular Endocrinology Research Center, Laval University Hospital (CHUL), 2705 Laurier Boulevard, Quebec, PQ G1V 4G2, Canada. E-mail: georges.pelletier@crchul.ulaval.ca
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Summary |
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There is evidence that estrogens can modulate the activity of prostate epithelial cells. To determine whether estradiol can have a direct influence on rat prostate, this study examined the effects of estradiol-17ß (E2) administered alone or in combination with dihydrotestosterone (DHT) to castrated rats for 3 weeks on prostate binding protein (PBP) C1 mRNA expression and androgen receptor (AR) localization. PBP C1 mRNA levels were measured by semi-quantitative in situ hybridization using a 35S-labeled cDNA probe. In intact animals, strong hybridization signal could be observed in prostate sections after 12 hr of exposure to Kodak X-Omat films. In castrated rats, no PBP C1 mRNA could be detected even with longer exposure times, an effect that was prevented by administration of DHT. E2 administered alone induced a detectable hybridization signal, and the concomitant administration of E2 and DHT induced an increase in PBP C1 mRNA that significantly exceeded that obtained in animals that received only DHT. In prostate epithelial cells of intact animals, AR immunostaining was restricted to the nucleus. In castrated animals the alveoli were decreased in size and the epithelial cells were atrophied. AR staining was weak and was detected in both cytoplasm and nucleus. DHT administration completely obviated the effect of castration on epithelial cell histology and on AR immunostaining distribution and intensity. Interestingly, E2 administration alone induced moderate hypertrophy of epithelial cells compared to the histological appearance of cells in untreated castrated rats. Moreover, in E2-treated animals the nuclear staining was much stronger than that detected in untreated castrated rats, whereas the cytoplasmic staining was not modified by the treatment. In animals that received both DHT and E2, the staining was similar to that seen in DHT-treated rats. These results suggest that E2 can influence the activity of rat prostate epithelial cells by mechanisms that remain to be fully clarified.
(J Histochem Cytochem 50:15171523, 2002)
Key Words: prostrate, estrogens, androgen receptor, prostate steroid-binding, protein, in situ hybridization, immunocytochemistry
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Introduction |
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THE PROSTATE is a highly androgen-dependent tissue (
During the past few years accumulating evidence suggests that estrogens can play a physiological role in male reproduction (. ERß expressed at high levels in rodent and primate prostate (
mRNA have also been reported in rat prostate (
expression could be detected.
There is some evidence that estrogen itself can exert an influence on prostate epithelial cell division and differentiation. Estrogen administration to castrated or hypophysectomized dogs induced glandular hyperplasia (
To study the involvement of estrogens in prostate epithelial cell functions, we evaluated the effects of E2 administered alone or in combination with DHT to castrated adult male rats on the expression of PBP C1 mRNA, as evaluated by in situ hybridization and the immunohistochemical localization of AR.
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Materials and Methods |
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Animals
Thirty adult male SpragueDawley rats (Charles River; Wilmington, MA) weighing 225250 g at the beginning of the experiments were housed under constant temperature (21 ± 1C) and lighting (light on from 0600 to 2000) regimens. They had free access to standard rat chow and tapwater. All the protocols were approved by the Laval University's Animal Welfare Committee.
Treatments and Tissue Preparation
Four groups of animals (six per group) were castrated via the scrotal route. One group of sham-operated rats was used as intact control. The castrated animals were treated twice daily with the vehicle, E2 (0.4 µg/kg bw), DHT (400 µg/kg bw), or DHT in combination with E2 for 3 weeks. The steroids were administered SC in 0.5 ml 1% (w/v) gelatin. The intact animals received only the vehicle (1% gelatin). The steroids were purchased from Steraloids (Wilton, NH). On the morning after the last day of the treatment, animals were perfused transcardially with 200 ml 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Ventral prostates were excised and postfixed in the same fixative for 48 hr at 4C. For immunocytochemistry, the tissues were embedded in paraffin. For in situ hybridization, the tissues were placed in 15% sucrose in 0.1 M phosphate buffer before being quickly frozen in isopentane chilled in liquid nitrogen. This experiment was duplicated and essentially the same results were obtained.
PBP C1 Probe Preparation
The plasmid containing cDNA, corresponding to the C1 peptide of PBP cloned in the Pst-1 site of pS64, was provided by Dr. M.G. Parker (London, UK). The Pst-1 restriction fragment was radiolabeled with [35S]-CTP (NEN Life Science Products; Boston, MA), as previously described (
In Situ Hybridization
Frozen sections (10 µm) were serially cut at 20C and mounted on gelatin- and poly-L-lysine-coated slides. In situ hybridization was performed as previously described (
Immunocytochemistry
AR immunostaining was performed on paraffin sections (two sections/glass slide) as previously described (
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Results |
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PBP C1 mRNA Expression
As shown in Fig 1A, in vehicle-treated, sham-operated animals, hybridization with the 35S-labeled PBP C1 cDNA probe induced a strong signal in prostate sections after 12 hr of exposure. Pretreatment of the sections with RNase before hybridization completely prevented any labeling (not shown). In castrated animals no detectable signal could be obtained even after longer exposure times (up to 7 days; not shown). As shown in Fig 1B and Fig 2, administration of DHT to castrated rats completely obviated the effects of castration, the mRNA levels being 18% above the levels measured in vehicle-treated, sham-operated animals (p<0.001). Administration of E2 induced a hybridization signal that could be detected after 12 hr of exposure, the measured PBP C1 mRNA levels corresponding to approximately 3% of the values obtained in intact animals (Fig 1C and Fig 2). Fig 1D and Fig 2 show the effect of the administration of both DHT and E2 on the mRNA levels, which exceeded by 16% (p<0.001) those observed in DHT-treated animals.
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AR Immunostaining
In prostate sections from vehicle-treated, sham-operated animals immunostained for AR localization, strong labeling was detected in nuclei of all secretory epithelial cells. The cytoplasm of the epithelial cells did not exhibit any labeling (Fig 3A). Other immunostained nuclei in the stroma surrounding the alveoli were also consistently observed. In castrated animals, as shown in Fig 3B, the alveoli were markedly reduced in size and appeared dispersed throughout the stroma, which was not modified. The epithelial cells had a cuboidal appearance, with markedly reduced cytoplasm and an increased nuclear-to-cytoplasmic ratio. In contrast to the observations in sham-operated rats, immunostaining was present in both cytoplasm and nuclei, with a marked reduction in nuclear labeling. The staining of stromal cells did not appear to have been modified by castration. As shown in Fig 3C, treatment with DHT completely reversed the effect of castration on the epithelial cells. The histology and AR localization were very similar to what has been observed in vehicle treated, sham-operated animals, with nuclei being strongly immunoreactive. In castrated animals treated with E2, the size of the alveoli was not modified, but the epithelial cells appeared hypertrophied compared to those observed in vehicle-treated castrated rats (compare Fig 3D and Fig 3B). The nucleus:cytoplasmic ratio appeared to be decreased by E2 treatment. Moreover, in the E2-treated animals the intensity of the reaction was very different from that observed in vehicle-treated castrated rats. The nuclei were strongly labeled and light cytoplasmic staining was present (Fig 3D). In the stroma, AR labeling was also stronger. In animals that received both DHT and E2, the results were very similar to those observed in animals treated only with DHT (Fig 3E).
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Discussion |
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The present results clearly demonstrate that 3-week administration of E2 can stimulate the mRNA expression of a prostate androgen-dependent protein, PBP C1, in adult castrated rats. The effect of E2 was weak but significant because no hybridization signal could be observed in untreated castrated rats. Moreover, E2 was also effective in stimulating PBP C1 mRNA levels when the activity of epithelial cells was maintained by DHT administration. These data strongly suggest that E2 can directly stimulate the activity of prostate epithelial cells in the absence or presence of circulating androgens.
The hypertrophy of epithelial cells after administration of E2 suggests that estrogens can directly stimulate the activity of secretory epithelial cells. These results are in agreement with several reports indicating that administration of estrogen to castrated or hypophysectomized animals could exert a stimulatory influence on prostate epithelial cells. In castrated or hypophysectomized dogs, estrogen induced hypertrophy of epithelial cells (
AR immunolocalization showed that, in intact animals, the staining was restricted to nuclei in epithelial cells. After castration there was a marked reduction in nuclear labeling and, contrary to what was observed in sham-operated rats, cytoplasmic labeling was consistently found. These results are in agreement with previous findings indicating a decrease in nuclear androgen-binding sites in ventral prostate in 2-week castrated rats (
Of great interest was the finding that E2 administration induced a marked increase in nuclear AR labeling and a decrease in cytoplasmic AR staining. This suggests that estrogens, even in the absence of circulating androgens, can activate AR, leading to transfer of the receptors from the cytoplasm to the nucleus. Such an activation of AR might result from direct interaction of E2 with AR. Other mechanisms, such as an increase in AR biosynthesis, might be involved in the increase in nuclear staining. We have recently observed that administration of estrogens to castrated rats induced a marked increase in prostate AR mRNA levels (unpublished). Using subcellular fractionation,
We have previously reported that administration of E2 during one week to castrated rats did not induce significant changes in PBP C1 mRNA levels (
It clearly appears, on the basis of the present experiments, that E2 administration can stimulate the expression of an androgen-dependent protein and interact with AR in rat prostate epithelial cells. The mechanism(s) of action of E2, which might involve interaction with ERß and/or AR, remains to be fully clarified. Other studies involving use of anti-estrogens and anti-androgens would help to clarify the exact role of estrogens in prostate regulation.
Received for publication February 27, 2002; accepted June 5, 2002.
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