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Journal of Histochemistry & Cytochemistry Effects of Estradiol on Prostate Epithelial Cells in the Castrated Rat
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Journal of Histochemistry & Cytochemistry
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Volume 50(11): 1517–1523, 2002
The Journal of Histochemistry & Cytochemistry
Effects of Estradiol on Prostate Epithelial Cells
in the Castrated Rat

Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL), and Laval University, Québec, Canada There is evidence that estrogens can modulate the activity of prostate epithe- lial cells. To determine whether estradiol can have a direct influence on rat prostate, thisstudy examined the effects of estradiol-17␤ (E2) administered alone or in combination withdihydrotestosterone (DHT) to castrated rats for 3 weeks on prostate binding protein (PBP)C1 mRNA expression and androgen receptor (AR) localization. PBP C1 mRNA levels weremeasured by semi-quantitative in situ hybridization using a 35S-labeled cDNA probe. In in-tact animals, strong hybridization signal could be observed in prostate sections after 12 hrof exposure to Kodak X-Omat films. In castrated rats, no PBP C1 mRNA could be detectedeven with longer exposure times, an effect that was prevented by administration of DHT.
E2 administered alone induced a detectable hybridization signal, and the concomitant ad-ministration of E2 and DHT induced an increase in PBP C1 mRNA that significantly exceededthat obtained in animals that received only DHT. In prostate epithelial cells of intact ani-mals, AR immunostaining was restricted to the nucleus. In castrated animals the alveoliwere decreased in size and the epithelial cells were atrophied. AR staining was weak andwas detected in both cytoplasm and nucleus. DHT administration completely obviated theeffect of castration on epithelial cell histology and on AR immunostaining distribution andintensity. Interestingly, E K E Y W O R D S
2 administration alone induced moderate hypertrophy of epithe- lial cells compared to the histological appearance of cells in untreated castrated rats. More- 2-treated animals the nuclear staining was much stronger than that detected in untreated castrated rats, whereas the cytoplasmic staining was not modified by the treat- ment. In animals that received both DHT and E 2, the staining was similar to that seen in DHT-treated rats. These results suggest that E 2 can influence the activity of rat prostate ep- ithelial cells by mechanisms that remain to be fully clarified.
(J Histochem Cytochem 50:1517–1523, 2002)
The prostate is a highly androgen-dependent tissue
ventral prostate (Heyns and De Moor 1977). This (Roy and Chatterjee 1995). In the human and rat protein consists of two subunits, each containing prostate, androgen receptors (ARs) have been local- polypeptide C1 and either polypeptide C2 or C3, all ized to epithelial secretory cells, stromal cells, and en- of which are under androgenic control (Parker et al.
dothelial cells in capillaries and large blood vessels 1980; Page and Parker 1982). To measure androgen- (Iwamura et al. 1994; El-Alfy et al. 1999; Pelletier dependent prostate activity, the expression of PBP C1 2000; Pelletier et al. 2000). Prostate steroid-binding mRNA in epithelial cells is a very suitable parameter protein (PBP) is the major secretory protein of the rat During the past few years accumulating evidence suggests that estrogens can play a physiological role in Correspondence to: Dr. Georges Pelletier, Oncology and Molec- male reproduction (Sharpe 1998; Simpson et al.
ular Endocrinology Research Center, Laval University Hospital 2000). It is well documented that prostate tissue from (CHUL), 2705 Laurier Boulevard, Quebec, PQ G1V 4G2, Canada.
several species contains estrogen receptors (ERs) E-mail: georges.pelletier@crchul.ulaval.ca (Tilley et al. 1980,1985; Swaneck et al. 1982; West et Received for publication February 27, 2002; accepted June 5, al. 1988). Recently a second ER, called ER␤, has been cloned from a rat prostate library (Kuiper et al. 1996), experiment was duplicated and essentially the same results and the original one is now designated as ER␣. ER␤ expressed at high levels in rodent and primate prostate(Kuiper et al. 1996,1997; Couse et al. 1997; Pelletier 2000; Pelletier and El-Alfy 2000; Pelletier et al. 2000).
The plasmid containing cDNA, corresponding to the C1 Low levels of ER␣ mRNA have also been reported in peptide of PBP cloned in the Pst-1 site of pS64, was provided rat prostate (Kuiper et al. 1997; Lou et al. 1998). In by Dr. M.G. Parker (London, UK). The Pst-1 restriction rat prostate, ER␤ was localized by in situ hybridiza- fragment was radiolabeled with [ S]-CTP (NEN Life Sci- tion and immunocytochemistry in epithelial cells in al- ence Products; Boston, MA), as previously described (Pelle-tier et al. 1988). As control, sections were treated with veoli (Prins and Birch 1997; Pelletier 2000; Pelletier et pancreatic RNase A (20 ␮g/ml; Boehringer Mannheim; al. 2000), whereas no ER␣ expression could be de- Mannheim, Germany) for 1 hr at 37C before hybridization.
There is some evidence that estrogen itself can exert an influence on prostate epithelial cell division and Frozen sections (10 ␮m) were serially cut at –20C and differentiation. Estrogen administration to castrated mounted on gelatin- and poly-l-lysine-coated slides. In situ or hypophysectomized dogs induced glandular hyper- hybridization was performed as previously described (Pelle- plasia (Leav et al. 1978; Tunn et al. 1979). In the No- tier et al. 1988). After hybridization, the sections were ex- ble rat, estrogen synergizes with androgens to induce posed to Kodak X-Omat films (Eastman Kodak; Rochester, glandular hyperplasia and dysplasia (Leav et al. 1989; NY) for 12 hr. For each experimental group, densitometric Lou et al. 1998). On the other hand, ER␤ knockout measurements of autoradiographs of whole sections (six sec- mice display local prostate hyperplasia with aging tions/rat) were obtained using an optical system coupled to a (Krege et al. 1998), suggesting that ER␤ might exert a Macintosh computer and image software (version 1.6.0 non- negative regulation on prostate growth.
FPU; W. Rasband, NIH, Bethesda, MD). The optical density To study the involvement of estrogens in prostate (OD) of the signal was measured under illumination. TheOD of each tissue was corrected for the average back- epithelial cell functions, we evaluated the effects of E2 ground signal. Comparisons of the OD between experimen- administered alone or in combination with DHT to tal groups was performed by an analysis of variance (Stat- castrated adult male rats on the expression of PBP C1 mRNA, as evaluated by in situ hybridization and theimmunohistochemical localization of AR.
AR immunostaining was performed on paraffin sections Materials and Methods
(two sections/glass slide) as previously described (Pelletier2000; Pelletier et al. 2000). An affinity-purified rabbit poly- clonal antibody reacting with rat AR (N-20; Santa Cruz Bio- Thirty adult male Sprague–Dawley rats (Charles River; technology, Santa Cruz, CA) was used at a concentration of Wilmington, MA) weighing 225–250 g at the beginning of 1 ␮g/ml. This antibody has been successfully used to localize the experiments were housed under constant temperature AR in several tissues, including the prostate (Pelletier and El- (21 Ϯ 1C) and lighting (light on from 0600 to 2000) regi- Alfy 2000; Pelletier et al. 2000). Control reaction was ob- mens. They had free access to standard rat chow and tapwa- tained by substituting preabsorbed antibody with an excess ter. All the protocols were approved by the Laval Univer- of the peptide used as an antigen (20 ␮g/ml). After immuno- staining the sections were counterstained with hematoxylin.
To avoid any variations related to the staining procedure, two sections from each of the 30 prostates (six prostates/ Four groups of animals (six per group) were castrated via group) were stained in the same run. In each run, immuno- the scrotal route. One group of sham-operated rats was used absorption controls (one for each experimental group) were as intact control. The castrated animals were treated twice also included. We thus proceeded to four runs. The results were evaluated by two independent investigators who were roids were administered SC in 0.5 ml 1% (w/v) gelatin. Theintact animals received only the vehicle (1% gelatin). Thesteroids 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) paraformalde- As shown in Figure 1A, in vehicle-treated, sham-oper- hyde in 0.1 M phosphate buffer (pH 7.4). Ventral prostates were excised and postfixed in the same fixative for 48 hr at ated animals, hybridization with the S-labeled PBP 4C. For immunocytochemistry, the tissues were embedded C1 cDNA probe induced a strong signal in prostate in paraffin. For in situ hybridization, the tissues were placed sections after 12 hr of exposure. Pretreatment of the in 15% sucrose in 0.1 M phosphate buffer before being sections with RNase before hybridization completely quickly frozen in isopentane chilled in liquid nitrogen. This prevented any labeling (not shown). In castrated ani- Effects of E2 on Prostate Epithelial Cells X-ray autoradiographs illustrating the expression of PBP C1 mRNA in rat prostate sections (exposure time 12 hr). (A) Vehicle-
treated intact (INT) rats. (B) DHT-treated castrated (CX) rats. (C) E2-treated castrated rats. (D) Castrated rats treated with both DHT and E2.
No detectable reaction could be obtained in sections from vehicle-treated castrated rats. Bars ϭ 4 ␮m.
mals no detectable signal could be obtained even after intact animals (Figures 1C and 2). Figures 1D and 2 longer exposure times (up to 7 days; not shown). As show the effect of the administration of both DHT shown in Figures 1B and 2, administration of DHT to and E2 on the mRNA levels, which exceeded by 16% castrated rats completely obviated the effects of cas- (pϽ0.001) those observed in DHT-treated animals.
tration, the mRNA levels being 18% above the levelsmeasured in vehicle-treated, sham-operated animals (pϽ0.001). Administration of E2 induced a hybridiza- In prostate sections from vehicle-treated, sham-oper- tion signal that could be detected after 12 hr of expo- ated animals immunostained for AR localization, sure, the measured PBP C1 mRNA levels correspond- strong labeling was detected in nuclei of all secretory ing to approximately 3% of the values obtained in epithelial cells. The cytoplasm of the epithelial cells those observed in animals treated only with DHT (Fig-ure 3E).
Discussion
The present results clearly demonstrate that 3-week
administration of E2 can stimulate the mRNA expres-
sion 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 epi-
thelial cells in the absence or presence of circulating
Effects of castration and DHT and E2 administration to castrated rats on PBP C1 mRNA levels as evaluated by semi-quanti- The hypertrophy of epithelial cells after administra- tative in situ hybridization. INT, intact animals; C, control (vehicle- treated). Results are expressed as a percentage of the control value 2 suggests that estrogens can directly stimu- (vehicle-treated intact animals). ***p Ͻ 0.001 DHT-treated cas- late the activity of secretory epithelial cells. These re- trated rats vs all the other experimental groups; ND, non-detect- sults are in agreement with several reports indicating that administration of estrogen to castrated or hypo-physectomized animals could exert a stimulatory in-fluence on prostate epithelial cells. In castrated or hy-pophysectomized dogs, estrogen induced hypertrophyof epithelial cells (Leav et al. 1978; Tunn et al. 1979; did not exhibit any labeling (Figure 3A). Other immu- Merk et al. 1980,1986; Kwan et al. 1982). Similarly, nostained nuclei in the stroma surrounding the alveoli in castrated rats, estrogens increase epithelial cell were also consistently observed. In castrated animals, height in the ventral prostate (Salander and Tisell as shown in Figure 3B, the alveoli were markedly re- 1976; Thompson et al. 1979; Timms and Chandler duced in size and appeared dispersed throughout the 1985). Moreover, it has been shown that, in the Noble stroma, which was not modified. The epithelial cells rat prostate, androgen-supported estrogen could be re- had a cuboidal appearance, with markedly reduced sponsible for epithelial proliferation and dysplasia cytoplasm and an increased nuclear-to-cytoplasmic ra- (Leav et al. 1989; Lou et al. 1998). Our data also tio. In contrast to the observations in sham-operated clearly show that, in DHT-treated animals, E2 could rats, immunostaining was present in both cytoplasm further increase PBP C1 mRNA expression. It thus ap- and nuclei, with a marked reduction in nuclear label- pears reasonable to hypothesize that estrogens can po- ing. The staining of stromal cells did not appear to tentiate the effects of androgens on prostate epithelial have been modified by castration. As shown in Figure cell activity. Recently, Yeh et al. (1998) have reported 3C, treatment with DHT completely reversed the ef- that E2 can activate androgen target genes in the pros- fect of castration on the epithelial cells. The histology tate via an interaction with the AR complex.
and AR localization were very similar to what has AR immunolocalization showed that, in intact ani- been observed in vehicle treated, sham-operated ani- mals, the staining was restricted to nuclei in epithelial mals, with nuclei being strongly immunoreactive. In cells. After castration there was a marked reduction in castrated animals treated with E2, the size of the alve- nuclear labeling and, contrary to what was observed oli was not modified, but the epithelial cells appeared in sham-operated rats, cytoplasmic labeling was con- hypertrophied compared to those observed in vehicle- sistently found. These results are in agreement with treated castrated rats (compare Figures 3D and Figure previous findings indicating a decrease in nuclear an- 3B). The nucleus:cytoplasmic ratio appeared to be de- drogen-binding sites in ventral prostate in 2-week cas- creased by E2 treatment. Moreover, in the E2-treated trated rats (Prins 1989). The presence of immunoreac- animals the intensity of the reaction was very different tive material in the cytoplasm of epithelial cells may from that observed in vehicle-treated castrated rats.
therefore reflect retention of AR in the cytoplasmic The nuclei were strongly labeled and light cytoplasmic compartment. It is well known that binding of a ste- staining was present (Figure 3D). In the stroma, AR roid (including androgens) to its receptor molecule re- labeling was also stronger. In animals that received sults in activation of the receptor in the cytoplasmic both DHT and E2, the results were very similar to compartment (Clark et al. 1992). The activated recep- Effects of E2 on Prostate Epithelial Cells Immunocytochemical localization of AR in rat prostate sections. (A) Vehicle-treated intact rats. Nuclear staining is ob-
served in all epithelial cells (E). (B) Vehicle-treated castrated rats.
The epithelial cells are atrophied. Both nucleus and cytoplasm are
weakly labeled. (C) DHT-treated castrated rats. The treatment has
completely obviated the effect of castration (compare to A). (D) E2-
treated castrated rats. The cytoplasm of epithelial cells (E) is larger
than that observed in untreated castrated rats. The nuclear stain-
ing is much stronger compared to that seen in untreated castrated
rats (B). (E) Castrated rats treated with both DHT and E2. Staining is
similar to that observed in DHT-treated rats (C). Bars ϭ 25 ␮m.
tor–steroid complex, which has a high affinity for var- mechanism(s) of action of E2, which might involve in- ious nuclear binding sites, then migrates to the nu- teraction with ER␤ and/or AR, remains to be fully cleus. In castrated rats that were treated with DHT, clarified. Other studies involving use of anti-estrogens no cytoplasmic staining could be detected, whereas and anti-androgens would help to clarify the exact strong nuclear staining similar to that observed in role of estrogens in prostate regulation.
sham-operated animals was present. The histology ofthe epithelial cells was also very similar to that ob-served in intact animals. This appears to be a morpho- Literature Cited
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Other mechanisms, such as an in- nase, 3␤-hydroxysteroid dehydrogenase and androgen receptor crease in AR biosynthesis, might be involved in the in- in the human prostate by in situ hybridization and immunocy- crease in nuclear staining. We have recently observed tochemistry. Endocrinology 140:1481–1491 that administration of estrogens to castrated rats in- Heyns W, De Moor P (1977) Prostatic binding protein: a steroid- binding protein secreted by rat prostate. Eur J Biochem 89:221– duced a marked increase in prostate AR mRNA levels (unpublished). Using subcellular fractionation, Blondeau Iwamura M, Abrahamsson P-A, Benning CM, Cockett ATK, Di et al. (1982) have reported that, in castrated rat pros- Santagnese PA (1994) Androgen receptor immunostaining andits tissue distribution in formalin-fixed, paraffin-embedded sec- tate, AR could be found in the cytosolic fraction and tions after microwave treatment. 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