Journal of Histochemistry and Cytochemistry, Vol. 50, 1517-1524, November 2002, Copyright © 2002, The Histochemical Society, Inc.


ARTICLE

Effects of Estradiol on Prostate Epithelial Cells in the Castrated Rat

G. Pelletiera
a Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL), and Laval University, Québec, Canada

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


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 50:1517–1523, 2002)

Key Words: prostrate, estrogens, androgen receptor, prostate steroid-binding, protein, in situ hybridization, immunocytochemistry


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

THE PROSTATE is a highly androgen-dependent tissue (Roy and Chatterjee 1995 ). In the human and rat prostate, androgen receptors (ARs) have been localized to epithelial secretory cells, stromal cells, and endothelial cells in capillaries and large blood vessels (Iwamura et al. 1994 ; El-Alfy et al. 1999 ; Pelletier 2000 ; Pelletier et al. 2000 ). Prostate steroid-binding protein (PBP) is the major secretory protein of the rat ventral prostate (Heyns and De Moor 1977 ). This protein consists of two subunits, each containing polypeptide C1 and either polypeptide C2 or C3, all of which are under androgenic control (Parker et al. 1980 ; Page and Parker 1982 ). To measure androgen-dependent prostate activity, the expression of PBP C1 mRNA in epithelial cells is a very suitable parameter (Pelletier et al. 1988 ).

During the past few years accumulating evidence suggests that estrogens can play a physiological role in male reproduction (Sharpe 1998 ; Simpson et al. 2000 ). It is well documented that prostate tissue from several species contains estrogen receptors (ERs) (Tilley et al. 1980 , Tilley et al. 1985 ; Swaneck et al. 1982 ; West et al. 1988 ). Recently a second ER, called ERß, has been cloned from a rat prostate library (Kuiper et al. 1996 ), and the original one is now designated as ER{alpha}. ERß expressed at high levels in rodent and primate prostate (Kuiper et al. 1996 , Kuiper et al. 1997 ; Couse et al. 1997 ; Pelletier 2000 ; Pelletier and El-Alfy 2000 ; Pelletier et al. 2000 ). Low levels of ER{alpha} mRNA have also been reported in rat prostate (Kuiper et al. 1997 ; Lou et al. 1998 ). In rat prostate, ERß was localized by in situ hybridization and immunocytochemistry in epithelial cells in alveoli (Prins and Birch 1997 ; Pelletier 2000 ; Pelletier et al. 2000 ), whereas no ER{alpha} 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 (Leav et al. 1978 ; Tunn et al. 1979 ). In the Noble rat, estrogen synergizes with androgens to induce glandular hyperplasia and dysplasia (Leav et al. 1989 ; Lou et al. 1998 ). On the other hand, ERß knockout mice display local prostate hyperplasia with aging (Krege et al. 1998 ), suggesting that ERß might exert a negative regulation on prostate growth.

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.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Animals
Thirty adult male Sprague–Dawley rats (Charles River; Wilmington, MA) weighing 225–250 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 (Pelletier et al. 1988 ). As control, sections were treated with pancreatic RNase A (20 µg/ml; Boehringer Mannheim; Mannheim, Germany) for 1 hr at 37C before hybridization.

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 (Pelletier et al. 1988 ). After hybridization, the sections were exposed to Kodak X-Omat films (Eastman Kodak; Rochester, NY) for 12 hr. For each experimental group, densitometric measurements of autoradiographs of whole sections (six sections/rat) were obtained using an optical system coupled to a Macintosh computer and image software (version 1.6.0 non-FPU; W. Rasband, NIH, Bethesda, MD). The optical density (OD) of the signal was measured under illumination. The OD of each tissue was corrected for the average background signal. Comparisons of the OD between experimental groups was performed by an analysis of variance (Statview ANOVA).

Immunocytochemistry
AR immunostaining was performed on paraffin sections (two sections/glass slide) as previously described (Pelletier 2000 ; Pelletier et al. 2000 ). An affinity-purified rabbit polyclonal antibody reacting with rat AR (N-20; Santa Cruz Biotechnology, Santa Cruz, CA) was used at a concentration of 1 µg/ml. This antibody has been successfully used to localize AR in several tissues, including the prostate (Pelletier and El-Alfy 2000 ; Pelletier et al. 2000 ). Control reaction was obtained by substituting preabsorbed antibody with an excess of the peptide used as an antigen (20 µg/ml). After immunostaining 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/group) were stained in the same run. In each run, immunoabsorption controls (one for each experimental group) were also included. We thus proceeded to four runs. The results were evaluated by two independent investigators who were unaware of the treatments.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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.



View larger version (130K):
[in this window]
[in a new window]
 
Figure 1. 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 = 4 µm.



View larger version (27K):
[in this window]
[in a new window]
 
Figure 2. Effects of castration and DHT and E2 administration to castrated rats on PBP C1 mRNA levels as evaluated by semi-quantitative in situ hybridization. INT, intact animals; C, control (vehicle-treated). Results are expressed as a percentage of the control value (vehicle-treated intact animals). ***p < 0.001 DHT-treated castrated rats vs all the other experimental groups; ND, non-detectable.

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).



View larger version (115K):
[in this window]
[in a new window]
 
Figure 3. Immunocytochemical localization of AR in rat prostate sections. (A) Vehicle-treated intact rats. Nuclear staining is observed 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 staining 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 = 25 µm.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Leav et al. 1978 ; Tunn et al. 1979 ; Merk et al. 1980 , Merk et al. 1986 ; Kwan et al. 1982 ). Similarly, in castrated rats, estrogens increase epithelial cell height in the ventral prostate (Salander and Tisell 1976 ; Thompson et al. 1979 ; Timms and Chandler 1985 ). Moreover, it has been shown that, in the Noble rat prostate, androgen-supported estrogen could be responsible for epithelial proliferation and dysplasia (Leav et al. 1989 ; Lou et al. 1998 ). Our data also clearly show that, in DHT-treated animals, E2 could further increase PBP C1 mRNA expression. It thus appears reasonable to hypothesize that estrogens can potentiate the effects of androgens on prostate epithelial cell activity. Recently, Yeh et al. 1998 have reported that E2 can activate androgen target genes in the prostate via an interaction with the AR complex.

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 (Prins 1989 ). The presence of immunoreactive material in the cytoplasm of epithelial cells may therefore reflect retention of AR in the cytoplasmic compartment. It is well known that binding of a steroid (including androgens) to its receptor molecule results in activation of the receptor in the cytoplasmic compartment (Clark et al. 1992 ). The activated receptor–steroid complex, which has a high affinity for various nuclear binding sites, then migrates to the nucleus. In castrated rats that were treated with DHT, no cytoplasmic staining could be detected, whereas strong nuclear staining similar to that observed in sham-operated animals was present. The histology of the epithelial cells was also very similar to that observed in intact animals. This appears to be a morphological confirmation that the activation of AR by circulating androgens leads to a translocation of the receptor to the nucleus.

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, Blondeau et al. 1982 have reported that, in castrated rat prostate, AR could be found in the cytosolic fraction and that the injection of DHT or E2 4 hr before sacrifice could increase nuclear AR concentrations. On the other hand, it cannot be totally excluded that cytoplasmic staining might result from artifactual redistribution or unbound AR, which might occur during the immunocytochemical procedure.

We have previously reported that administration of E2 during one week to castrated rats did not induce significant changes in PBP C1 mRNA levels (Pelletier et al. 1988 ). It therefore appears that longer exposure to E2 is required to positively modulate PBP C1 mRNA in castrated animals. Because androgens can rapidly (within 12 hr) stimulate PBP C1 mRNA in castrated rats, it is unlikely that the effect of E2 might be related to an activation of AR although, as mentioned above, interaction of E2 with AR might explain the changes in AR staining distribution. We have recently observed that chronic (3-week) administration of E2 to castrated rats could decrease ERß expression in the prostate (unpublished data). Because, in ERß knockout mouse, hyperplasia of prostate epithelium occurs with aging, it has been proposed that ERß might negatively regulate epithelial cell activity in the prostate (Weihua et al. 2001 ). Such an effect on ERß might explain the stimulatory influence of E2 that occurs after 3 weeks of administration.

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.
  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Blondeau JP, Baulieu EE, Robel P (1982) Androgen-dependent regulation of androgen nuclear receptor in the rat ventral prostate. Endocrinology 110:1926-1932[Abstract]

Clark JH, Schrader WT, O'Malley BW (1992) Mechanisms of action of steroid hormones. In Wilson J, ed. Textbook of Endocrinology. Philadelphia, WB Saunders, 35-90

Couse JF, Lindzey JKG, Gustafsson JA, Korach KS (1997) Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology 138:4613-4621[Abstract/Free Full Text]

El-Alfy M, Luu–The V, Huang XF, Berger L, Labrie F, Pelletier G (1999) Localization of type 5 17ß-hydroxysteroid dehydrogenase, 3ß-hydroxysteroid dehydrogenase and androgen receptor in the human prostate by in situ hybridization and immunocytochemistry. Endocrinology 140:1481-1491[Abstract/Free Full Text]

Heyns W, De Moor P (1977) Prostatic binding protein: a steroid-binding protein secreted by rat prostate. Eur J Biochem 89:221-230[Abstract]

Iwamura M, Abrahamsson P-A, Benning CM, Cockett ATK, Di Santagnese PA (1994) Androgen receptor immunostaining and its tissue distribution in formalin-fixed, paraffin-embedded sections after microwave treatment. J Histochem Cytochem 42:783-788[Abstract/Free Full Text]

Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M et al. (1998) Generation and reproductive phenotypes of mice lacking estrogen receptor ß. Proc Natl Acad Sci USA 95:15677-15682[Abstract/Free Full Text]

Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA (1997) Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors a and b. Endocrinology 138:863-870[Abstract/Free Full Text]

Kuiper GGJM, Enmark E, Pelto-Huiko M, Nilsson S, Gustafsson JA (1996) Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:2925-2930

Kwan PW, Merk FB, Leav I, Ofner P (1982) Estrogen-mediated exocytosis in the glandular epithelium of prostates in castrated and hypophysectomized dogs. Cell Tissue Res 226:689-693[Medline]

Leav I, Merk FB, Kwan PW, Ho SM (1989) Androgen-supported estrogen enhanced epithelial proliferation in the prostates of intact Noble rats. Prostate 15:23-40[Medline]

Leav I, Merk FB, Ofner P, Goodrich G, Kwan PW, Stein BM, Sar M et al. (1978) Bipotentiality of response to sex hormones by the prostate of castrated or hypophysectomized dogs. Direct effects of estrogen. Am J Pathol 93:69-92[Abstract]

Lou KM, Leav I, Ho SM (1998) Rat estrogen receptor-apha and beta and progesterone receptor mRNA expression in various prostatic lobes and microdissected normal and dysplastic epithelial tissues of the Noble rats. Endocrinology 139:404-427

Merk FB, Leav I, Kwan PW, Ofner P (1980) Effects of estrogen and androgen on the ultrastructure of secretory granules and intercellular junctions in regressed canine prostate. Anat Rec 197:111-132[Medline]

Merk FB, Walrhol MJ, Kevan PW, Leav I, Alrag J, Ofna P, Pinkus G (1986) Multiple phenotypes of prostatic glandular cells in castrated dogs after individual or combined treatment with androgen and estrogen. Lab Invest 54:442-453[Medline]

Page MJ, Parker JG (1982) Effect of androgen on the transcription of rat prostatic binding protein genes. Mol Cell Endocrinol 27:343-355[Medline]

Parker MG, White R, Williams JG (1980) Cloning and characterization of androgen-dependent mRNA from rat ventral prostate. J Biol Chem 255:6996-7001[Abstract/Free Full Text]

Pelletier G (2000) Localization of androgen and estrogen receptors in rat and primate tissues. Histol Histopathol 15:1261-1270[Medline]

Pelletier G, El-Alfy M (2000) Immunocytochemical localization of estrogen receptors alpha and beta in the human reproductive organs. J Clin Endocrinol Metab 85:4835-4840[Abstract/Free Full Text]

Pelletier G, Labrie C, Labrie F (2000) Localization of oestrogen receptor a, oestrogen receptor b and androgen receptors in the rat reproductive organs. J Endocrinol 165:359-370[Abstract/Free Full Text]

Pelletier G, Labrie C, Simard J, Duval M, Martinoli MG, Zhao HF, Labrie F (1988) Effects of sex steroids on the regulation of C1 peptide of rat prostatic steroid binding protein mRNA levels evaluated by in situ hybridization. J Mol Endocrinol 1:213-223[Abstract]

Prins GS (1989) Differential regulation of androgen receptors in the separate rat prostate lobes: androgen independent expression in the lateral lobe. J Steroid Biochem 33:319-326[Medline]

Prins GS, Birch L (1997) Neonatal estrogen exposure up-regulates estrogen receptor expression in the developing and adult rat prostate lobes. Endocrinology 138:1801-1809[Abstract/Free Full Text]

Roy AK, Chatterjee B (1995) Androgen action. Crit Rev Eukaryot Gene Expr 5:157-176[Medline]

Salander H, Tisell LE (1976) Effects of megestrol on oestradiol induced growth of the prostatic lobes and the seminal vesicles in castrated rats. Acta Endocrinol (Copenh) 82:213-224[Medline]

Sharpe RM (1998) The roles of oestrogen in the male. Trends Endocrinol Metab 9:371-378

Simpson E, Rubin G, Clyne C, Robertson K, O'Donnell L, Jones M, Davis S (2000) The role of local estrogen biosynthesis in males and females. Trends Endocrinol Metab 11:184-188[Medline]

Swaneck GE, Alvarez JM, Sufrin G (1982) Multiple species of estrogen binding sites in the nuclear fraction of the rat prostate. Biochem Biophys Res Commun 106:1441-1447[Medline]

Thompson SA, Rowley DR, Heidger PM (1979) Effects of estrogen upon the fine structure of epithelium and stroma in the rat ventral prostate gland. Invest Urol 17:83-89[Medline]

Tilley WD, Horsfall DJ, McGee MA, Henderson DW, Marshall VR (1985) Distribution of oestrogen and androgen receptors between the stroma and epithelium of the guinea-pig prostate. J Steroid Biochem 22:713-719[Medline]

Tilley WD, Keightley DD, Marshall VR (1980) Oestrogen and progesterone receptors in benign prostatic hyperplasia in humans. J Steroid Biochem 13:395-399[Medline]

Timms BG, Chandler JA (1985) The effects of estradiol-17 beta on the ultrastructure and subcellular distribution of zinc in the prostatic epithelium of castrated rats. Prostate 6:61-79[Medline]

Tunn U, Senge T, Schenck B, Neumann F (1979) Biochemical and histological studies on prostates in castrated dogs after treatment with androstanediol, oestradiol and cyproterone acetate. Acta Endocrinol (Copenh) 91:373-384[Medline]

Weihua Z, Makela S, Andersson LC, Salmi S, Saji S, Webster JI, Jensen EV et al. (2001) A role for estrogen receptor beta in the regulation of growth of the ventral prostate. Proc Natl Acad Sci USA 98:6330-6335[Abstract/Free Full Text]

West NB, Roselli CE, Resko JA, Greene GL, Brenner RM (1988) Estrogen and progestin receptors and aromatase activity in Rhesus monkey prostate. Endocrinology 88:2312-2322

Yeh S, Miyamoto H, Shima H, Chang C (1998) From estrogen to androgen receptor: a new pathway for sex hormones in prostate. Proc Natl Acad Sci USA 95:5527-5532[Abstract/Free Full Text]