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Endocrinology Vol. 142, No. 9 4066-4075
Copyright © 2001 by The Endocrine Society

ARTICLES

Increased Androgen Receptor Expression Correlates with Development of Age-Dependent, Lobe-Specific Spontaneous Hyperplasia of the Brown Norway Rat Prostate

Partha P. Banerjee1, Subhadra Banerjee1 and Terry R. Brown

Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Dr. Terry R. Brown, Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens are essential for development and differentiated function, as well as proliferation and survival of cells within the prostate gland. Age-related changes in the hormonal milieu, marked by a decrease in the serum androgen to estrogen ratio may contribute to the evolution of pathological changes, such as benign prostatic hyperplasia and carcinoma of the prostate gland, in older men. A similar phenomenon occurs in Brown Norway rats, in which the serum testosterone to estradiol ratio declines with age, and despite the lower serum testosterone level, age-dependent prostatic hyperplasia develops in the dorsal and lateral lobes, but not in the ventral lobe. To evaluate a role for changes in androgen action in the evolution of prostatic hyperplasia, we compared the immunostaining intensity of androgen receptor in the different prostate lobes from young (4 months of age) and old (24 months of age) Brown Norway rats. Androgen receptor immunostaining was present in the nuclei of all epithelial cells and some stromal cells throughout the prostatic ducts of each lobe from both young and old rats. Whereas androgen receptor immunostaining intensity decreased in luminal epithelial cells of the ventral prostate from old rats, it increased in luminal epithelial cells of the dorsal and lateral lobes from old rats, when compared with young rats. To validate immunocytochemical studies, Western blot analyses were performed. The total tissue level of androgen receptor decreased by 30% in the ventral lobe of old rats, whereas tissue levels of androgen receptor increased 2.7-fold and 1.3-fold in the dorsal and lateral lobes, respectively, of old rats. Similarly, the percentage of epithelial cells staining positive for the proliferation marker, proliferating cell nuclear antigen, was increased approximately 2-fold in the dorsal and lateral lobes as a function of older age. The presence of higher levels of androgen receptor and increased number of proliferating cell nuclear antigen-positive cells in the dorsal and lateral lobes of old rats suggest that changes in androgen receptor levels may be related to the lobe-specific proliferation of cells that occurs with increasing age. Additional evidence for lobe-specific regulation of androgen receptor expression was obtained from Western blots and by immunocytochemistry following castration. Androgen receptor levels in the ventral and dorsal lobes, but not the lateral lobe, of young and old rats were down-regulated in the absence of testicular androgen. However, nuclear immunostaining of androgen receptor returned by 7–10 d after castration in the ventral and dorsal lobes in the continued absence of androgen. Moreover, up-regulation of the androgen receptor level occurred more rapidly in the ventral and dorsal lobes of old, compared with young, castrated rats. Taken together, these results suggest that lobe-specific and age-dependent differences in the regulation of androgen receptor expression might lead to changes in tissue androgen responsiveness and the consequent development of lobe-specific hyperplasia in the Brown Norway rat prostate gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS PLAY A central role in the regulation of growth of the mammalian prostate gland, having the dual capacity to stimulate proliferation and inhibit death of the glandular epithelial cells (1, 2). In the adult mammalian prostate gland, these rates are normally balanced so as to achieve homeostasis of growth (1). However, overgrowth of the prostate frequently occurs in aging men in the form of benign prostatic hyperplasia (BPH) or prostatic carcinoma, and as hyperplasia in aging dogs. Spontaneous, as well as hormonal and chemically induced, hyperplasia and prostatic carcinoma develop in some strains of rats (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Androgen action is also believed to be involved in the development of prostatic disease (see review; Ref. 14). Age-dependent changes in the hormonal milieu, marked by a decrease in the serum testosterone to estradiol ratio, when coupled with alterations in the concentrations of nuclear steroid receptors, may contribute to the evolution of pathological changes observed in BPH and carcinoma of the prostate gland among older men (see review; Ref. 15). In Brown Norway rats, the serum testosterone to estradiol ratio also declines with age, and despite the lower serum testosterone level, age-dependent and spontaneous prostatic hyperplasia develops in the dorsal and lateral lobes of the prostate, but not in the ventral lobe (16). The lobe-specific, age-dependent hyperplasia can be further enhanced by administration of pharmacological doses of testosterone (17).

Investigation of overgrowth and its mechanisms in the rodent prostate gland are complicated by the heterogeneous cell population and regional structural differences that exist within the different anatomic lobes (18, 19, 20, 21, 22, 23, 24). Unlike the prostate in humans and dogs, the rodent prostate gland is composed of four distinct anatomical lobes: ventral, dorsal, lateral, and anterior (17). Within each lobe, a tree-like branching system of glandular ducts is represented by a central proximal duct that initially branches into a series of intermediate ducts and subsequently into a more highly branched pattern of distal ducts (21). Regional differences in cell type within the proximal, intermediate, and distal ducts, as well as differences in hormonal responsiveness exist along the proximal-distal axis (19, 21, 23, 25, 26, 27, 28). Our previous work demonstrated that spontaneous age-related cellular hyperplasia in the dorsal and lateral lobes of the Brown Norway rat prostate was primarily composed of epithelial cells localized to the more distal regions of the ducts (16). As shown in other reports, androgen receptors are expressed along the entire prostatic ducts in all three lobes from adult rats (29, 30). In response to the administration of exogenous testosterone to Brown Norway rats, a generalized hypertrophy of epithelial cells along the entire proximal-distal ductal axis was observed in all three prostatic lobes of young and old rats (17). However, epithelial cell hyperplasia was only present in the distal region of ducts within the dorsal and lateral lobes of old rats, and the extent of this hyperplasia was markedly enhanced by androgen treatment (17). Therefore, we hypothesized that age-dependent hyperplasia in the dorsal and lateral lobes of Brown Norway rats might occur in relation to age-dependent and lobe-specific differences in androgen receptor expression. To test our hypothesis, we examined the immunostaining of androgen receptor throughout the prostatic ducts in the ventral, dorsal, and lateral lobes and compared androgen receptor levels determined from immunoblots for each lobe in young and old rats. Herein, we report that lobe-specific differences in the regulation of androgen receptor expression and age-dependent changes in levels of the androgen receptor are correlated with the development of lobe-specific hyperplasia in the Brown Norway rat prostate gland.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young (4 months of age) and old (24 months of age) male Brown Norway rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) under special arrangement with the National Institute on Aging (NIH, Bethesda, MD). The rats were housed under standard conditions with food and water ad libitum. Castration was performed via the abdominal route under ether anesthesia. Epididymides were removed along with the testes. Rats were killed at 1, 2, 3, 4, 7, and 10 d after castration. Animal protocols were approved by the Animal Care and Use Committee of The Johns Hopkins University School of Hygiene and Public Health.

Serum testosterone concentrations
Trunk blood was collected, and serum was stored frozen (-20 C) until assayed. Testosterone concentrations were determined by specific RIA according to the manufacturer’s instructions (Diagnostics Systems Laboratories, Inc., Webster, TX). The sensitivity of the testosterone assay was 0.05 ng/ml.

Dissection of prostatic lobes
The entire urogenital complex was removed from rats, and the ventral, dorsal, and lateral lobes were separated under a dissection microscope as described previously (24). Each lobe was divided into left and right portions to preserve the distal and proximal aspects and weighed. Prostatic fluid was expressed from the left portion of each lobe before snap-freezing the tissue in liquid nitrogen for subsequent determination of androgen receptor level by Western blot analysis. The right portion of each lobe was fixed in neutral buffered paraformaldehyde and embedded in paraffin for androgen receptor and proliferating cell nuclear antigen (PCNA) immunolocalization.

Immunohistochemical localization of androgen receptor
The immunocytochemical procedure was a modification of our earlier procedure (27) with the additional treatment of tissue sections at 90 C for 10 min in citrate buffer [10 mM sodium citrate (pH 6.0)] for antigen retrieval. Nonspecific binding was blocked by incubation of sections with 10% normal goat serum in PBS for 30 min at room temperature. Slides were incubated in a humidified chamber at 4 C for 16–18 h with an affinity purified rabbit polyclonal antibody, raised against a peptide (AR 21) containing the first 21 amino acids of the rat and human androgen receptor (a gift from Dr. Gail Prins, University of Illinois, Chicago, IL). The antibody was diluted to 1 µg/ml in 1% crystalline grade BSA in PBS. The reaction sites were visualized by incubating the tissue sections with biotinylated second antibody, avidin-biotin-peroxidase complex, and diaminobenzidine reagent (Vector Laboratories, Inc., Burlingame, CA). Negative control slides were prepared in an identical manner, except that the primary antibody was replaced with similar concentrations of rabbit IgG. The localization of immunoreactive androgen receptor was observed by light microscopy (Carl Zeiss, Oberkochen, Germany); the microscopic images were recorded on Kodak 64T color slide film (Kodak, Rochester, NY) and scanned in Adobe Photoshop.

Immunohistochemical staining of PCNA and quantification of PCNA-positive cells
The immunocytochemical staining for PCNA was performed as described previously (27). In brief, tissue sections were deparaffinized, rehydrated, digested with 0.025% pronase, treated to remove endogenous peroxidase activity, and blocked for nonspecific binding. Anti-PCNA (Upstate Biotechnology, Inc., Lake Placid, NY) was diluted to 5 µg/ml in 1% crystalline grade BSA in PBS, and slides were incubated in a humidified chamber at 4 C for 16–18 h. The reaction sites were then visualized by incubating the tissue sections with biotinylated second antibody, avidin-biotin-peroxidase complex, and diaminobenzidine reagent (Vector Laboratories, Inc.), and counterstained with 0.5% methyl green. Negative control slides were prepared in an identical manner, except that the primary antibody was replaced with similar concentrations of rabbit IgG. The percentages of PCNA-positive cells throughout the prostatic ducts were determined by enumerating PCNA-positive cells and methyl green-positive cells in randomly selected areas, with approximately 1000–2000 cells counted for each lobe of five rats per group. Thus, at least 5000 cells were counted for each lobe for each age group of rats.

Preparation of cell and tissue extracts for Western blot analysis
Frozen tissue samples and pellets of LNCaP and PC3 human prostate cancer cells (American Type Culture Collection, Manassas, VA) were homogenized in tissue lysis buffer [10 mM Tris-HCl (pH 7.4), containing 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM benzamidine, 0.28 U/ml aprotinin, 50 µg/ml leupeptin, and 0.7 µg/ml pepstatin]. DNA was quantified in an aliquot of tissue homogenate by the Hoechst fluorometric method described previously (31). The tissue homogenates and cell extracts were clarified by centrifugation at 14,000 x g for 20 min at 4 C. An aliquot of each sample was used for the determination of protein content using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA). The clarified supernatants were mixed (1:1) with 2x Laemmli buffer [100 mM Tris-HCl (pH 6.8), containing 10% 2-mercaptoethanol, 4% SDS, and 20% glycerol], transferred to a boiling water bath for 5 min, rapidly frozen on dry ice, and stored at -70 C until use.

Samples with equivalent amounts of protein or DNA were subjected to SDS-PAGE on a 10% acrylamide gel, run under reducing conditions, according to the method described by Laemmli (32). After separation, proteins were electrophoretically transferred at 500 mA for 2 h at 4 C to Hybond nitrocellulose membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the method of Towbin et al. (33). The membrane was initially incubated for 1 h with Tris-buffered saline [TBS; 20 mM Tris-HCl (pH 7.6), with 137 mM NaCl] containing 0.1% Tween 20 and 5% nonfat dry milk to block nonspecific binding. Subsequently, the membrane was incubated for an additional 2 h at room temperature in the presence of androgen receptor antibody (0.5 µg/ml) or anti-ß-actin (1:8000 dilution, clone AC-15; Sigma, St. Louis, MO) with frequent agitation. The membrane was washed with TBS-Tween 20, incubated with horseradish peroxidase-labeled secondary antibody (1:3000 dilution; Amersham Pharmacia Biotech) for 1 h at room temperature, and washed with TBS-Tween 20. Antibody binding sites were visualized on Hyperfilm (Amersham Pharmacia Biotech) by exposure for either 5 min (androgen receptor) or 1 min (ß-actin) using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Molecular weight markers (Life Technologies, Inc.) were run on each gel to confirm the molecular size of the androgen receptor.

Statistical analysis
Data are expressed as the mean ± SEM. Statistical differences within treatment groups were determined by one-way ANOVA. Differences between individual groups were determined with the Scheffé’s F-test (P < 0.05). Statistical differences between young and old groups of rats were compared by t test (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum testosterone concentrations
The mean serum testosterone concentration in intact Brown Norway rats 4 months of age was 1.45 ± 0.08 ng/ml and decreased to 0.92 ± 0.02 ng/ml in rats 24 months of age (P < 0.05). In both young and old rats, serum testosterone levels were at the lower limit of assay sensitivity (approximately 0.05 ng/ml) by 1 d after castration and reached undetectable levels (<0.05 ng/ml) by the second and subsequent days during the experimental period up to 10 d after castration.

Specificity of androgen receptor antibody in immunohistochemistry and Western blot
To determine specificity of the androgen receptor antibody, rat prostatic tissue sections were incubated with either androgen receptor antibody alone (Fig. 1AGo) or with androgen receptor antibody neutralized by androgen receptor synthetic peptide (Fig. 1BGo). Distinct nuclear staining was observed in the epithelial cells of each rat prostatic lobe. When androgen receptor antibody was preincubated with a 10-fold excess of synthetic androgen receptor peptide, the immunostaining disappeared completely. Western blot analysis of protein extracts prepared from LNCaP cells showed a single 110-kDa protein band (Fig. 1CGo), demonstrating specificity of the antibody for the androgen receptor of appropriate molecular weight.



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  		Figure 1. Specificity of androgen receptor antibody. A, Androgen receptor antibody (PG-21-25A) shows immunoreactivity in adult Brown Norway rat ventral prostate tissue section. B, Androgen receptor antibody was preabsorbed with 10-fold excess of androgen receptor peptide before immunostaining of adult Brown Norway rat ventral prostate. The absence of immunostaining demonstrates the specificity of this antibody. C, LNCaP cell extract on a Western blot, probed with androgen receptor antibody, shows a single protein band of 110 kDa. The positions of the molecular mass markers (kilodaltons) are indicated.

 
Immunolocalization of androgen receptor in young and old ventral, dorsal, and lateral prostatic lobes
Our goal was to compare androgen receptor levels in different prostatic lobes from young and old rats in an attempt to correlate our findings with the occurrence of lobe-specific cellular hyperplasia. Therefore, we examined androgen receptor protein localization throughout the prostatic ducts of the ventral, dorsal, and lateral lobes. It is difficult to show distinct cellular structure coincident with immunolocalization of androgen receptor in an entire prostatic duct. Instead, we divided the prostatic ducts into three representative segments (distal, intermediate, and proximal) based on their proximity to the urethra. In the ventral lobe from young rats, columnar epithelial cells lining the ducts of the distal and intermediate segments showed intense nuclear staining for androgen receptor (Fig. 2Go, A and B). Very few stromal cells were androgen receptor positive in these segments. Prominent androgen receptor staining was also present in the cuboidal epithelial cells of the proximal segment (Fig. 2CGo). Some stromal cells in the proximal segment were also androgen receptor positive. When androgen receptor staining intensity was compared for epithelial cells in the same three segments of the ventral lobe from young and old rats, a lower staining intensity was observed for each ventral lobe segment from old rats (Fig. 2Go, D–F). The intensity of androgen receptor staining in stromal cells did not change with age. The most striking difference in staining intensity was observed in the intermediate segment where the majority of the epithelial cells had very little immunostaining for androgen receptor, and some epithelial cells were completely devoid of any staining (Fig. 2EGo).



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  		Figure 2. Immunohistochemical localization of androgen receptor in tissue sections of ventral, dorsal, and lateral prostatic lobes in young and old Brown Norway rats. A, B, and C are the distal, intermediate, and proximal segments, respectively, of young rat ventral lobe. D, E, and F are the distal, intermediate, and proximal segments, respectively, of old rat ventral lobe. G, H, and I are the distal, intermediate, and proximal segments, respectively, of young rat dorsal lobe. J, K, and L are the distal, intermediate, and proximal segments, respectively, of old rat dorsal lobe. M, N, and O are the distal, intermediate and proximal segments, respectively, of young rat lateral lobe. P, Q, and R are the distal, intermediate, and proximal segments, respectively, of old rat lateral lobe.

 
In contrast to the ventral lobe of young rats (Fig. 2Go, A–C), androgen receptor staining was less intense in the prostatic ducts throughout the dorsal lobe of young rats (Fig. 2Go, G–I). However, as seen for the ventral lobe, epithelial cells were the primary cell type that exhibited androgen receptor immunostaining within each segment. In this lobe, some stromal cells were also positive for androgen receptor expression. When segments of the dorsal lobe from young and old rats were compared, androgen receptor staining intensity was greater in all three segments of old rats (Fig. 2Go, J–L). These differences were more distinct in the distal and intermediate ductal segments where epithelial cells formed infoldings with crowded nuclei (Fig. 2Go, J and K), representative of the epithelial cell hyperplasia that occurs in the dorsal lobe of old rats.

Like the dorsal lobe, androgen receptor staining intensity was lower throughout the prostatic ducts of the lateral lobe (Fig. 2Go, M–O) from young rats, when compared with the ventral lobe. However, the nuclear staining intensity for androgen receptor was greater in all three segments of the lateral lobe as a function of age (Fig. 2Go, P–R). Similar to the ventral and dorsal lobes, epithelial cells were the predominant cells that were positive for androgen receptor expression in the lateral lobe, as relatively few stromal cells were stained. The occurrence of age-dependent epithelial cell hyperplasia is also evident in the distal segment of the lateral lobe from old rats (Fig. 2PGo).

Quantification of androgen receptor levels in young and old ventral, dorsal, and lateral prostatic lobes
To validate immunostaining results, we used Western blot analyses to semiquantitatively determine the levels of androgen receptor protein in ventral, dorsal, and lateral prostatic tissues from young and old rats. A single immunoreactive protein of approximately 110 kDa was detected in all rat prostate tissue extracts, as well as extracts from LNCaP cells (positive control). As expected, extracts from PC-3 cells were negative for androgen receptor and served as a negative control. When androgen receptor levels were compared, relative to levels of ß-actin, in the ventral lobe of young and old rats, we found that the level was 30% lower in old rats (Fig. 3Go, A and B). By contrast, androgen receptor levels were 2-fold and 1.3-fold higher in the dorsal (Fig. 3Go, C and D) and lateral (Fig. 3Go, E and F) lobes, respectively, of old rats, compared with young rats.



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  		Figure 3. Western blot analysis of androgen receptor levels in prostate tissue extracts from young and old rats. Experimental results for androgen receptor (upper band) and ß-actin (lower band) expression in the ventral, dorsal, and lateral lobes are shown in A, C, and E, respectively. Lanes 1–3 represent three rats in each age group. LNCaP and PC-3 cell extracts were used as the positive and negative controls for androgen receptor. Protein extracts obtained from equal amounts (5 µg) of tissue DNA were loaded in each lane, except LNCaP and PC-3 where 10 µg protein were loaded. Positions of the molecular mass markers (kilodaltons) are included. Quantitative analyses of androgen receptor protein normalized for ß-actin levels in the same tissue samples from the ventral, dorsal, and lateral lobes are shown in B, D, and F, respectively. Values are the mean ± SEM from three different rats. *, Significantly different from young rats (P < 0.05).

 
Proliferative activity of prostatic cells in young and old ventral, dorsal, and lateral prostatic lobes
To understand the proliferative activity of cells in the ventral, dorsal, and lateral lobes of young and old rats, and its association with androgen receptor immunostaining, we examined PCNA labeling in prostate cells. Epithelial cells were the predominant cells that were positive for PCNA immunostaining (data not shown). In the ventral lobe of young and old rats and the dorsal and lateral lobes of young rats, PCNA-positive cells were observed throughout the length of the prostatic ducts. However, in the dorsal and lateral lobes of old rats, PCNA-positive cells were primarily restricted to the regions of the gland where epithelial cell hyperplasia was evident (data not shown). Quantitative analyses did not reveal any difference in the percentages of PCNA-positive cells in the ventral lobe from young and old rats (Fig. 4Go). By contrast, the percentages of PCNA-positive cells were higher in the dorsal and lateral lobes from old rats compared with young rats, but this difference was statistically significant only in the dorsal lobe.



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  		Figure 4. Percentages of PCNA-positive cells in the ventral, dorsal, and lateral lobes of young and old Brown Norway rats. Values are the mean ± SEM from five different rats. {blacksquare}, Young rat prostatic lobes; {square}, old rat prostatic lobes. *, Significantly different from young rats (P < 0.05).

 
Regulation of androgen receptor levels in young and old ventral, dorsal, and lateral prostatic lobes
To understand the regulation of androgen receptor expression in the ventral, dorsal, and lateral lobes from young and old rats following castration, we used Western blot analyses to quantify the levels of androgen receptor in tissue extracts. All samples were normalized for ß-actin expression within each sample and for cell number based on DNA content due to differences in apoptotic cell death between individual lobes dependent on age of the animal and time following castration. The androgen receptor level was down-regulated in the ventral lobe of young rats by 1 d after castration, coincident with the fall in serum testosterone concentration (Fig. 5Go, A and B). Androgen receptor levels in the ventral lobe decreased significantly by 2 d and further decreased by 3 and 4 d in castrated rats. However, by 7 d after castration when serum testosterone levels were undetectable, the androgen receptor level was significantly up- regulated, compared with levels observed 3 and 4 d after castration. The androgen receptor level was maintained between 7 and 10 d after castration, but did not return to the level of intact rats. In the ventral lobe of old rats (Fig. 5Go, C and D), the androgen receptor level was also down-regulated by 2–3 d after castration, but up-regulation of the androgen receptor was detected as early as 4 d after castration. Moreover, the androgen receptor level returned to the level of intact old animals by 7–10 d after castration.



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  		Figure 5. Western blot analysis of androgen receptor expression in tissue extracts from each prostatic lobe of intact, 1-, 2-, 3-, 4-, 7-, and 10-d postcastrated young and old rats. Equal amounts (5 µg) of DNA were loaded in each lane. Positions of the molecular mass markers (kilodaltons) are included. Representative blots from the ventral lobe of young (A) and old (C) rats, from the dorsal lobe of young (E) and old (G) rats and from the lateral lobe of young (I) and old (K) rats are shown. Quantitative analyses of androgen receptor protein were obtained by normalization for ß-actin levels in the same tissue samples. The relative levels of androgen receptor expression are shown for the ventral lobe of young (B) and old (D) rats, for the dorsal lobe of young (F) and old (H) rats and for the lateral lobe of young (J) and old (L) rats. Values are the mean ± SEM from three different rats. *, Significantly different from intact controls (P < 0.05).

 
Similar to the ventral lobe in young rats, androgen receptor levels in the dorsal lobe were down-regulated in young rats by 2–4 d after castration (Fig. 5Go, E and F). Subsequently, the androgen receptor level was up-regulated by 7 d after castration and returned to the level of intact rats by 10 d after castration. In the dorsal lobe of old rats (Fig. 5Go, G and H), androgen receptor levels were down-regulated by 2–3 d after castration, however, the relative decrease was less than occurred in the dorsal lobe of young rats during the same period. By 4 d after castration in old rats, androgen receptor levels were up-regulated and then returned to the intact control level by 7–10 d after castration. In contrast to the effects of castration on androgen receptor expression in the ventral and dorsal lobes, androgen receptor levels in the lateral lobe of young (Fig. 5Go, I and J) and old (Fig. 5Go, K and L) rats were unaffected throughout the 10-d period following castration.

To confirm our observations from Western blot analyses, we also determined the localization of androgen receptors before and after castration in the ventral, dorsal, and lateral lobes by immunohistochemistry (Fig. 6Go). As expected, distinct nuclear staining of androgen receptor was evident in epithelial cells of the ventral, dorsal, and lateral lobes from young rats (Fig. 6Go, A–C). Androgen receptor nuclear staining intensity decreased by 3 d after castration in the ventral and dorsal lobes, but no change was observed in the lateral lobe (Fig. 6Go, D–F). By 7 d after castration (Fig. 6Go, G–I), distinct staining of androgen receptor returned in nuclei of epithelial cells in the ventral and dorsal lobes, however, the staining intensity for the ventral lobe from castrate rats was less intense than observed in the same lobe of intact rats. In addition, more cytoplasmic staining was evident in the epithelial cells of castrate rats. In the lateral lobe, nuclear androgen receptor staining intensity remained unchanged 3 and 7 d after castration. Although, the results shown are for 3 and 7 d after castration, no further changes were evident at 10 d following castration (data not shown). Parallel results for androgen receptor immunocytochemical localization and expression were observed for the ventral, dorsal, and lateral lobes from old rats following castration (data not shown).



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  		Figure 6. Immunohistochemical localization of androgen receptor in tissue sections of the ventral, dorsal, and lateral prostate lobes of intact and castrated young rats. A, B, and C are the ventral, dorsal, and lateral prostatic lobes, respectively, of intact young rat. D, E, and F are the ventral, dorsal, and lateral prostatic lobes, respectively, of 3-d postcastrated young rat. G, H, and I are the ventral, dorsal, and lateral prostatic lobes, respectively, of 7-d postcastrated young rat.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The prostate maintains its ability to respond to androgens throughout life. During pubertal maturation, testicular synthesis and secretion of androgens provide the stimulus for growth of the prostate to its normal adult size. The proliferative response of cells within the prostate to androgens is dependent on expression of the intracellular androgen receptor. Following growth of the prostate to its adult size, a balance between cell proliferation and cell death is established so that no further growth occurs. However, later in life in several species, including man, dog, and some strains of rats, cellular hyperplasia occurs despite a decrease in testicular androgen production and a fall in the peripheral levels of androgen reaching the prostate. The imbalance in cell death and cell proliferation that leads to the age-dependent prostatic hyperplasia may be related to an increased sensitivity of the prostate to androgen. One hypothesis to explain this phenomenon would be that nuclear androgen receptor expression changes with age in those cells that contribute to the evolution of cellular hyperplasia. In the human, androgen receptor levels in the prostate remain high throughout aging (34, 35, 36). In fact, there is evidence to suggest that nuclear androgen receptor levels may be higher in hyperplastic prostate tissue than in normal controls (34). Age-related increases in the relative estrogen level, as well as other factors, may increase androgen receptor expression in the aging prostate (37, 38), leading to further growth or a decrease in cell death (39), despite decreasing levels of androgen in the peripheral circulation and normal levels of dihydrotestosterone in the prostate.

We examined levels of androgen receptor expression and its regulation by androgen in the different lobes of young and old rats. The results presented herein demonstrate that enhanced androgen receptor protein expression is lobe-specific and correlates with our observations of age-dependent hyperplasia in the dorsal and lateral lobes of the Brown Norway rat prostate. As reported previously for other rat strains (30, 40, 41, 42) and now confirmed in the Brown Norway rat, androgen receptor is expressed in all three lobes of the prostate gland, predominantly in epithelial cells along the proximal to distal axis of the prostatic ducts. In our study of Brown Norway rats, different levels of androgen receptor expression were evident in the ventral, dorsal, and lateral lobes based on immunocytochemical staining intensity and semiquantitative Western blot analyses. Androgen receptor staining in nuclei of the ventral lobe from young rats was more intense than in the dorsal and lateral lobes. This result corroborates earlier reports of marked differences in androgen receptor binding activity among the three lobes, with ventral > lateral >= dorsal (41). Moreover, the lateral lobe responds to hormones in ways that differ significantly from the ventral and dorsal lobes (30). In the present study, we found that androgen receptor staining intensity decreased as a function of age in the ventral lobe, but the staining intensity increased in the dorsal and lateral lobes with age. Our results confirm previous reports that androgen receptor levels were decreased in the ventral lobe of aging AXC rats, but are contrary to the observation in the same report of a 50% decrease in the nuclear androgen receptor content in the dorsolateral prostate (43). Our results are supported by Western blot analyses showing decreased androgen receptor protein levels in the ventral lobe, but increased levels in the dorsal and lateral lobes, as a function of age. The age- dependent increase in androgen receptor expression correlated with an increased percentage of proliferating cells detected by PCNA immunostaining of epithelial cells in the dorsal and lateral lobes. These findings suggest an association between androgen receptor expression and cell proliferation that may be involved in the lobe-specific hyperplasia in the Brown Norway rat that occurs with increasing age.

An intriguing aspect of our study in Brown Norway rats is the androgen dependency of androgen receptor expression in the ventral and dorsal lobes following castration. Immediately after castration (1–3 d after castration) androgen receptor expression was down-regulated in the ventral and dorsal lobes. In the ventral lobe, the loss of androgen receptor expression is undoubtedly correlated with apoptosis of luminal epithelial cells (28), the primary cells in which the androgen receptor is expressed. By contrast, relatively few luminal epithelial cells die in the dorsal lobe (28), yet androgen receptor expression also decreased immediately following castration. Interestingly, androgen receptor expression was up-regulated within 7–10 d following castration in the ventral and dorsal lobes, despite the death of many luminal epithelial cells in the ventral lobe and the continued absence of testicular androgens. Moreover, this up-regulation occurred faster in the ventral and dorsal lobes from old rats, compared with young rats. By contrast, androgen receptor expression in the lateral lobe was androgen independent because its level of expression did not change following castration.

Our results from castration experiments in Brown Norway rats demonstrate the inherent complexity that exists in the androgen-dependent autoregulation of androgen receptor expression in the rat prostate. A series of earlier studies by Prins et al. (30, 40, 42) with prostates of young adult Sprague Dawley rats also showed lobe-specific autoregulation of the androgen receptor. Saturation ligand binding and immunocytochemical assays revealed a fall in androgen receptor to very low levels in the ventral and dorsal lobes within 7 d after castration, whereas androgen receptor levels in the lateral lobe were unchanged. Conversely, androgen receptor mRNA levels in all three lobes were increased following castration and decreased on testosterone replacement. This discordant response of androgen receptor protein and mRNA levels to androgen manipulation is unlike the autoregulation of other members of the steroid receptor family where alterations in protein and mRNA levels parallel each other in response to their cognate ligand (44, 45, 46, 47, 48, 49). These results suggest that prostatic androgen receptor regulation is complex and is likely to be regulated by different cellular mechanisms in each lobe.

The assumption in castration studies is that serum and tissue levels of testosterone and dihydrotestosterone are dramatically decreased such that any significant biologic effect of androgen is ablated. We previously reported that serum testosterone levels were significantly reduced in old Brown Norway rats (16) and the ratio of serum testosterone to dihydrotestosterone is maintained at approximately 10:1 for both ages. In preliminary studies of prostate tissue levels (pg steroid/ug DNA) for these steroids, dihydrotestosterone represents approximately four times the amount of intratissue testosterone present in each lobe of young and old rats, and hence there were no obvious age-dependent differences in the dihydrotestosterone to testosteorne ratio in each of the prostatic lobes that might affect androgen receptor expression. Serum testosterone levels were undetectable (<0.05 ng/ml) in castrated Brown Norway rats within 2 d of castration. Kyprianou and Isaacs (50) reported similar results for serum testosterone levels in Copenhagen rats, whereas serum dihydrotestosterone levels only decreased by 50% as long as 20 wk after castration. Moreover, they reported that intraprostatic levels of testosterone and dihydrotestosterone remained at 40% and 20%, respectively, as long as 7 d following castration. The residual levels of androgen in serum and tissue were undetectable following simultaneous castration and adrenalectomy. In the same study, however, the lower residual androgen level in the ventral prostate following castration was insufficient to inhibit cell atrophy, prevent apoptosis or affect the rate of DNA synthesis. We, too, observed a high rate of apoptosis in the ventral prostate of Brown Norway rats following castration. However, differences in the sensitivity of specific responses may exist between the ventral, dorsal, and lateral lobes. We have observed two such responses in the Brown Norway rat model, as epithelial cell apoptosis (28) and androgen receptor expression are differentially affected among the three lobes following castration.

These findings serve to reemphasize a role for other factors, in addition to androgen, in the regulation of androgen receptor expression in the prostate gland. At this time, we do not know the other factor(s), aside from testosterone that may be involved in the regulation of androgen receptor expression in the ventral, dorsal, and lateral lobes. The different lobes of the rat prostate may, however, represent a useful model to understand the regulation of androgen receptor expression. The presence of androgen receptors in the prostate following castration and in the absence of endogenous ligand may not preclude transcriptional activity of the receptor in prostatic cells. Culig and co-workers (51, 52, 53) have shown that growth factor signaling pathways for IGF-I, keratinocyte growth factor, and interleukin 6, as well as other signaling pathways that function through protein-protein interactions, such as activator protein-1 or Ets transcription factors, may influence androgen receptor transcriptional activity.

In the Brown Norway rat, the age-dependent hyperplasia is specific to the lateral and dorsal lobes. The dorsal and lateral lobes in the rat are most homologous to the human prostate, being similarly derived from the same prostatic buds arising in the urogenital sinus (54). The transition zone of the human prostate in which histologic BPH is observed is similarly derived from the urogenital sinus (55, 56). Although the morphology of human BPH are often referred to in the literature as a proliferation of stromal cells, both human and canine BPH is most often characterized by varying degrees of complexity involving glandular epithelial cell proliferation accompanied by elaborate branching of alveoli with increased papillary projections (55, 56, 57). Hyperplasia in the dorsal and lateral lobes of the Brown Norway rat is primarily due to epithelial cell proliferation with evidence of a more convoluted ductal architecture (16). By contrast, cellular hyperplasia is not observed in the rat ventral prostate, which has no identifiable homology with the male primate prostate and develops from the urogenital sinus independent of the dorsal and lateral lobes. Like the human prostate, the dorsolateral lobes of the rat prostate are rich in citrate and accumulate high levels of zinc (58, 59).

Although the molecular mechanism(s) for age-dependent and lobe-specific hyperplasia remains elusive at the present time, the aging Brown Norway rat provides a model for investigating potential mechanisms. Taken together, our studies provide the basis for further examination of androgen receptors in lobe-specific hyperplasia in Brown Norway rat prostate. More specifically, the different lobes of the prostate in the aging Brown Norway rat prostate provide a model system in which to study the complexity of androgen- dependent and -independent regulation of androgen receptor expression and function.


   Acknowledgments
 


   Footnotes
 
This work was supported by NIH Grant PO1-AG08321 (to T.R.B.) and American Federation for Aging Research Grant A98120 (to P.P.B.).

1 Present address: Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, D.C. 20007. Back

Abbreviations: BPH, Benign prostatic hyperplasia; PCNA, proliferating cell nuclear antigen; TBS, Tris-buffered saline.

Received December 29, 2000.

Accepted for publication May 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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