* School of Pharmacy and
Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53706
Received May 15, 2000; accepted August 2, 2000
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ABSTRACT |
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Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; rat, prostate; development; impaired epithelial differentiation; 5-dihydrotestosterone-forming enzymes; testicular testosterone production.
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INTRODUCTION |
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Another possible explanation for some of the seemingly antiandrogenic effects of TCDD, particularly with respect to prostate development, is that in utero and lactational exposure inhibits the formation of 5-dihydrotestosterone (DHT). The rat ventral prostate begins to arise before birth from the prostate anlagen in the urogenital sinus region. Prostatic mesenchyme, under the influence of androgenic stimulation, induces the formation of epithelial buds that will eventually develop into secretory glands (Hayward et al., 1996
). In order for this process to occur, however, circulating testosterone from the fetal testes must be enzymatically converted to DHT within the nascent prostate (George and Peterson, 1988
). The development of the seminal vesicle and epididymis, like that of the prostate, requires androgenic stimulation. However, unlike the prostate, the seminal vesicle and epididymis do not require DHT for normal development (Clark et al., 1993
; George and Peterson, 1988
; Hayward et al., 1996
). If DHT synthesis is globally inhibited during the period from gestational day (GD) 14 to GD 16, normal prostate differentiation and growth will be prevented (Clark et al., 1993
), the normal course of the urethral opening to the penis tip will be deflected, and a hypospadia will result (Anderson and Clark, 1990
). However, penis development appears normal and hypospadias are not seen in response to TCDD exposure (Mably et al., 1992b
). It remains possible, though, that a local decrease in DHT synthesis activity may well play a role in decreasing ventral prostate growth, as in utero and lactational TCDD exposure decreased ventral prostate DHT concentration (Roman et al., 1995
).
Prior to and just after birth, the predominant circulating androgen in fetal male rat blood is testosterone (Corpechot et al., 1981). This hormone is converted to DHT by two NADPH requiring isozymes of 5
-reductase (5
-R), which are the products of different genes, are distinguishable in enzyme assays by their different Michaelis constants and pH optima, and appear to play different roles in ventral prostate androgen metabolism (Berman et al., 1995
; Moore and Wilson, 1976
; Normington and Russell, 1992
; Span et al., 1995
). The type-1 5
-R isozyme has a Michaelis constant in the micromolar range, a pH optimum in vitro of 6.9, and is the only form found in the ventral prostate epithelium. In contrast, the type-2 5
-R isozyme has a Michaelis constant in the nanomolar range, pH optimum in vitro of 5.5, and is found in the developing ventral prostate exclusively in the developing mesenchyme and stroma (Berman et al., 1995
; Span et al., 1995
). Since androgen signaling in the developing fetal prostate initially occurs via the interaction of androgens with receptors in the mesenchyme, which in turn relay a signal to the epithelium, it appears that the type-2 5
-R isozyme is the enzyme form required for this early androgenic activity (Berman et al., 1995
; Chung and Cunha, 1983
). The type-1 5
-R isozyme, on the other hand, with its higher Km, appears to play a catabolic role, which primarily facilitates the elimination of testosterone from the ventral prostate, rather than activating androgen receptors.
Shortly after birth, the androgenic prohormone 5-androstane-3
, 17ß-diol (3
-Diol) transiently becomes the predominant circulating androgen in postnatal rat plasma until just before puberty (Corpechot et al., 1981
). This precursor is converted to DHT in the developing ventral prostate by the action of 3
-hydroxysteroid dehydrogenase (3
-HSD) (Penning et al., 1996). Unlike 5
-R, 3
-HSD catalyzes a thermodynamically reversible reaction and prefers NAD rather than NADP as the cofactor. Because the enzyme reaction is thermodynamically reversible, 3
-HSD can act as a molecular switch that provides a buffering action to resist changes in the intracellular DHT concentration (Penning et al., 1996).
Androgen receptors first appear in rat ventral prostate epithelial cells on GD 19 (Hayward et al., 1996). Early ventral prostate epithelial development (before PND 19) occurs under the inductive influence of the mesenchyme and involves mesenchymal androgen receptors, which when activated, elicit production of paracrine growth factors such as keratinocyte growth factor, which binds to and activates receptors on epithelial cells (Thomson et al., 1997
). While epithelial androgen receptors are not required for prostatic development, they are absolutely necessary for secretory function (Donjacour and Cunha, 1993
). In addition to androgen-receptor expression, ventral prostate secretory activity requires the formation of luminal epithelial cells from cellular precursors, which presumably are basal cells that are induced along a differentiation pathway by the presence of circulating androgens (Bonkhoff and Remberger, 1996
; Robinson et al., 1998
).
Previous results indicated that in utero and lactational TCDD exposure inhibits epithelial-bud formation, decreases the number of luminal epithelial cells, decreases luminal epithelial-cell androgen receptor-protein expression, and decreases androgen-responsive mRNA expression in the rat ventral prostate (Roman and Peterson, 1998; Roman et al., 1998
). Therefore, the present study was undertaken in order to determine whether these effects might be contributed to by decreased testosterone production by the fetal and early neonatal testis, or decreased postnatal DHT synthesis, or altered androgen metabolism within the ventral prostate. Furthermore, prostatic binding protein subunit C3 (C3) mRNA and protein expression were assayed as indicators of the in vitro response of ventral prostates from TCDD and vehicle-exposed male offspring to testosterone, 3
-Diol, and DHT. Luminal epithelial cell differentiation, as judged by the immunohistochemical localization of androgen receptors, cytokeratin 18, and C3 were also assayed in these cultures. The results of the current study indicate that the TCDD-induced decrease in ventral prostate growth cannot be explained by decreased perinatal androgen production in the fetal testis, or by a decreased conversion of the precursor androgens testosterone and 3
-Diol to the active androgen DHT within the PND 14 to PND 32 ventral prostate. However, TCDD-induced decreases in androgen responsiveness, androgen receptor expression, and luminal cell development suggest an interference with prostate differentiation that may be caused by TCDD acting directly on the developing prostate to alter gene expression and/or growth factor activity.
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MATERIALS AND METHODS |
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Testicular androgen content, human chorionic gonadotropin (hCG)-stimulated testosterone synthesis, and plasma testosterone concentration were evaluated at GDs 18 and 20 and 2 h after birth. For prenatal androgen measurements, dams were sacrificed on GD 18 or 20 by light CO2 overdose followed by decapitation. Uteri were removed and placed on ice, and fetuses were excised sequentially and held on ice until sacrifice. Fetuses and 2-h-old neonates were initially sexed by visual inspection of anogenital distance (AGD; distance between the anus and the genital tubercle, which is longer in males than in females), and sex determination was confirmed by gonadal inspection using a dissecting microscope. Implantation sites were counted after staining of the uterus with 10% (v/v) ammonium sulfide, and percent survival was calculated as number of live offspring at time of sacrifice/number of implantation sites x 100. This study was conducted in 3 consecutive blocks involving 15 to 24 dams each. In each block, dams were dosed with either TCDD or vehicle, and all litters were sacrificed on either GD 18, or 20, or at 2 h after birth. Overall n values were 713 litters per treatment per time point.
For analysis of postnatal ventral prostatic 5-R and 3
-HSD activities, dams were allowed to deliver and all litters were culled to 10 pups on PND 1 in order to normalize lactational TCDD exposure across litters. Male offspring were sacrificed on PNDs 14, 21, and 32, while the remaining offspring were weaned at PND 21. After weaning, all males from the same litter were housed in 1 or 2 cages (maximum 4 rats per cage). This study consisted of 4 blocks, each involving 23 litters per treatment. Overall n values were 35 litters per treatment per time point.
For analysis of postnatal ventral-prostate androgen metabolism and androgen responsiveness in organ culture, dams were allowed to deliver and litters were culled to 10 pups in order to normalize lactational TCDD exposure across litters. Male offspring were sacrificed on PNDs 11 and 18. This study consisted of 1 block involving 20 litters. Overall n values were 3 litters per treatment per time point.
Necropsy and in vitro testis incubations.
Prior to sacrifice by decapitation, male offspring were weighed and, at the 2-h time point, AGD and crown-rump length were measured using a hand-held micrometer. Trunk blood was collected in heparinized capillary tubes, pooled within each litter, centrifuged for 10 min at 12,000 x g at 4°C, and plasma was stored in polypropylene tubes at 80°C. Testes were excised, weighed, and stored in a 24-well culture plate (Corning, Corning, NY) in 0.5 ml ice-cold 0.9% saline, until all offspring from a single litter had been processed. Subsequently, approximately one testis per litter was bisected and frozen immediately in DMEM/F-12 (medium, Sigma, St. Louis, MO) for determination of intratesticular testosterone content. All remaining testes were transferred to a fresh 24-well culture plate containing 25 µl medium per well and bisected with fine dissecting scissors. Twenty-five µl of the appropriate dilution of hCG (Sigma, St. Louis, MO) was then added to each well, followed by 450 µl medium. Final hCG concentrations were 0, 1, 2, 4, 8, 16, 32, 64, and 128 mIU/ml. Testes were incubated on an orbital shaker (150 rpm) for 3 h at 37°C in 5% CO2/95% O2. At the end of the incubation, testes and medium were collected and frozen at 20°C until assay.
Testosterone radioimmunoassay.
Testicular parenchyma and incubation medium were homogenized for 30 s (Tissumizer; Tekmar, Cincinnati, OH). After homogenization, the Tissumizer probe was rinsed with 1 ml fresh medium, and the combined sample and rinse were disrupted for 10 s, using an immersion sonicator (Laboratory Supplies Co. Inc, Hicksville, NY). Testis homogenates (100 µl) and plasma samples (300 µl) were extracted twice with 10 volumes of anhydrous ethyl ether and organic phases were combined and dried in a Speed Vac concentrator (Savant Instruments, Farmingdale, NY). Samples were reconstituted in ethanol. Recovery of [3H]-testosterone from representative spiked samples ranged from 8090% for testis homogenates and plasma. Standards and samples were assayed in triplicate for testosterone by radioimmunoassay (Robinson et al., 1975). Unlabelled testosterone was purchased from Steraloids (Wilton, NH); [3H]-testosterone was purchased from Amersham (Arlington Heights, IL), and testosterone antiserum was purchased from Endocrine Sciences (Calabasa, CA). The range of quantitation for the RIA was 101000 pg/tube.
5-reductase and 3
-hydroxysteroid dehydrogenase activities.
Ventral prostates removed from vehicle- and TCDD-exposed offspring on postnatal days (PNDs) 14, 21, and 32 were immediately frozen in liquid nitrogen, and were subsequently stored at 80°C until use. PNDs 21 and 32 ventral prostates were thawed on ice and bisected so that one lobe was used to measure the type-1 5-R isozyme and 3
-HSD activities. The remaining ventral prostate lobe was used to measure the activity of the type-2 5
-R isozyme. Ventral prostates were homogenized in a buffer system that contained 116 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 10 mM sodium phosphate salts, 10 mM sodium citrate salts, 2 mM EDTA, 4 mM dithiothreitol (DTT), and 5% (v:v) glycerol. The prostate lobe intended for the analysis of type-1, 5
-R and 3
-HSD activities was homogenized in 2 ml of the buffer at pH 6.9, whereas the prostate lobe intended for the analysis of type 2 5
-R activity was homogenized in 1 ml of the buffer at pH 5.5. On PND 14 type 1 5
-R activity was not detectable and separate ventral prostates had to be used for the analysis of type-2 5
-R and 3
-HSD activities.
To assay the type-1 and type-2 5-R activities, run buffers were prepared that contained 116 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 10 mM sodium phosphate salts, and 10 mM sodium citrate salts. Type-1 5
-R activity was assayed in tubes that contained 50 µl of run buffer and 50 µl of homogenate in the presence of 0.5 µM [3H]-testosterone and 2 mM NADPH at pH 6.9. Type-2 5
-R activity was assayed in similar tubes that contained 0.05 µM [3H]-testosterone at pH 5.5. The testosterone concentration was lower than that used for the type-1 5
-R assay so that the differing Michaelis constants between the 2 isozymes could be used to increase assay selectivity (Span et al., 1995
). 3
-HSD activity was determined in 40 mM trizma buffer (pH 8.6) in the presence of 0.5 µM 3H-3
-Diol, 2 mM NAD and 50 µl of homogenate in 100 µl of total volume. In each assay (5
-R and 3
-HSD) the reactions, in duplicate tubes, were stopped at 10, 20, 30, 40, 50, and 60 min by the addition of 10 µl of 3 N NaOH. Zero-time tubes were formed by adding the NaOH to prevent reaction before adding the homogenate. Very little metabolism of the radioactive substrates occurred in the zero-time tubes. All samples were stored at 20°C until they were extracted.
All samples were diluted with 0.9% saline to 1 ml and were extracted once with 2 ml of chloroform:methanol (2:1) and once with chloroform. Organic phases from each sample were combined and evaporated at 45°C under a gentle steam of nitrogen. The aqueous phase of each sample was prepared for liquid scintillation counting to determine the radioactivity. The dried organic phases were redissolved in 40 µl of methanol that contained non-radiolabeled testosterone, dihydrotestosterone, 3-, 3ß-Diols, and either androstenedione (5
-R assays) or androstanedione (3
-HSD assay). The reconstituted samples (20 µl) were applied to the preabsorbant region of a 19-channel silica gel TLC plate (J. T. Baker, Phillipsburg, NJ). TLC plates were developed with toluene:95% ethanol (94:6) and the bands for the carrier steroids were visualized after staining with anisaldehyde spray reagent (Sigma, St. Louis, MO). The zones for each steroid, and for the determination of background radioactivity were scraped into scintillation vials, such that every lane was counted from the preabsorbant area to a point just beyond the farthest migrating steroid. The results from each vial were then considered in the analysis either as a known steroid, or as background.
Analysis included a subtraction of the background counts and where necessary, the inclusion of polar metabolites recovered in the aqueous phase. In the 5-R assays the extraction efficiency was
90% and constant for all time points. However, in the 3
-HSD assay, there were increasingly greater amounts of radioactivity recovered in the aqueous phase as assay time increased. These were considered to be polar metabolite(s) (mostly of the substrate, 3
-Diol) and they were quantified and included in the analysis. Initial reaction velocities for both 5
-R isozymes and for 3
-HSD were determined by computer-assisted numerical and graphical analysis using the slope of the tangent line that passed through the origin in the applicable time-course curve for DHT. These initial velocities were then used to determine the specific activity of each 5
-R isozyme and 3
-HSD relative to the total protein concentration in each homogenate. Protein concentrations were determined by a modification of the Lowry protein assay (Bensadoun and Weinstein, 1976
).
Ventral prostate organ cultures.
Ventral prostates were obtained from vehicle- and TCDD-exposed offspring on PNDs 11 and 18. The ventral prostate from each rat was immediately placed into DMEM (GIBCO, Grand Island, NY) with 5% of charcoal-dextran-stripped fetal bovine serum (Summit, Ft. Collins, UT) and cultured at 37°C in an atmosphere of 95% air and 5% carbon dioxide. On PND 11 whole prostates were cultured, whereas one-half of the prostate from each rat was sufficient on PND 18. After 24 h the medium was removed and new DMEM that contained 5% charcoal-dextran-stripped fetal bovine serum supplemented with graded concentrations of androgenic steroids was added. Each culture was then exposed to 109 M, 108 M, or 107 M concentrations of [3H]-testosterone, [3H]-DHT, or [3H]-3-Diol for 48 h. The harvesting dates for the cultures initiated on PND 11 and PND 18 then coincided with PND 14 and PND 21, respectively. The tissue culture medium was stored at 20°C prior to solvent extraction and TLC analysis of steroid content (as above). Total RNA was isolated from each cultured ventral prostate and separated by electrophoresis on a 1.4% agarose gel that contained formaldehyde (Krumlauf, 1996
). The RNA was transferred from the gel to a nylon membrane, which was probed for prostatic-binding protein C3 subunit and cyclophillin by using [32P]-labeled DNA probes. Triplicate determinations were made for each concentration of each steroid, and band intensities were evaluated by using commercially available phosphoimage analysis equipment and software (Molecular Dynamics, Sunnyvale, CA). The C3 subunit band intensities from each sample were normalized to the appropriate cyclophillin band intensity. To evaluate the effects of in vivo TCDD exposure on the expression of the C3 subunit protein, a separate set of ventral prostates was cultured for 24 h in the absence of steroids, and then for 48 h in the presence of 108 M 3
-Diol. This steroid was chosen because it is the predominant circulating androgen in rat blood on PNDs 1121 (Corpechot et al., 1981
).
Immunohistochemistry.
Cultured ventral prostates were fixed in Bouin's solution overnight and embedded in paraffin. Tissue sections (5 µm) were evaluated for androgen receptor, prostatic binding protein subunit C3 (C3), or cytokeratin 18 (CK 18) expression by an immunohistochemical avidin-biotin-coupling (ABC) technique (Loeffler and Peterson, 1999). Briefly, sections were deparaffinized and rehydrated through a graded series of alcohol concentrations to PBS. Androgen receptor and C3 antigenicity was recovered by boiling sections in 5% urea (ICN, Aurora, OH) for 30 min, while CK 18 antigenicity was restored by digesting sections with 0.25% trypsin (Difco Labs, Detroit, MI) at 37°C for 30 min. Nonspecific binding was blocked by incubation for 20 min with 2% heat-inactivated goat serum (Vector Labs, Burlingame, CA) in PBSG (0.05 M phosphate buffered saline and 0.1% gelatin, pH 7.3). The primary antibody for the androgen receptor was a rabbit polyclonal antibody directed against a human androgen receptor fusion protein (NH27, kindly provided by Dr. A. Mizokami, University of Occupational and Environmental Health, Osaka, Japan). A rabbit polyclonal antibody directed against the C3 subunit was kindly provided by Dr. T. C. Shao, VA Medical Center, Houston, TX). A commercially available mouse monoclonal antibody directed against CK 18 (Sigma, St. Louis, MO) was used to assess the functional differentiation of ventral prostate luminal epithelial cells. Primary antibodies were diluted in the PBSG-goat serum-blocking solution as follows: anti-androgen receptor 1:1,200, anti-C3 1:20,000, and anti-CK 18 1:200. These were then incubated over the sections in a humid chamber at 4°C overnight. Immunochemical-positive cells were visualized by using the Elite Vectastain ABC kit according to the manufacturer's protocol (Vector Labs, Burlingame, CA). Sections were counterstained for 1 min in Mayer's hematoxylin, rinsed in running tap water until clear, dehydrated through a graded series of alcohol concentrations, cleared with xylene, and mounted with Permount. Negative controls, in which primary antibody was replaced with normal rabbit/mouse IgG, showed no nonspecific staining.
Statistical analysis.
Statistical analysis of data was performed using either the SAS statistical package (SAS Institute, Cary, NC) or Statistica/w 5.0 (Statsoft, Tulsa OK). Data presented are means ± SE; when multiple animals from a single litter were analyzed, data presented are litter means ± SE, so that each n represents an independent litter. Dam body weights were analyzed by repeated-measures analysis of variance (ANOVA), and survival was analyzed by Chi-square analysis. Fetal offspring body and testis weights, plasma testosterone concentrations, and intratesticular testosterone content were analyzed by Student's (homogeneous variance) or Cochran's (heterogeneous variance) t-test, whereas hCG-stimulated androgen production data were analyzed by 2-way ANOVA. Normalized prostatic binding protein-C3 subunit expression data obtained from ventral prostate organ cultures that were stimulated by 3 different androgens were analyzed by ANOVA. Steroid concentration was considered nested for each treatment within the stimulating steroid category. Levene's test was used to assess homogeneity of variance; when necessary, square root transformations were performed. Pup-body-weight data, and prostatic-binding protein-C3 data for PND 14 did not pass Levene's test, and were therefore analyzed by the Kruskal-Wallis procedure. All other statistical analyses were done using Student's t-test. Significance was set at p < 0.05.
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RESULTS |
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Testosterone Measurements
Plasma testosterone concentrations in control animals decreased by approximately 50% between GDs 18 and 20, and increased 5-fold between GD 20 and at 2 h after birth (Table 2). This later time point corresponds to the time of the expected postnatal testosterone surge. The same pattern of early postnatal changes in plasma testosterone concentration was observed in TCDD-exposed animals. In contrast to results reported previously (Mably et al., 1992a
), GD 15 exposure to 1.0 µg TCDD/kg did not decrease plasma testosterone concentrations at any of the time points examined; in fact, testosterone concentrations in TCDD-exposed animals ranged from 98 to 118% of control (Table 2
). Similarly, no significant decreases in intratesticular testosterone content were detected, as values from TCDD-exposed animals ranged from 87124% of control on a per testis basis (Table 2
). Even though testes from TCDD-exposed offspring were smaller than those from control offspring on GD 20 (Table 1
), there was no effect of in utero and lactational TCDD exposure at any time when intratesticular testosterone content was expressed on a testis-weight basis (Table 2
).
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On PNDs 14, 21, and 32, the specific activity of 3-HSD in all ventral prostates examined, regardless of TCDD-exposure, was at least 20-fold greater than that of the 5
-R type-2 isozyme (compare ordinate on Figs. 2B and 2C
). In addition, the ventral prostates removed from TCDD-exposed rats on PND 14 exhibited a 60% reduction in the amount of 3
-HSD specific activity, when compared to that in vehicle-exposed rats (Fig. 2C
). The magnitude of the TCDD exposure-induced decrease in 3
-HSD-specific enzyme activity, which was not statistically significant on PND 14, was reduced when the prostates were obtained on PND 21, and there was no difference between vehicle and TCDD-exposed rats in the specific activity of 3
-HSD on PND 32.
Androgen Metabolism
Ventral prostates obtained from vehicle- and TCDD-exposed rats on PNDs 11 and 18 were not exposed to any steroid for the 24 h in organ culture, and subsequently were exposed to [3H]-testosterone, [3H]-3-Diol, or [3H]-DHT for 48 h. At the end of the androgen exposure period, the radiolabeled steroid products were extracted from the culture medium on PNDs 14 and 21, respectively, and separated by TLC. While multiple radiolabeled products were recovered in the medium, the results indicate that the activities of 5
-R and 3
-HSD in the organ-cultured ventral prostates were apparently not inhibited by TCDD exposure. After 48 h of exposure to [3H]-testosterone or [3H]-3
-Diol, similar amounts of DHT were recovered from the culture medium of ventral prostates from vehicle- and TCDD-exposed offspring on PNDs 14 and 21 (Figs. 4 and 5
). In addition, similar amounts of DHT were recovered when vehicle- and TCDD-exposed ventral prostates were exposed to [3H]-DHT in organ culture. This suggests that TCDD exposure did not substantially enhance the ventral prostate's ability to metabolize DHT in organ culture. Indeed after 48 h, most of the [3H]-DHT was recovered from the culture medium in both treatment groups as the unmetabolized substrate on PND 14 (Fig. 4
). Similar amounts of DHT remained, and equal amounts of polar metabolites were formed by ventral prostates of both treatment groups after 48 h of exposure to [3H]-DHT that ended on PND 21 (Fig. 5
). Thus, there was never as much as a 2-fold difference between the vehicle- and TCDD-exposed ventral prostate cultures in the amount of DHT recovered, with any substrate at either time point. Therefore, these results do not suggest that there would be any substantial decrease in the amount of DHT available to vehicle- and TCDD-exposed prostates in the presence of similar concentrations of the DHT precursors. Rather, these results complement those of the direct enzyme assays for 5
-R and 3
-HSD activity. Taken together, all results support the conclusion that the effect of in utero and lactational TCDD exposure on ventral prostate development in the Holtzman rat is not associated with any lasting reduction in its ability to form DHT during the postnatal period, although there was a small decrease in 3
-HSD activity on PND 14 that was not statistically significant.
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Immunohistochemistry of the Cultured Vventral Prostates for Prostatic Binding Protein-C3 Subunit
Sections from vehicle-exposed prostates that had been cultured in the presence of 3-Diol were evaluated for immunohistochemical C3 expression. Less than one-half of the ducts from vehicle-exposed offspring examined on "PND 14" displayed the C3 subunit (Fig. 9A
). Staining was largely confined to epithelial cells, but in some cases the ductal lumens were also stained. In contrast to the vehicle-exposed ventral prostates, however, exposure of TCDD-treated ventral prostates to 108 M 3
-Diol stimulated little or no C3 staining in the epithelium or ductal lumens, even though numerous unstained epithelial ducts were present (Fig. 9B
). The periductal smooth muscle sheaths appeared to be thicker in the TCDD-exposed ventral prostates than in the vehicle-exposed prostates already described. This last observation is consistent with previous in vivo results (Roman et al., 1998
).
Vehicle-exposed ventral prostates obtained on PND 18 and cultured with 3-Diol to "PND 21" exhibited increased ductal development and more extensive staining for the C3 subunit compared to the corresponding results on "PND 14." Exposure to 108 M 3
-Diol caused a majority of ducts to exhibit epithelial C3 (Fig. 9C
). Unlike the results from "PND 14", however, most ductal lumens were also stained, indicating that most ducts were secreting prostatic binding protein. Results from sections of similarly cultured ventral prostates from TCDD-exposed rats corresponded to the previous analysis of cytokeratin-18 expression in that C3 staining was found in relatively few ducts, and such staining was more diffuse (Fig. 9D
). In most ducts, a majority of the periluminal epithelial cells did not stain for either cytokeratin-18 or -C3, and prostatic binding protein-C3 secretion into the lumen was not nearly as prevalent as it was in the vehicle-exposed organs. In addition, the smooth muscle sheaths of ducts in sections from TCDD-exposed ventral prostates appeared to be thicker than those sheaths from vehicle-exposed prostates. These results compliment the Northern blot analyses, and indicate that TCDD-exposed prostatic epithelium synthesized less C3 protein in response to 3
-Diol stimulation in organ culture than did prostatic epithelium from vehicle-exposed control rats.
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DISCUSSION |
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The lack of effect of TCDD on perinatal androgen production was somewhat unanticipated considering that TCDD exposure of adult male rats has been shown to decrease plasma testosterone concentrations by interfering with gonadotropin-stimulated testicular testosterone production (Kleeman et al., 1990; Moore et al., 1985
). Differences between responses of the perinatal and adult testes to TCDD exposure may be due to a dosage effect. The ED50 for decreased plasma androgen concentrations in the adult rat is 15 µg TCDD/kg, whereas the dose of TCDD used in the present study was 1.0 µg TCDD/kg maternal body weight. In Long Evans rats, about 0.15% of a maternal dose of 1.0 µg TCDD/kg administered on GD 15 is present in the fetal compartment on GD 16 (Hurst et al., 1996
). The approximately 300-g pregnant Holtzman dams in the present study were dosed on average with 0.3 µg TCDD. If Holtzman rats are similar to Long Evans rats, about 0.00045 µg of TCDD would have been distributed between each of about 10 fetuses. Since the body weights of GD 16 Holtzman fetuses are approximately 0.5 g, a rough estimate of the actual dose to each GD 16 fetus in this study is 0.09 µg/kg. Fetal body burdens later in gestation would most likely be lower than at GD 16, considering the rate of growth of the fetus. This estimated GD 16 fetal body burden is considerably lower than the NOAEL for decreased plasma androgen concentrations in adulthood (6.25 µg/kg, Moore et al. 1985).
Effects of in Utero and Lactational TCDD Exposure on Ventral Prostate Androgen Metabolizing Enzymes
The mechanism by which in utero and lactational TCDD exposure impairs development of the ventral prostate does not involve decreased circulating testosterone concentrations during the period just prior to, or immediately after, birth. However, testosterone is not the primary proximal mediator of ventral prostate development. To be effective in the ventral prostate mesenchyme, circulating testosterone must be converted in situ to DHT (George and Peterson, 1988; Wilson and Lasnitzki, 1971
). Prior to birth, testosterone is the predominant androgen in the fetal circulation (Corpechot et al., 1981
), and it is converted to DHT by 2 isozymes of 5
-R within the developing prostate (Berman et al., 1995
).
In the fetal rat, the type-2 isozyme, which is expressed exclusively in mesenchymal cells, is probably the most important of the 2 isozymes for androgenic signaling (Berman et al., 1995). In the present study, the anticipated response was a decrease in 5
-R activity following in utero and lactational exposure to TCDD. However, no TCDD exposure-induced decrease in the activity of either 5
-R isozyme was detected in the ventral prostate postnatally and similar results have been obtained for the dorsolateral prostate (Theobald et al., in press). In the adult rat, but not the fetal rat, this isozyme is subjected to feed-forward control, a mechanism by which type-2 5
-R activity can increase in response to DHT (Berman et al., 1995
). Therefore, it does not seem likely that the increases in this enzyme activity, which occurred in TCDD-exposed male offspring on PNDs 14, 21, and 32, could reflect decreased ventral prostate DHT levels, even if the amount of the type-2 isozyme expressed were to be considered androgen-responsive at these early postnatal time points.
Shortly after birth, 3-Diol, rather than testosterone, becomes the predominant circulating androgen, and remains so almost until the rat reaches puberty (Corpechot et al., 1981
). Thus, in the early postnatal period, most of the prostatic DHT content is derived from 3
-Diol by the action of 3
-HSD. Since the activity of this enzyme, unlike that of 5
-R is readily reversible, it can act as a molecular switch which controls the amount of DHT available for receptor signaling (Penning et al., 1996). In the early postnatal period, up to PND 32, ventral prostate homogenates contained substantially more 3
-HSD activity than 5
-R activity, which indicates the importance of this enzyme. In addition, the ability of neonatal ventral prostate homogenates to form polar metabolites over a period of 60 min when [3H]-3
-Diol is used as a substrate, but not during a similar 1-h incubation when [3H]-testosterone is used as a substrate, indicates that the ventral prostate is geared up for the elimination of 3
-Diol, but not testosterone. This observation again suggests the relative importance of the role that 3
-Diol plays within the neonatal ventral prostate, compared to that of testosterone.
On PND 14, the earliest day examined for enzyme activities in ventral prostate homogenates, in utero and lactational TCDD exposure transiently reduced the conversion of 3-Diol to DHT to about 40% of its value in vehicle-exposed offspring. Since previous results demonstrated a 37% decrease in ventral prostate DHT concentration (ng/g tissue wet weight) on PND 21 in response to in utero and lactational TCDD exposure (Roman et al., 1995
), it is possible that reductions in 3
-HSD activity, or a delay in the onset of its expression might have functional significance. Thus, greater effects on 3
-HSD activity than those observed on PND 14 could be present just after birth. However, the ventral prostates were quite small on PND 14 and the activity of 3
-HSD was low and difficult to measure. Therefore, an accurate assay of this enzyme activity during the first week after birth was not considered to be technically feasible. By PND 32, however, 3
-HSD activity (the present study) and ventral prostate DHT concentration (Roman et al., 1995
) have returned to control values.
It is not clear whether the observed alterations in 5-R and 3
-HSD activities reflect alterations in the enzyme composition of individual cells, or changes in the cell type composition of the developing prostate. Even though the activity of 3
-HSD in the rat ventral prostate was measured in the direction of DHT formation, no distinction could be made between similar enzyme activities that are catalyzed by different proteins. This is significant because the rat testis and ventral prostate each contain at least one 3
-HSD isozyme that also has retinol dehydrogenase activity (Biswas and Russell, 1997
; Hardy et al., 2000
), and the relative importance of each different 3
-HSD isozyme for androgen signaling has not been clarified. Nevertheless, in utero and lactational TCDD exposure causes alterations in cell type differentiation and composition within the developing prostate, which could have affected the activity measurements. The mechanistic relationships between these changes in androgen responsiveness, androgen metabolism, and epithelial differentiation in the developing prostate have not been fully characterized. However, some of these important developmental changes begin to take place during the period between birth and PND 14, the earliest time point examined in the present study.
Effects of in Utero and Lactational TCDD on Androgen Responsiveness
Ventral prostates, removed from TCDD-exposed offspring and cultured for 48 h in the presence of androgens, ending on PND 14, expressed significantly less C3 mRNA and protein than did androgen-treated ventral prostates removed from vehicle-exposed offspring. Similar to the effect of perinatal TCDD exposure on 3-HSD activity, there is evidence that this effect of TCDD may be transient, as recovery from the decrease in expression of this androgen-regulated secretory protein had started by PND 21. Similarly, in the dorsolateral prostate of rats exposed in utero and via lactation to TCDD, decreases in the expression of testosterone, DHT, and 3
-Diol-stimulated probasin mRNA expression in organ culture were observed (Theobald et al., 2000). Thus, the TCDD-induced decrease in androgen responsiveness is not unique to the ventral prostate as it occurs in other lobes as well.
Concomitant with the decreases in androgen responsiveness of the ventral prostate, which were indicated by reductions in C3 expression, androgen receptor levels in the periductal epithelium were also reduced following in utero and lactational TCDD exposure. It is possible that decreased androgen receptor levels may be the result of decreased exposure of the ventral prostate to circulating androgens, or to decreased conversion of precursors to DHT within the organ. This is because androgens can modulate the amount of androgen receptor in the rat embryonic urogenital tract either by inducing the proliferation of androgen responsive cells or by increasing androgen-receptor levels in individual cells (Bentvelsen et al., 1994, 1995
). However, other factors also influence the expression of rat ventral-prostate androgen receptors during embryonic development. Exposure of newborn rats to estrogens, for example, results in a permanent inhibition of androgen receptor expression in ventral-prostate luminal epithelial cells (Prins and Birch, 1995
). Since the administration of flutamide, an antiandrogen to pregnant rats, on GDs 1222 can decrease urogenital sinus androgen receptor expression in male offspring (Bentvelsen et al., 1994
), it is possible that TCDD could decrease postnatal androgen receptor expression by altering either androgen or estrogen signaling within the developing prostate. While the TCDD-induced decrease in epithelial cell androgen receptor expression was observed following organ culture, a similar effect has been observed in ventral prostates removed from TCDD-exposed rat offspring and evaluated without organ culture (Roman et al., 1998
). The present results advance our understanding of this effect because the decrease in androgen receptor expression was observed despite the fact that the cultured ventral prostates were exposed to normal physiological levels of exogenous androgens, including DHT, during the culture period. The decrease in androgen-receptor expression appears to have been imprinted, such that the critical period for androgen exposure to permit the development of androgen receptor-positive cells, and the effect of in utero and lactational TCDD exposure, most likely occur during fetal development. However, the ability of in utero TCDD exposure to potentially alter this imprinting by inhibiting type-2 5
-R expression in the fetal prostate could not be determined by measurements of enzyme activity because of technical difficulties caused by the small size of the organ and low activity level in normal rats. Genetic studies of type-2 5
-R mRNA expression in vehicle- and TCDD-exposed rats will have to be used to answer this question.
Effects of in Utero and Lactational TCDD on Luminal Cell Cytodifferentiation
The expression of cytokeratin 18 in the prostate epithelium is specific for luminal cells (Hayward et al., 1996). These are the only cells in the ventral prostate that synthesize and secrete C3 in response to androgenic stimulation. As TCDD exposure inhibited epithelial cell-androgen receptor expression and prostatic-binding protein C3 secretion in ventral prostates exposed to exogenous androgens in organ culture, it was unclear whether these responses reflect the development of atypical luminal cells, or a failure of luminal cells to develop. In the same organ-cultured prostates, it was found that in utero and lactational TCDD exposure caused an inhibition or delay of cytokeratin-18 expression in the postnatal prostate. This result indicates that TCDD exposure blocked the formation of luminal cells from their cellular precursors, which presumably are basal cells (Evans and Chandler, 1987
; Bonkhoff and Remberger, 1996
). A similar effect of in utero and lactational TCDD exposure has been described in ventral prostates that were not subjected to organ culture (Roman et al., 1998
). These results suggest the possibility that the central lesion caused by in utero TCDD exposure on prostate development involves an alteration in the differentiation pathway between basal cells and luminal cells, which is controlled by changes in gene expression, and modulated by the influence of hormones and growth factors, even though the mechanisms for causing this cellular transition are not understood.
The relative importance of direct effects of TCDD on gene expression in cells of the developing prostate, in contrast to the potential effects of TCDD exposure on endocrine or exocrine factors that modulate such gene expression cannot be determined, given the current state of knowledge. While the bulk of available information suggests that the ability of the testis to produce androgens, and the ability of the postnatal ventral prostate to form DHT from circulating androgens are not impaired, it is also true that the ability of TCDD to influence the formation of DHT in the fetal and postnatal prostate prior to PND 14 could not be determined by the methods used in the present study. The developing fetal prostate epithelium, which lacks androgen receptors, is somewhat coaxed along by its interactions with the androgen-responsive mesenchyme, making mesenchymal androgen responsiveness crucial to prostatic epithelial development. Therefore, it remains possible that TCDD exposure may inhibit androgen action and epithelial differentiation within a critical window that occurs during fetal prostate development. That is, TCDD may alter prostate development by acting directly on the fetal prostate. The effects of such direct prostatic TCDD exposure could be either to alter gene expression, or to change the expression of growth factors that may be regulated by posttranscriptional control mechanisms (Chang et al., 1999). Both mechanisms are consistent with in utero TCDD exposure causing an inhibition or delay in the ability of differentiating epithelial cells to respond to androgenic hormones without a decrease in androgenic-hormone concentrations. On the other hand, it is possible that TCDD could delay luminal epithelial cell development by interfering with estrogen signaling. As a result of whatever hormonal- or growth factor-mediated process is affected, a prostate that has been exposed to TCDD in utero and/or via lactation may develop fewer of the luminal epithelial cells that are responsible for secretory function later in life.
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ACKNOWLEDGMENTS |
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NOTES |
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1 Current address: Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, NIH, 6B/309, Bethesda, MD 20892.
2 Current address: Breeding Technology Laboratory, Department of Animal Science, Osaka Prefectural Agricultural and Forestry Research Center, 442, Shakudo, Habikino, Osaka 583-0862, Japan.
3 To whom correspondence should be addressed at the School of Pharmacy, University of Wisconsin, 425 N. Charter St., Madison, WI 53706. Fax: (608) 265-3316. Email: repeterson{at}pharmacy.wisc.edu.
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