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Estrogen Receptor-ß Messenger Ribonucleic Acid Ontogeny in the Prostate of Normal and Neonatally Estrogenized Rats¹

Departments of Urology (G.S.P., M.M., C.W., W.C., L.B.) and Physiology and Biophysics (G.S.P.), University of Illinois College of Medicine, Chicago, Illinois, 60612; and Center for Biotechnology and Department of Medical Nutrition, Karolinska Institute (G.K., J.-Å.G.), Huddinge, Sweden

Address all correspondence and requests for reprints to: Gail S. Prins, Ph.D., Department of Urology, M/C 958, 820 South Wood Street, Chicago, Illinois 60612. E-mail: gprins{at}uic.edu

	Abstract

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Neonatal exposure to estrogens permanently alters rat prostate growth and epithelial differentiation leading to prostatic dysplasia on aging. The effects are lobe-specific, with the greatest response observed in the ventral lobe. Recently, a novel estrogen receptor (ER) complementary DNA was cloned from the rat prostate and termed ER-ß (ERß) due to its high homology with the classical ER. The protein possesses high affinity for 17ß-estradiol, indicating that ERß is an alternate molecule for mediating estrogenic effects. Importantly, ERß messenger RNA (mRNA) was localized to rat prostatic epithelial cells, which contrasts with the stromal localization of ER in the rat prostate. The present study was undertaken to determine the ontogeny of ERß mRNA expression in the rat prostate lobes and to examine the effects of early estrogen exposure on prostatic ERß expression. Male rat pups were given 25 µg estradiol or oil on days 1, 3, and 5; were killed on day 1, 3 (oils only), 6, 10, 30, or 90; and prostate lobes were frozen. Longitudinal sections were processed for in situ hybridization using an ³⁵S-labeled antisense mRNA probe corresponding to a 400-bp EcoRI-AccI fragment in the 5'untranslated region of rat ERß complementary DNA. Image analysis was used to quantitate silver grains. In addition, total RNA was isolated from the ventral prostate (VP) and used for semiquantitative RT-PCR. Results from in situ hybridization revealed that at birth, ERß was equivalently expressed at low levels in both mesenchymal and epithelial cells in oil-treated rats. From day 1 onwards, expression in all stromal cells slowly and significantly declined, so that in the control adult prostate, stromal ERß mRNA was slightly above background. In the oil-treated control rats, epithelial ERß mRNA increased to moderate levels between days 6–10 in the VP and days 10–15 in the dorsal and lateral lobes as cells began differentiation and ducts lumenized. A further significant increase in ERß message was observed at day 30, which indicates that full epithelial ERß expression may require the completion of functional differentiation. By day 90, expression levels were maximal and similar between the lobes. RT-PCR substantiated this developmental increase in ERß between days 1–90. Neonatal exposure to estrogens did not have an immediate effect on prostatic ERß mRNA levels as determined by in situ hybridization and RT-PCR. However, the marked increase in epithelial cell expression at day 30 observed in the control VP was dampened in the VP of animals exposed neonatally to estrogens. By day 90, the VP of estrogenized rats possessed low ERß message levels compared with the high expression in oil controls. In contrast, the dorsal and lateral lobes of neonatally estrogenized rats possessed high levels of ERß mRNA at day 90, equivalent to controls. The present data demonstrate that ERß mRNA expression in the rat prostate is developmentally regulated, and that neonatal estrogen can affect this expression in the adult VP. Because the effect of neonatal estrogens was not immediate, the data imply that early estrogen exposure may not directly autoregulate ERß expression, and suggests that the adult effects on ERß mRNA expression may be indirect. The differences in ERß mRNA imprinting in the separate lobes may account for or reflect the lobe-specific neonatal estrogen imprints previously observed in the rat prostate.

	Introduction

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EXPOSURE of rats to exogenous estrogens during the neonatal period causes marked developmental abnormalities in the prostate gland and permanent alterations in its growth, secretory function, and activational response to androgens during adulthood (1, 2, 3, 4). This process, referred to as neonatal imprinting or developmental estrogenization, is associated with an increased incidence of prostatic metaplasia, dysplasia, and tumor formation with aging (5, 6, 7). Accordingly, the rodent model is used to evaluate the role of early exogenous and endogenous estrogen exposure as a potential predisposing factor for prostatic disease later in life. Work from our laboratory has shown that the neonatal estrogen imprint is mediated in part by a permanent loss in androgen receptor expression, and that this effect is lobe-specific, with the ventral lobe exhibiting the most pronounced response (4, 8, 9). Although all regions of the rat prostate exhibit early loss of normal androgen receptor expression after estrogen exposure on neonatal days 1–5, the proximal and central ducts of the ventral and dorsal lobes appear to bear these permanent imprints into adulthood, whereas the lateral prostate recovers from the estrogen exposure. Because the affected regions possess increased numbers of basal cells, reduced numbers of luminal epithelial cells, a significant delay in epithelial differentiation, and altered secretory gene expression (9), we have proposed that neonatal estrogenization blocks certain epithelial cells from entering a normal differentiation pathway. However, the mechanism whereby estrogen transmits this effect on the prostate gland is unresolved. Although some of the effects of estrogen may be indirectly mediated through alterations in circulating androgens and peptide hormones (10, 11), a direct response at the level of the prostate has also been documented (8).

The rat prostate gland is rudimentary at birth and undergoes developmental morphogenesis during the first 15 days of life (12). Recently, we showed that expression of the estrogen receptor- (ER) (13) was confined to mesenchymal cells in the urogenital sinus and proximal regions of the budding prostate lobes at birth, and that this expression declined with morphogenesis (14). Importantly, ER was never observed in epithelial cells of untreated animals using immunocytochemical techniques. Neonatal estrogenization resulted in a marked autoinduction of ER expression at the protein and messenger RNA (mRNA) level in periductal smooth muscle cells along the length of the prostatic ducts. Thus neonatal estrogen up-regulates its cognate receptor and amplifies the estrogenic effect. Based on this data, we concluded that mesenchymal and smooth muscle cells are the initial targets of estrogen action in the developing prostate, and postulated that stromal-derived paracrine factors may mediate the estrogenic effects on the adjacent epithelium (7).

Recently, a novel member of the steroid receptor superfamily was cloned from a rat prostate complementary DNA (cDNA) library and termed ERß due to its high homology with ER (15, 16). Because the ERß protein possesses high affinity for 17ß-estradiol, it is possible that ERß may be an alternate molecule for mediating estrogenic effects within the prostate gland. Using in situ hybridization, ERß mRNA was localized to the epithelium of the adult rat prostate (15), which indicates that this molecule regulates a different cell population than ER. To better understand its potential role in mediating the neonatal estrogen imprint on the prostate gland, we herein characterized the ontogeny of ERß mRNA expression in the rat prostate gland and examined the effects of early estrogen exposure on its expression pattern in the separate prostate lobes.

	Materials and Methods

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Animals
All rats were handled in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals. Timed pregnant female Sprague-Dawley rats were purchased from Charles-River (Indianapolis, IN) and housed individually in a temperature (21 C) and light (14 h light/10 h dark) controlled room. Rats were fed Purina rat chow (Ralston-Purina, St Louis, MO) ad libitum. They were monitored daily for delivery of pups, and the day of birth was designated as day 0. Pups were sexed according to anogenital distance, and female pups were removed. All males from a single mother were assigned to one of two treatment groups given sc injections of either 25 µg estradiol benzoate in 25 µl sesame oil or oil alone on neonatal days 1, 3, and 5. Animals were weaned on day 25 and subsequently housed two or three per cage. Pups from both treatment groups were killed by decapitation on day 6, 10, 30, 35, or 90. In addition, oil control pups were killed on days 1 and 3. Accessory sex gland complexes were quickly removed and placed in ice-cold PBS. Prostatic complexes or individual lobes were microdissected at 4 C using watchmakers’ forceps under a dissecting microscope. No enzymes were used during the dissection process. The tissues used for in situ hybridization were arranged on a nylon square, covered with OCT compound, frozen in liquified propane, and subsequently stored in liquid nitrogen. The tissues used for RT-PCR were snap frozen in liquid nitrogen and stored at -196 C.

In situ hybridization
A 400-bp antisense ERß complementary RNA (cRNA) probe corresponding to an EcoRI-AccI fragment of the 5'untranslated region (UTR) of the ERß was used for in situ hybridization. This fragment was subcloned into a Bluescript KS plasmid and, after linearization with EcoRI, T3 polymerase was used to transcribe ³⁵S-labeled antisense cRNA probe via the Riboprobe kit (Promega Corp, Madison, WI) with [³⁵S]UTP (40 mCi/ml; Amersham, Arlington Heights, IL). ³⁵S-Labeled sense cRNA was transcribed from a linearized AccI template using T7 polymerase and used as a control to define background levels of silver grains on sections adjacent to those probed with antisense cRNA. Two other ERß cRNA antisense and sense probes corresponding to the 5'UTR-N-terminal region (360 bp; AccI-PstI) and the 3'UTR region (300 bp; XBaI-EcoRI) were also employed in this study with similar results as obtained with the 5'UTR probe.

Frozen prostate complexes or individual lobes were mounted on precooled chucks (-20 C) in a Reichert-Jung cryostat (Leica, Deerfield, IL). Whenever possible, individual lobes were sectioned longitudinally to reveal the proximal-distal orientation. Prostate sections (6 µm) from days 1, 3, 6, 10, 30, and 90 oil control rats and from days 6, 10, 30, and 90 estrogen-treated rats were thaw-mounted on a single positive charged slide (Superfrost Plus, Fisher Scientific, Itasca, IL) to allow direct comparison of silver grain intensity over time and between treatment groups. A minimum of three rats at each time point for oil and estrogen-treatment were examined. The slides were fixed in 4% formaldehyde for 5 min at room temperature and acetylated for 10 min (0.25% acetic anhydride, 0.1 M triethanolamine, 0.9% sodium chloride, pH 8). Following a rinse in 2x SSC, the sections were dehydrated in ascending alcohol. Ninety microliters heat-denatured hybridization solution (50% formamide, 0.25 M NaCl, 1x Denhardt’s solution, 10% dextran sulfate, 25 µg yeast transfer RNA, 500 µg total yeast RNA, 100 µg sheared salmon DNA, 50 mM dithiothreitol, 0.05% sodium thiosulfate, 0.25% SDS) containing 20 x 10⁶ cpm/ml ERß cRNA probe was applied to each slide, and the slides were incubated for 16–20 h at 60 C in a humidified container. Slides were washed in a series of 2x SSC rinses and treated with RNase for 30 min at 37 C. Slides were rinsed in SSC under increasing stringency conditions with a final wash in 0.1x SSC at 60 C. After dehydration in alcohol, the slides were apposed to BioMax MR film (Kodak, Rochester, NY) for 7 days. The slides were then dipped in 1:1 Kodak NTB-3 emulsion and exposed for 2–4 weeks at 4 C before developing. The slides were counterstained with cresylviolet, dehydrated with alcohol, cleared with xylene, and cover slipped with Permount (Sigma Chemical Co., St. Louis, MO). Only background hybridization signal was detected on control slides that were incubated with radiolabeled sense strand RNA probes or in negative control tissue (spleen). Photographs of control and treated tissues were taken from the same slide using Kodak 125 X-Plus film.

To more accurately estimate the expression of ERß by particular cell types, an image analysis program written through Interactive Data Language was used to quantitate the number of silver grains per cell in control and treated prostates processed on the same slide. Twenty to thirty epithelial and stromal cells per field were outlined with the aid of a computer mouse, the in situ hybridization image was digitized, and pixels per unit area for each cell type were quantitated. Ten separate fields were analyzed for each section. Results are expressed as mean ± SEM. ANOVA was used to compare silver grain number over time, between cell types, and between treatment groups. The Schiff test was used to determine groups with significant differences.

RT-PCR
ERß and RPS16, a ubiquitous ribosomal RNA, were reverse transcribed and coamplified to obtain semiquantitative results. Total RNA was isolated from prostate tissue using guanidinium thiocyanate-chloroform extraction (RNA STAT-60; Tel-Test, Friendswood, TX). Two micrograms total RNA was reverse transcribed at 48 C for 50 min in 100 µl PCR reaction buffer (Perkin-Elmer, Norwalk, CT) with 10 mM deoxynucleotide triphosphates and 25 mM MgCl₂ through use of the reverse primers (see below) and 400 U of murine leukemia virus reverse transcriptase (Promega). The forward primers (see below), 2.5 U of Taq DNA polymerase (Perkin-Elmer), and 10 µCi [-³²P]deoxycytidine triphosphate (Amersham) were added to the reaction mix, and the sample was overlaid with light mineral oil. Amplification was carried out for 30 cycles by incubation at 94 C for 1 min, 55 C for 2 min, and 72 C for 3 min with a final extension at 72 C for 4 min in a Perkin-Elmer 9600 thermal cycler. The radiolabeled cDNAs were separated on 4% NuSieve/agarose (3:1) gel (FMC, Rockland, ME), and specific radioactive bands were quantitated on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) after transfer of emitted radioactivity on a phosphor plate. The intensity of the ERß signal was normalized to that of the ribosomal protein RPS16 internal control.

For ERß, the forward primer (ERß-5'primer: 5'-AAAGCCAAGAGAAACGGTGGGCAT-3') and reverse primer (ERß-3'primer: 5'-GCCAATCATGTGCACCAGTTCCTT-3') produced a 204-bp product corresponding to nucleotides +1018 to +1221 in the ERß hinge region (15). For RPS16, the forward primer (RPS16–5' primer: 5'-TCCAAGGGTCCGCTGCAGTC-3') and reverse primer (RPS16–3'primer: 5'-CATTCACCTTGATGAGCCCAT-3') produced a 100-bp product corresponding to nucleotides +59 to +158 (17). It was determined for each product that amplification for 30 cycles fell within the linear range with respect to the amount of input RNA. In addition, it was shown that coamplification of the two products for 30 cycles produced equivalent amounts of ERß and RPS16 as individual amplification of each product for 30 cycles. To confirm the identity of the ERß product, restriction digestion was undertaken. The 204-bp product was incubated with either HaeIII (1 U/µg DNA) or SmaI (1 U/µg DNA) for 22 h at room temperature. The digested products were separated on a 4% NuSieve/agarose gel, and the product sizes were compared with a molecular weight ladder. HaeIII digestion yielded two fragments of 145 and 59 bp as expected, whereas the SmaI digestion yielded the predicted 173- and 31-bp fragments (data not shown), confirming that the 204-bp amplification product was ERß.

RT-PCR was performed on ventral prostate tissue from days 1 (oil), 10, 35, and 90 from oil control and estrogen-treated rats in four to nine replicates. Comparisons between oil and estrogen treatment were always performed in parallel. The mean ± SEM of the relative values of ERß mRNA (normalized to RPS16) were obtained for each time point and treatment, and ANOVA followed by the Schiff test was used to determine statistical significance.

	Results

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Ontogeny of ERß mRNA
The ontogeny of ERß mRNA expression was followed in the prostate lobes using in situ hybridization from day 1 through day 90, which encompasses morphogenesis, pubertal growth, and adulthood. Figure 1 shows representative low-power images of the ventral prostate on days 1, 10, 30, and 90, whereas Fig. 2 compares silver grain intensity between day 1 and day 90 at high power. Graphic representation of silver grain counts per cell obtained with an image analysis system is shown in Fig. 3. Specific hybridization signal, visualized as bright silver grains present with the antisense cRNA probe (Figs. 1A and 2A) over the background signal seen with the sense cRNA probe (Figs. 1C and 2C) was present in low and equal amounts in epithelial and mesenchymal cells on neonatal day 1. From day 1 onwards, expression in all stromal cells slowly and significantly declined, so that in the adult prostate, stromal ERß mRNA was slightly above background levels (Fig. 3). In contrast, epithelial cell ERß message expression increased with development and growth. The first significant increase in expression was observed in epithelial cells from luminized ducts at days 6 and 10 in the ventral prostate (Fig. 1, D-F) and between days 10–15 in the dorsal and lateral lobes. A further marked increase in ERß message expression was noted at day 30 in all three lobes (Fig. 1, G-I). At day 90, maximal expression of ERß mRNA was realized in the epithelial cells (Figs. 1, J-L and 2, D-F). In the adult prostate lobes, a gradient in signal intensity for ERß mRNA was observed (Fig. 4). ³⁵S-Antisense labeling for ERß mRNA was strongest in the distal tips, moderate in the central ducts, and significantly lower in the proximal duct. Minimal background levels of silver grains with no ductal gradient were observed with ³⁵S-labeled sense RNA hybridized on adjacent sections (data not shown). The dorsal and lateral lobes exhibited the same ontogenic pattern, silver grain intensities, and expression gradient as the ventral prostate (data not shown).

View larger version (160K): [in this window] [in a new window]   Figure 1. ERß mRNA ontogeny in rat ventral prostate lobe. In situ hybridization for ERß message is shown on day 1 (A–C), day 10 (D–F), day 30 (G-I), and day 90 (J-L) of life. Sections shown with darkfield illumination in A, D, G, and J were hybridized with 35S-labeled antisense RNA complementary to ERß mRNA, whereas their adjacent sections were hybridized with 35S-labeled sense cRNA to measure background signal (C, F, I, and L). For histological detail, brightfield images of A, D, G, and J are shown in B, E, H, and K, respectively. To allow for comparisons of silver grain density, tissues from each time point were processed on a single glass slide. Photographs shown are from a 4-week exposure to emulsion. Low specific signal for ERß mRNA was observed in day 1 prostate exposed to 35S-labeled antisense cRNA (A), and equivalent numbers of silver grains were observed over epithelial and mesenchymal cells. As ducts lumenized at day 10 (arrows), increased intensity of silver grains was observed over epithelium exposed to antisense [35S]cRNA (D) as compared with signal in day 1 prostate. At day 30, a further increase in silver grains was found in epithelial cells as compared with day 10, whereas stromal cell signal for ERß was low (G). Maximal numbers of silver grains were found in epithelial cells at day 90 following exposure to 35S-labeled antisense cRNA (J), whereas stromal cell signal was not above background counts observed with 35S-labeled sense cRNA (L). Magnification: x33. Cresyl violet counterstain was used.

View larger version (159K): [in this window] [in a new window]   Figure 2. High-power images comparing in situ hybridization signal for ERß mRNA between day 1 (A-C) and day 90 (D-F) of life in ventral prostate lobe. Cellular details are visualized in brightfield images (A and D), whereas darkfield images at same locations (B and E) revealed specific silver grain signal on sections exposed to 35S-labeled antisense cRNA for ERß. Adjacent sections were hybridized to 35S-labeled sense cRNA to reveal background signal (C and F). To allow for comparisons of silver grain density, tissues from both time points were processed on same glass slide. Specific signal observed in epithelial cells at day 1 is quite low relative to ERß mRNA present in adult epithelium. Magnification: x330. Cresyl violet counterstain was used.

View larger version (24K): [in this window] [in a new window]   Figure 3. Silver grain counts per cell in ventral prostate sections hybridized with 35S-labeled antisense RNA complementary to ERß mRNA. An image analysis system was used to digitize and quantitate silver grains as described in Materials and Methods. Hatched bars represent mesenchymal/stromal cell counts, open bars represent counts from epithelial cells in nonlumenized ducts, and solid bars represent counts in epithelial cells from lumenized ducts. In day 30 and 90 prostates, epithelial counts were taken from an equal mixture of central ducts and distal tips. Bars represent mean ± SEM; a, P < 0.05 vs. days 3–90 for stromal cells b, P < 0.05 vs. day 1 epithelial cells; c, P < 0.001 vs. days 1, 3, 6, and 10 epithelial cells; d, P < 0.001 vs. days 1–30 epithelial cells.

View larger version (55K): [in this window] [in a new window]   Figure 4. A gradient in silver grain intensity was observed in epithelial cells along ductal length of adult day 90 prostate lobes. Representative photographs were taken of distal tip (A), central duct (B), and proximal duct (C) from a single ventral prostate sectioned along its longitudinal axis and hybridized with 35S-labeled antisense RNA complementary to ERß mRNA. Strongest signal was observed in distal tip, and signal intensity declined toward proximal duct. Adjacent sections probed with 35S-labeled sense RNA produced minimal background silver grains with no apparent gradient. D, Silver grain counts per unit area of epithelium in distal, central, and proximal regions using an image analysis system. Bars represents mean ± SEM of counts taken from three separate ventral lobes processed on one glass slide; a, P < 0.05 vs. distal region.

View larger version (37K): [in this window] [in a new window]   Figure 5. Semiquantitative analysis of ERß mRNA levels in rat ventral prostate from days 1–90 of life using RT-PCR. A, Representative gel of PCR products separated in 4% NuSieve/agarose and stained with ethidium bromide. Upper band is 204-bp ERß fragment and lower band is 100-bp fragment of RPS16 internal control. Weak ERß signal seen on day 1 shows a steady increase through day 90. B, PhosphorImager plate of representative gel seen in A shows radioactivity emitted from 32P-labeled cDNA products corresponding to ERß and RPS16. PhosphorImager system was used to quantitate radioactivity within each lane. C, Graphic representation of ERß mRNA ontogeny determined from six separate RT-PCR experiments and quantitated with PhosphorImager. ERß signal was normalized to RPS16 signal in each lane. Bars represent mean ± SEM of six separate reactions; a, P < 0.025 vs. day 1; b, P < 0.001 vs. day 1; c, P < 0.002 vs. day 10; d, P < 0.05 vs. day 35.

View larger version (182K): [in this window] [in a new window]   Figure 6. In situ hybridization for ERß mRNA in day 10 ventral prostate lobe of rats treated neonatally with oil (A–C) or estradiol benzoate (D–F). Brightfield images (A and D) and darkfield images (B and E) are shown of sections hybridized with 35S-labeled antisense RNA complementary to ERß mRNA. Arrows point to epithelial ducts that have begun to lumenize. Intense silver grains are found over epithelial cells in both treatment groups, and a lower amount of silver grains is observed over surrounding stromal cells. Adjacent sections hybridized with radiolabeled sense cRNA are shown in C and F and provide background level of silver grains. To allow for comparisons of silver grain density, tissues from both treatments were processed on same glass slide. Photographs shown are from a 4-week exposure to emulsion. Magnification: x133. Cresyl violet counterstain was used.

View larger version (92K): [in this window] [in a new window]   Figure 7. ERß mRNA in ventral prostate lobes of day 90 rats. A, Oil-treated control prostate hybridized with 35S-labeled antisense RNA complementary to ERß mRNA. Photograph was taken at distal-central duct interface, and heavy silver grain deposits are observed over epithelium. B, Neonatal estrogen-exposed prostate hybridized with radiolabeled antisense cRNA on same glass slide as tissue shown in A. Amount of silver grains over epithelium at distal-central duct interface was markedly reduced as compared with control prostate. Adjacent sections hybridized with 35S-labeled sense RNA showed minimal background silver grain levels, similar to that observed in Figs. 1 and 2. Magnification: x133. C, Silver grain counts per unit area of epithelium in day 90 prostates from oil-treated rats and estrogen-exposed (neoEB) rats using an image analysis system. Bar represents mean ± SEM of counts taken from three separate tissues in each treatment group; a, P < 0.001 vs. oil controls.

View larger version (39K): [in this window] [in a new window]   Figure 8. ERß mRNA levels as determined by RT-PCR in ventral prostates of rats exposed neonatally to oil or estradiol and killed on day 1, 10, 35, or 90 of life. A, PhosphorImager plate of a representative reaction shows radioactivity emitted from 32P-labeled cDNA products corresponding to a fragment of ERß (204 bp) and RPS16 (100 bp). At day 10, ERß mRNA levels in control and estrogenized prostates were similar. By day 35, ERß message in oil control prostate increased, whereas estrogenized prostate expressed low levels. At day 90, control levels of ERß mRNA were high, whereas estrogenized ventral lobe continued to express low levels. B, Graphic representation of ERß mRNA in ventral prostates of oil controls (solid bar) and neonatally estrogenized rats (hatched bar) on days 10, 35, and 90 of life. PhosphorImager system was used to quantitate radioactivity within each lane of an agarose gel, and ERß signal was normalized to RPS16 signal in each lane. Bar represents mean ± SEM of four to six separate reactions; a, P < 0.05 between oil and neonatal estrogen treatment on day 35; b, P < 0.005 between oil and neonatal estrogen treatment on day 90.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It is interesting to note that an expression gradient of ERß mRNA exists along the ductal axis of the adult prostate lobes with high amounts in the distal tips and significantly lower levels in the proximal ducts. Microdissection techniques, as employed in this study, have demonstrated that there is significant histological, structural, and functional heterogeneity in prostatic epithelium along the length of the branched ductal network from its origin in the urethra to the distal branched termini. Proliferative activity (DNA synthesis) occurs primarily at the distal tips (19, 20), whereas apoptosis has been observed primarily in the proximal prostatic ductal regions of intact animals (21, 22). Secretory protein expression has been demonstrated in epithelial cells in the distal tips and central prostatic ducts but not in the proximal regions of the prostate lobes (21, 23). Because it was determined that there is no gradient in androgen receptor expression or 5-reductase activity along the ductal length (24), the underlying mechanisms that regulate these heterogeneous androgen-driven activities along the prostatic ducts are not well understood. One possibility is that it may be attributed to variation in stromal cell organization along the ductal length (24, 25). Although the role of the ERß protein in the prostate is totally undetermined, it is a member of a superfamily of transcription factors that regulate gene expression in a variety of cells. That there may be an expression gradient of this transcription factor within prostatic epithelial cells along the ductal axis is an important consideration in evaluating heterogeneity in the prostatic ductal system.

Steroid autoradiography has been used in the past to localize specific binding sites for [³H]estradiol within prostate tissue. Theoretically, this approach should identify both ER as well as ERß, because both protein receptors have equivalent affinity for estradiol-17ß (16). Interestingly, epithelial localization of [³H]estradiol was observed in the adult rat dorsal prostate (26) and the hamster ventral prostate (27), albeit at weak levels as compared with specific androgen binding sites. Using tissue fractionation to separate epithelial and stromal cells, [³H]estradiol binding sites have also been found in the epithelial cell fraction of rat ventral prostate at one third the amount of stromal sites (28) or at equivalent amounts in both fractions (29) using sucrose density gradients. Thus, there is a precedence in the literature for ER in epithelial cells of adult prostate tissue. In contrast, specific [³H]estradiol localization was only observed in stromal cells of the developing mouse prostate and not in epithelial cells using autoradiography (30). It is possible that the low level of epithelial ERß mRNA observed in the day 1 prostate in the present study is either not translated into a functional protein at that stage, or that the level of ERß protein is below the limit of detection for autoradiography.

Because the present study showed that ERß message is present in the prostatic epithelial and mesenchymal cells at day 1 of life, it is possible that some of the neonatal estrogenic effects are mediated through this molecule. However, it is important to keep in mind that the ERß signal at day 1 is extremely low when compared with day 90 expression levels, and although the possibility exists that this low amount of message is translated into a functional receptor, its levels would most likely be minimal. Because the present findings also demonstrate that neonatal estrogen exposure does not autoregulate the ERß message at the time of estrogen exposure, any direct effects of estrogen mediated through ERß must be transmitted through low levels of this molecule.

Localization of ERß mRNA in prostatic epithelial cells, as opposed to stromal localization for ER, indicates that this estrogen-liganded transcription factor may activate a different set of genes than ER in response to estrogenic stimulation. In total, the estrogenized developing prostate has several potential mechanisms for mediating estrogen action through both ER and ERß. First, the up-regulated ER may directly influence mesenchymal and smooth muscle cells following neonatal estrogen exposure (14). Alternatively, estrogen action through stromal ER may indirectly affect epithelial cells by altering stromal-derived paracrine factors, which have been shown to be essential for normal epithelial differentiation and morphogenesis (31). In addition, estrogens may activate mesenchymal and epithelial ERß. In the later scenario, ERß, activated by estrogens, may directly affect epithelial differentiation and/or proliferation by altering specific genes in epithelial cells. Thus, actions of estrogens in the developing prostate epithelium need not be solely an indirect effect through the stromal compartment as previously believed.

A delayed effect of neonatal estrogen exposure on ERß expression was observed in the ventral prostate lobe in the present study, because the increased expression normally found in control animals at days 35 and 90 did not occur in the estrogenized rats. Because prostatic ERß appears to be under androgen regulation, one factor that may contribute to the reduced ERß expression is the reduced circulating testosterone levels following developmental estrogenization. Adult testosterone levels in our animal model were half the normal values following neonatal estrogen exposure (8). Another factor that would contribute to the reduced ERß mRNA levels is the differentiation defects in epithelial cells that occur following developmental estrogenization. Our previous work has shown that neonatal estrogen blocked cytodifferentiation and nearly eliminated androgen receptor and prostate binding protein expression in the ventral lobe (4). Because the ontogeny data described herein indicate that elevated ERß expression in the prostate is a function of cytodifferentiation and functional differentiation of the epithelium, we postulate that reduced ERß expression following neonatal estrogen exposure is a function of the differentiation defects. The lack of an effect on lateral prostate ERß mRNA levels is easily explained because differentiation defects are not observed in that lobe following early estrogen exposure. The dorsal lobe, on the other hand, does exhibit estrogen imprinting of the epithelium but to a far lesser degree than observed in the ventral prostate. Thus, dorsal lobe androgen receptor levels are half of normal values following neonatal estrogen treatment, and some secretory genes show minimal response to this hormone (probasin) or exhibit amplified expression (SVS II). Thus estrogen effects on dorsal lobe ERß would correspond to the probasin response in that region.

In summary, the present findings document the ontogeny of ERß mRNA in the rat prostate lobes, and demonstrate that it is a developmentally regulated gene that is expressed at high levels in epithelial cells following differentiation. This transcription factor may therefore play a role in the differentiated activities of the epithelial cell such as regulating secretory activity. Because estrogen did not affect ERß message levels for several days following exposure, the present data indicate that estrogens do not autoregulate this gene in the prostate gland. Finally, the present findings suggest that neonatal estrogen imprinting may be mediated, in part, through low levels of ERß in the neonatal mesenchymal and epithelial cells.

	Footnotes

 
¹ This work was supported by NIH Grants DK-40890 (to G.S.P.) and DK-09653 (to C.W.).

Received August 19, 1997.

	References

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