Brief exposure of male rats to estrogens during the neonatal period interrupts normal prostate development, alters epithelial cell differentiation, and predisposes this gland to hyperplasia and severe dysplasia analogous to prostatic intraepithelial neoplasia (PIN) with aging. Previous work demonstrated that the reduced growth, secretory activity, and androgen sensitivity that are observed in the adult ventral lobe are a function of reduced androgen receptor (AR) levels. Down-regulation of AR protein was found to occur immediately following neonatal exposure to estradiol benzoate (EB) and persist through adulthood and aging, indicating a permanent imprint on the ability of the prostate to express normal AR levels. To determine the intracellular mechanism of AR down-regulation by estrogens, the present study examined the effect of neonatal EB on AR gene transcription, mRNA levels, protein translation, and protein degradation in the d 10 ventral prostate glands. Nuclear run-on assays showed no alteration in AR gene transcription following exposure to EB on d 1-5 compared with controls. In situ hybridization and quantitative (q) RT-PCR revealed no difference in mRNA levels in the stromal or epithelial cells in response to estrogen exposure which, taken together, indicate that estrogen down-regulation of AR is mediated at the posttranscriptional level. AR translation was assessed with an in vitro transcription-translation assay in the presence of prostatic lysates from oil and estrogen-exposed animals, and no treatment effect was noted. AR degradation was examined in an in vitro assay validated with adult intact and castrate prostates. Prostatic lysates from intact rats initiated AR degradation with a t1/2 of 2.31 h, whereas proteins from castrate rats accelerated AR degradation to a t1/2 of 1.34 h (P < 0.001). Prostatic lysates from control d 10 prostates induced AR degradation with a t1/2 of 1.49 h, whereas estrogenized prostates increased AR degradation to a t1/2 of 1.11 h (P < 0.001). Proteosome inhibitors MG132 and ALLnL were able to reverse AR degradation induced by prostatic lysates from adult intact and castrate rats as well as from developing and estrogenized prostates, indicating that AR degradation was mediated through the proteosome pathway. Furthermore, the proteosome-mediated AR degradation in the estrogenized d 10 prostate was associated with a marked suppression of Akt phosphorylation that has been linked to AR degradation in other systems. Taken together, the present data show that exposure to neonatal estrogens down-regulates AR protein levels in the ventral prostate gland by accelerating AR degradation, which is mediated through the proteosome pathway.

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-beta (ER beta) due to its high homology with the classical ER alpha. The protein possesses high affinity for 17beta-estradiol, indicating that ER beta is an alternate molecule for mediating estrogenic effects. Importantly, ER beta messenger RNA (mRNA) was localized to rat prostatic epithelial cells, which contrasts with the stromal localization of ER alpha in the rat prostate. The present study was undertaken to determine the ontogeny of ER beta mRNA expression in the rat prostate lobes and to examine the effects of early estrogen exposure on prostatic ER beta expression. Male rat pups were given 25 microg 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 ER beta 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, ER beta 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 ER beta mRNA was slightly above background. In the oil-treated control rats, epithelial ER beta 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 ER beta message was observed at day 30, which indicates that full epithelial ER beta 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 ER beta between days 1-90. Neonatal exposure to estrogens did not have an immediate effect on prostatic ER beta 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 ER beta 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 ER beta mRNA at day 90, equivalent to controls. The present data demonstrate that ER beta mRNA expression in the rat prostate is developmentally regulated, and that neonatal estrogen...

Differential autoregulation of androgen receptors (AR) has been previously described for the separate lobes of the rat prostate gland. While AR are up-regulated by testosterone in the ventral, dorsal, and LP1 lateral lobes, the epithelial cells of the LP2 lateral ducts show continued expression of the AR protein following androgen withdrawal. To determine the mechanism of this differential autologous regulation, the present study examined the autoregulation of AR mRNA in the separate regions of the rat prostate gland. Northern blot analysis revealed that AR mRNA levels are down-regulated by androgens in all prostate lobes, since their levels increase following castration and decrease upon testosterone replacement. In situ hybridization confirmed that the increase in AR mRNA levels immediately following androgen withdrawal is due to increased transcripts per cell. When normalized to DNA content, the AR mRNA elevation upon androgen withdrawal was transient, and the value returned to control levels in the ventral and dorsal lobes within three days, while the elevation of AR message in the lateral lobe was prolonged. Quantitative reverse transcriptase-polymerase chain reaction studies revealed that elevated AR mRNA levels in the prolonged absence of androgens were confined to the LP2 ducts of the lateral lobe. Nuclear run-on experiments showed no alteration in AR gene transcription two days after castration in the ventral, dorsal, or LP1 lateral lobes when compared to the values in intact rats, indicating that posttranscriptional mechanisms are involved in AR mRNA autoregulation. In contrast, the AR gene transcription rate doubled in the lateral LP2 ducts. The elevated AR mRNA levels in the LP2 ducts due to increased AR gene transcription following castration may, in part, explain the continued expression of AR protein in that region in the absence of testosterone. However, the mechanism whereby AR translation becomes uncoupled from its AR mRNA levels in the ventral and dorsal lobes after hormone withdrawal remains unclear. In summary, the present data demonstrate that differences exist in AR mRNA regulation within the different regions of the rat prostate gland. These differences may begin to explain differential autoregulation of the AR protein in the separate prostate lobes.

Brief administration of estrogen to newborn rats permanently restricts prostatic growth and testosterone sensitivity in adulthood. Previous work demonstrated that neonatal exposure to estradiol benzoate produced lobe-specific imprints in prostatic androgen receptor (AR) expression. Epithelial cell AR was markedly reduced or absent in the adult ventral and dorsal lobes, which correlated with a lack of epithelial differentiation and responsiveness. While the lateral lobe also showed reduced growth and testosterone responsiveness after neonatal estradiol benzoate, normal cell differentiation and AR levels were observed within the adult epithelium. To determine the impact that these receptor imprints have on the functional capacity of adult tissue, we herein examined the expression of lobe-specific, androgen-dependent, or androgen-responsive secretory genes in prostates of rats given neonatal estradiol benzoate and directly compared this with epithelial cell AR using histological techniques. Sprague-Dawley rat pups were given 25 micrograms estradiol benzoate or oil on days 1, 3, and 5 and killed on day 90. Prostatic mRNA was analyzed using Northern blots and in situ hybridization. Ventral lobe mRNA was hybridized with a prostate binding protein (PBP) cDNA probe, while lateral and dorsal mRNA were hybridized with RWB (seminal vesicle secretory protein or SVS-II), probasin, and DP1 cDNA probes. Sections adjacent to those used for in situ hybridization were stained for AR by immunocytochemistry. Neonatal estradiol benzoate significantly reduced ventral lobe PBP message on Northern blots, and this was not restored with adult testosterone administration. There was a direct correlation between epithelial cell AR and PBP expression, in that PBP message and protein were only present in epithelial AR-positive cells and were absent in all AR-negative epithelium. In the lateral prostate, probasin expression was unaffected by neonatal estradiol benzoate, whereas RWB was slightly reduced using Northern analysis. By in situ hybridization, these messages were observed at normal levels in lateral lobe epithelial cells of estrogenized rats, which directly correlated with the presence of AR in those cells. In the dorsal prostate, different response patterns to neonatal estradiol benzoate were found for the three secretory genes analyzed. On Northern blots, DP1 message significantly declined, probasin mRNA was modestly suppressed, and RWB expression was significantly elevated compared to those in control tissue. In situ hybridization revealed that RWB expression in estrogenized dorsal lobes was amplified in AR-positive epithelial cells, whereas AR-negative cells appeared unaltered. In summary, prostatic functional activity after neonatal estradiol benzoate exposure is affected in a lobe-specific manner, which correlates with the AR imprints in the separate lobes.