Effect of androgen ablation on prostatic cell differentiation in dogs

This study was conducted to gain further insight into the role of androgen in maintaining a balanced prostate gland growth in dogs. Effects of castration on prostatic cell were assessed by comparing the expression level of high molecular weight cytokeratin (HMW), alpha-actin, and vimentin in intact and castrated dogs. Mature dogs were castrated while they were under general anesthesia and were killed after 1 month. Mature prostate gland structures from intact dogs are characterized by the presence of differentiated columnar secretary epithelial cells and progenitor basal cells that are located within acini and ducts embedded in a thin fibromuscular tissue. Basal cells were distinguished from secretory epithelial cells by HMW cytokeratin immunostaining, which is expressed specifically by basal cells but not by epithelial cells. Castration-induced secretory epithelial cell death, leave the basal cells intact to form a continuous layer lining the atrophied acini. However, the survived basal cells lost their capacity to differentiate to secretory epithelial cells. In addition, androgen ablation induced remarkable reorganization of the cellular components of the fibromuscular compartment. In intact dogs, this compartment of prostate gland is composed mainly of differentiated smooth muscles and scattered mesenchymal muscles as reflected by the high and low actin and vimentin expressions, respectively. Castration for 1 month induced a progressive shift toward mesenchymal cells, which appeared to occupy most of the fibromuscular compartment. Based on these findings, it appears that androgen acts to maintain a steady state of prostate gland by driving the differentiation of prostatic cells and by maintaining its fully differentiated state.     

Altered levels of angiopoietin 1 and tie 2 are associated with androgen-regulated vascular regression and growth in the ventral prostate in adult mice and rats

The involution of the rat ventral prostate gland after castration could be caused by primary changes in the vasculature. To explore the mechanisms, we studied the effects of castration and testosterone treatment on the vasculature in the ventral prostate in adult rats and mice. Androgen receptor expression, vascular morphology, and the expression of angiopoietin (ang) 1 and 2 and their receptor tie 2 were examined 1, 3, and 7 d after castration and after testosterone treatment of castrated animals using stereological methods, immunohistochemistry, laser capture microdissection, and Western blotting. One day after castration, the percentage of blood vessels covered with smooth muscle actin, endothelial cell proliferation, and vascular volume had decreased, whereas endothelial cell apoptosis had increased. Simultaneously, ang 1 and tie 2 protein levels decreased. Nuclear expression of androgen receptor was observed not only in glandular and stroma smooth muscle cells but also in the mural cells of prostate arteries and veins and was markedly down-regulated already 1 d after castration. Testosterone administration of castrated mice and rats reversed all the observed effects. At the mRNA level, tie 2 was exclusively, but ang 1 predominantly, expressed in the stroma, compared with the epithelial compartment. Local delivery of soluble tie 2 during testosterone-stimulated growth, inhibited vascular maturation and increased vascular volume and leukocyte infiltration compared with controls. We conclude that androgens may regulate the prostate vasculature by direct effects on mural vascular cells and by influencing the secretion of the angiopoietins, in above all, the stroma cells.     

Testosterone-stimulated growth of the rat prostate may be driven by tissue hypoxia and hypoxia-inducible factor-1alpha

Testosterone-stimulated growth of the ventral prostate (VP) in castrated rats is preceded by angiogenesis, but the mechanisms coordinating vascular and tissue growth are unknown. Adult rats were castrated and some treated with testosterone. Tissue hypoxia was studied morphologically using the hypoxia marker pimonidazole (Hypoxyprobe), hypoxia-inducible factor-1 (HIF-1) alpha, vascular endothelial growth factor (VEGF), and carbonicanhydrase 9 (CA-9) levels by western blotting and quantitative RT-PCR. In the intact untreated prostate, most glands were unstained by the hypoxia marker but already 1 day after castration most epithelial cells in the VP were stained. Seven days after castration prostate glands were apparently normoxic again, and HIF-1alpha, VEGF, and CA-9 were decreased. Treatment of 7-day castrated rats with testosterone resulted in increased epithelial hypoxyprobe staining and increased HIF-1alpha, VEGF, and CA-9 levels. The transient increase in tissue hypoxia after testosterone treatment is probably caused by a temporary mismatch between oxygen consumption and supply. Treatment of prostate epithelial cells in vitro under normoxic conditions also increased HIF-1alpha, and this could be blocked if epidermal growth factor receptor (EGFR) signaling was blocked with gefitinib. In vivo gefitinib could, however, not block the testosterone induced increase in HIF-1alpha. Testosterone may thus induce HIF-1alpha and its downstream angiogenesis promoting genes by at least two mechanisms, hypoxia and EGFR signaling. Transient epithelial cell hypoxia could by rapidly increasing HIF-1alpha and VEGF be an essential coordinator of testosterone-stimulated vascular and glandular growth.     

Interaction of testosterone with inhibin alpha and betaA subunits to regulate prostate gland growth

Testosterone regulation of prostate gland growth has been shown to involve reciprocal interaction with inhibin and activin. This study was therefore conducted to correlate the effect of testosterone on prostate gland proliferation and differentiation with the level of expression of inhibin α and βA subunits. Immature dogs were treated with testosterone for 0, 3, 7, and 14 days and prostate gland growth was assessed by morphological and immunohistological localization of differentiation and proliferation markers. The results showed that testosterone treatment resulted in an initial significant increase in PCNA proliferation index by days 3 and 7, followed by a significant decrease by day 14 post-treatment. Interestingly, the decrease of cell proliferation was associated with structural and biochemical changes characteristic of glandular and stromal differentiation of the prostate gland. These changes include progressive glandular ductal canalization and inter-ductal stroma differentiation which were apparent from a gradual shift from vimentin expression to vimentin and α-actin expression. Testosterone also had a differential effect on inhibin α and β subunits. Although testosterone treatment resulted in significant and constant inhibition of α subunit mRNA expression, it resulted in a significant increase of βA mRNA expression by day 3, followed by a decrease by days 7 and 14. These results indicated that testosterone acts first to drive proliferation of undifferentiated prostatic cells and then to maintain a low proliferation turnover of differentiated cells. Because it has been shown that activin is an antagonistic regulator of androgens, the attenuated stimulatory effect of testosterone on cell proliferation by day 14 might be mediated, at least in part, by interplay between testosterone and activin.     

Mechanism of androgen action on cell proliferation: AS3 protein as a mediator of proliferative arrest in the rat prostate

Androgens control the proliferation of their target cells first by increasing cell proliferation and later by inhibiting the proliferation of those same cells. Recently, we reported that the AS3 protein mediates the androgen-induced quiescence in androgen-target human cell lines. Our aims were to investigate the expression of the AS3 protein in the rat prostate in situ and in human cells in culture. Adult rats were separated into four groups (intact, castrated, castrated plus 3-d testosterone propionate replacement, and castrated plus 7-d testosterone propionate replacement). S9 cells expressing a tetracycline-regulated sense AS3 were also used. AS3 was expressed in the nuclei of over 90% of the epithelial cells and about 40% of the smooth muscle cells of the intact rat prostate. AS3 was not expressed in castrated rats or during the proliferative phase of androgen-induced regeneration. It was expressed in intact and castrated animals when the prostate has reached adult organ size. The AS3 protein was not expressed in cells that incorporate bromodeoxyuridine. These data suggest that AS3 is a mediator of the proliferative arrest in the normal rat prostate in situ and human prostate cell lines and that its expression is androgen-induced.     

Adrenomedullin gene is abundantly expressed and directly regulated by androgen in the rat ventral prostate

A gene-expression screen, looking for androgen response genes in the rat ventral prostate, has identified adrenomedullin (AM), a 52-amino acid pluripotent peptide hormone, first isolated from pheochromocytoma. Northern blot analysis demonstrates that the level of expression in the prostate is reduced at least 25-fold by castration, with the majority of the decrease occurring in the first day, and that androgen replacement in seven-day castrated rats stimulates expression to supernormal levels, with the majority of the increase occurring within 14 h. The level of expression in the prostate is at least 50-fold higher than in the adrenal gland and cardiac atria, tissues previously reported to have the highest level of expression in the rat. In prostate organ culture, androgen was able to induce AM expression; and this induction resists protein synthesis inhibition, indicating that AM is a direct androgen response gene in the prostate. In situ hybridization of normal rat prostate tissue showed that AM expression is localized in the epithelial cells. Our analysis demonstrates that AM, a multifunctional peptide hormone, is abundantly expressed and directly regulated by androgen in the prostate epithelial cells. Thus, AM has the potential to play a crucial function in androgen action in the prostate.     

Early effects of castration on the vascular system of the rat ventral prostate gland

Recent studies have found that blood flow to the rat ventral prostate gland is drastically reduced at an early time after castration. These observations caused us to reevaluate the effects of castration on the various cell populations of the ventral prostate, especially those in the prostatic vascular system. Sections of ventral prostate glands obtained at different times after castration were analyzed using the TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick END labeling) staining method to quantify apoptosis in different cell types. The results of this analysis showed a significant increase in TUNEL staining of prostate endothelial and (nonendothelial) stromal cells as early as 12 h postcastration that continued to 24 h after castration. In contrast, TUNEL labeling of prostate epithelial cells was not significantly increased compared with control values until 72 h after castration. The use of dual immunohistochemical staining procedures (anti-CD31 for endothelial cells or antismooth muscle actin for smooth muscle cells combined with TUNEL labeling) allowed us to confirm that the TUNEL-positive vascular cells at these early times after castration were endothelial in nature, whereas smooth muscle cells surrounding the prostate glands or portions of the afferent vascular endothelium were rarely TUNEL labeled. Electron microscopic evaluation of ventral prostate tissues at 48 h after castration provided further morphological evidence for the occurrence of apoptosis in prostate endothelial cells. Finally, the Lendrum-Fraser histochemical procedure used to identify fibrin leakage in tissues with vascular damage was applied to sections of the ventral prostate gland. This stain revealed diffuse fibrin accumulation in periglandular areas outside the capillaries and blood vessels in prostates from 24-h castrated rats, but not in prostates of sham-operated rats. Our results confirm an early effect of castration on the vascular system of the rat ventral prostate identified by increased apoptosis of endothelial cells and vascular leakiness. As these changes temporally precede the loss of epithelial cells, we propose that they may be causal rather than incidental to regression of the rat ventral prostate after castration.     

Steroid-sensitive gene-1 is an androgen-regulated gene expressed in prostatic smooth muscle cells in vivo

Steroid-sensitive gene-1 (SSG1) is a novel gene we cloned, found regulated by 17beta-estradiol in the rat uterus and mammary gland, and over-expressed in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. We show here that SSG1 mRNA and protein expression are regulated by androgens in the rat ventral prostate. Increases in SSG1 mRNA levels were detected by Northern blotting after 24 h and reached a 27-fold peak 96 h following castration, relative to SSG1 mRNA expression in sham-operated rats. Dihydrotestosterone or testosterone supplementation of castrated rats prevented this rise in SSG1 mRNA. In contrast with SSG1 mRNA expression, SSG1 protein was decreased 16-fold 2 weeks following castration but was at control levels in the prostates of castrated rats receiving dihydrotestosterone or testosterone. Although SSG1 is regulated by androgens in vivo, treatment of LnCap cells with dihydrotestosterone, cyproterone acetate or flutamide did not result in the regulation of SSG1 protein levels in vitro. Immunofluorescence studies show that SSG1 is mainly expressed in prostatic smooth muscle cells. These results indicate that SSG1 is an androgen-regulated gene that is expressed in the smooth muscle component of the rat ventral prostate in vivo.     

Increased androgen receptor expression correlates with development of age-dependent, lobe-specific spontaneous hyperplasia of the brown Norway rat prostate

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.     

Androgen receptor expression of proliferating basal and luminal cells in adult murine ventral prostate.

Maintenance of the size and differentiated function of the adult prostate is dependent on testicular androgens. In this study, simultaneous androgen receptor (AR) immunohistochemistry and [(3)H]thymidine labelling was used to characterise the proliferating epithelial cells of the murine ventral prostate. Proliferation in the adult prostate was more prevalent in the basal cell population with 1.8&percent; AR-negative cells labelled with [(3)H]thymidine as compared with 0.7% AR-expressing luminal cells. Three weeks following castration of mice, the atrophied prostate contained rudimentary glands composed of both luminal and basal cells with the proportion of AR-expressing basal cells reduced from 50 to 25%. Administration of testosterone enanthate to castrated mice induced a recapitulation of the prostate gland that was preceded by up-regulation of AR expression in basal cells to normal adult levels (50% AR-positive cells) by 12 h following testosterone injection. Proliferation of AR-positive luminal cells peaked at 48 h (22.8%) while proliferation of AR-negative basal cells peaked at 96 h (6.1%) following testosterone administration. These results suggest that distinct populations of luminal and basal cells are resistant to castration-induced involution of the prostate but remain responsive to direct or indirect testosterone effects and recapitulate the gland following administration of testosterone.     

Vascular endothelial growth factor (VEGF164) ameliorates intestinal epithelial injury in vitro in IEC-18 and Caco-2 monolayers via induction of TGF-beta release from epithelial cells

OBJECTIVE: VEGF is a glycoprotein with various (e.g. angiogenic) activities. So far, research has focused on its angiogenic properties. VEGF receptors are localized on epithelial cells of patients with inflammatory bowel disease (IBD) and also on Caco-2 and IEC-18 cells. Our aim was to evaluate the role of VEGF on intestinal epithelial cell (IEC) migration and proliferation by utilizing an established in vitro model. METHODS: IEC-18 and Caco-2 monolayers were wounded with a razor blade as described previously. Cells were incubated in medium w/o rat VEGF(164). After 24 h, migration was assessed by counting cells across the wound edge. Migration was blocked with neutralizing TGF-beta(1) antibodies. IEC proliferation was assessed using the MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) test. Semi-quantitative changes of the TGF-beta(1) mRNA expression were evaluated before and after stimulation of the cells with VEGF(164) by RT-PCR. Statistical analysis was performed with ANOVA and the Wilcoxon test. RESULTS: VEGF(164) significantly induced epithelial cell migration in Caco-2 and IEC-18 cells compared to control. TGF-beta(1) antibodies completely abolished this VEGF-induced cell migration. TGF-beta(1) mRNA significantly increased in IEC-18 and Caco-2 cells after stimulation with VEGF. VEGF significantly inhibited epithelial cell proliferation in IEC-18 and in Caco-2 cells, indicating that the observed effects on cell migration were not due to any proliferate effects. CONCLUSION: VEGF effects on epithelial cell migration play an important part in epithelial cell restitution by maintaining mucosal homeostasis after mucosal injury. This effect is mediated by TGF-beta(1). Our results obtain another possible role for increased VEGF levels in the intestinal mucosa of patients with IBD as reported recently by others.     

Androgen regulation of canine prostatic arginine esterase mRNA using cloned cDNA

Canine prostatic arginine esterase complementary DNA has been cloned in pPBS27, a new cloning vector. The relative abundance of androgen-regulated mRNA in intact dog prostate was reflected by the finding that a high proportion of the clones in the cDNA library hybridized strongly by plaque or colony hybridization with a poly(A)+ RNA probe from intact dog prostate but not with a poly(A)+ RNA probe from castrated dog prostate. One clone carrying a 400 base pairs cDNA insert was selected for further studies. Translation of the hybrid-selected RNA in a cell-free system resulted in the production of a 31 kDa peptide immunoprecipitable by antibodies against arginine esterase. This identification was confirmed by partial sequence analysis of the cDNA revealing an encoding protein with high homology to known kallikreins. Northern blot analysis of poly(A)+ and total RNA showed that arginine esterase mRNA had an approximate size of 1.0 kb which corresponded to a major androgen-regulated RNA species that could be observed after denaturing agarose gel electrophoresis of prostatic poly(A)+ RNA from intact dogs. Dot-blot analysis showed that dogs which had been castrated 3 weeks before had more than 100-fold lower arginine esterase mRNA level than intact dogs or castrated dogs treated with Depo-testosterone.     

Regulation of Apoptosis in the Prostate Gland by Androgenic Steroids

The prostate gland requires androgenic steroids for its appropriate embryological formation and postpubertal growth and, once at adult size, remains dependent on a continuous supply of androgens for its vitality and function. A reduction of the levels of circulating androgens will rapidly induce apoptosis of the cells of the prostate, leading to extensive glandular regression. Studies of rodent models of prostate response to castration have shown that there are some remarkable changes in the gene activity of prostate epithelial cells leading up to apoptosis. There is now evidence for a critical cell signaling pathway, regulated by c-fos expression, necessary for castration-induced apoptosis, as well as evidence that this signaling initiates an abrupt and transient alteration in the synthesis of fas antigen, p53, bax and bcl-2 proteins in the androgen receptor-expressing prostate epithelial cells, the cellular compartment that appears to be the most affected by castration. However, more recent studies suggest that these castration-induced effects on the prostate epithelial cells might be, at least in part, an indirect response to a critical reduction in blood flow to the prostate gland that precedes the onset of epithelial cell apoptosis. The castration effects on blood flow to the prostate gland seem to be related to vascular degeneration associated with apoptosis of a subset of prostate endothelial cells.     

Gonadotropin-releasing hormone desensitization preferentially inhibits expression of the luteinizing hormone beta-subunit gene in vivo

In this study we investigated changes in steady state cytoplasmic mRNA levels for LH subunits in pituitaries of male rats desensitized by continuous infusion of GnRH in vivo. Seven days of GnRH infusion (340 micrograms/day) reduced (P less than 0.01) LH beta mRNA levels in intact adult male rats and prevented the LH beta mRNA rise observed after castration. In contrast, common alpha mRNA doubled (P less than 0.05) in intact rats, and the elevated alpha mRNA after 7 days castration was unchanged. Serum and pituitary LH levels were suppressed below values of intact controls. Fourteen days of GnRH infusion (290 micrograms/day) further reduced LH beta mRNA levels in both intact and castrated male rat pituitaries. alpha mRNA levels in intact rat pituitaries were unchanged by 14 days of GnRH infusion, while in castrated rats there was a 23% (P less than 0.05) decrease, though values were still twice those of intact controls. As at 7 days, serum and pituitary LH were suppressed. Infusion of a superagonist analog (Buserelin) at a dose of 14 micrograms/day for 28 days reduced LH beta mRNA to 15% of intact control values in both castrated and intact rats. Common alpha mRNA was significantly (P less than 0.05) increased in intact rats and reduced by 13% (P less than 0.05) in castrates by superagonist infusion. These results were similar to those produced by 20- to 30-fold higher doses of native GnRH. GnRH and agonist analog effects were specific since no changes were observed in other mRNA species (GH, PRL, actin). These results indicate that in GnRH-desensitized gonadotropes LH beta gene expression is inhibited, and this may largely explain the reduced LH biosynthesis. However, there is a differential effect of continuous GnRH or agonist analog treatment on LH subunit gene expression, with a time-dependent stimulation of common alpha gene expression in intact rats. This may be caused by a stimulatory interaction between GnRH and progestagens at the level of the gonadotrope. Thus, common alpha gene expression is less tightly coupled than that of LH beta to GnRH action.     

Prostatic involution in rats induced by a novel 5 alpha-reductase inhibitor, SK&F 105657: role for testosterone in the androgenic response.

Within the prostate, androgen stimulates glandular cell secretion and proliferation while inhibiting glandular cell death. Due to its predominant nuclear localization, higher affinity for the androgen receptor, and more than 10-fold higher intracellular concentration than testosterone, dihydrotestosterone (DHT), not testosterone, appears to be the active intracellular androgen within the prostate of intact male hosts. The issue has remained unanswered, however, whether testosterone itself, without irreversible conversion to DHT by the 5 alpha-reductase enzyme, is capable of androgenic effects in the prostate. To address this issue, a novel dead end (i.e. product) inhibitor of the 5 alpha-reductase enzyme, SK&F 105657, was administered to intact or castrated male rats treated with either exogeneous testosterone or DHT. When administered twice a day orally at 25 mg/kg.dose, SK&F 105657 reduced the prostatic DHT content of either intact or castrated rats maintained with exogeneous testosterone to the same low level as that produced by surgical castration. Unlike castration, however, such SK&F 105657 treatment increased the prostatic testosterone content by more than 5-fold. The decrease in prostatic DHT coupled with a raise in testosterone are specifically due to the in vivo inhibition of the 5 alpha-reductase activity, since they were not observed in castrated rats maintained with exogeneous DHT. Treatment of intact or castrated male rats with exogeneous testosterone and oral SK&F 105657 (25 mg/kg, twice daily) resulted in a substantial inhibition of prostatic secretion, an inhibition of prostatic glandular cell proliferation, and an increase in prostatic glandular cell death. The magnitude of the changes, however, was not as great as that observed after surgical castration. The results are, however, specific for 5 alpha-reductase inhibition, since they were not observed in castrated rats given exogeneous DHT. These results demonstrate that if the prostatic testosterone content is elevated to sufficient levels, androgenic effects are induced without a requirement for an elevation in prostatic DHT content. Thus, the conversion of testosterone to DHT appears to function as a means of amplifying androgenic stimulation in the prostate.     

Canine prostate carcinoma: epidemiological evidence of an increased risk in castrated dogs

The present retrospective study investigated the frequency of prostate carcinoma (PCA) among prostate abnormalities in dogs and determined whether castration influences the incidence of PCA in dogs. During the years 1993-1998, 15,363 male dogs were admitted to the Utrecht University Clinic of Companion Animals, and of these dogs 225 were diagnosed with prostatic disease. In addition, another 206 male dogs were diagnosed as having prostatic disease based on cytologic examination of aspiration biopsies submitted by referring veterinarians. Benign prostatic hyperplasia was diagnosed in 246 dogs (57.1%), prostatitis in 83 dogs (19.3%), and PCA in 56 dogs (13%). Dogs with PCA were significantly older (mean age=9.9 years) than dogs with other prostatic diseases (mean age=8.4 years). The Bouvier des Flandres breed had an increased risk (odds ratio (OR)=8.44; 95% CI 4.38-16.1) of having PCA. Castration (26/56) increased the risk (OR=4.34; 95% CI 2.48-7.62) of PCA. The mean age at diagnosis of PCA in castrated dogs and in intact male dogs was not significantly different. The interval between castration and onset of prostatic problems was highly variable, suggesting that castration does not initiate the development of PCA in the dog, but it does favour tumor progression.     

The metaplastic effects of estrogen on mouse prostate epithelium: proliferation of cells with basal cell phenotype

The exogenous administration of estrogens to male mice alters the hypothalamic-pituitary-gonadal axis and reduces androgen levels, leading to a regression of the prostatic epithelium. As well, a specific direct response to estrogens is the induction of epithelial squamous metaplasia. The aims of this study were to identify the process by which the prostatic epithelium is transformed in intact adult male mice using the synthetic estrogen, diethylstilbestrol. A comparison of the effects of diethylstilbestrol in the three lobes revealed a hierarchy of response, with the anterior lobe being the most responsive, the dorsolateral lobe less responsive, and the ventral lobe the least responsive. The effect of castration was used to distinguish between the epithelial responses to estrogen administration and androgen deprivation. The results demonstrate that transformation of the epithelium involved proliferation of cells with a basal cell phenotype, the onset of cytokeratin 10 expression, up-regulation of progesterone receptor expression, and loss of the cell cycle inhibitor, p27(Kip1) expression; none of these changes was observed after castration. Mice lacking functional estrogen receptor alpha failed to respond, demonstrating a requirement for estrogen receptor alpha in the epithelium and/or stroma to mediate the proliferative response to estrogen in the prostate gland.     

Regional specificity of testosterone regulation of proopiomelanocortin gene expression in the arcuate nucleus of the male rat brain

Endogenous opioid peptides have been implicated as mediators in the negative feedback action of gonadal steroids on GnRH secretion. We have previously demonstrated that testosterone stimulates POMC gene expression in neurons of the arcuate nucleus. However, the wide distribution and variety of actions attributed to the numerous arcuate POMC neurons suggest that these cells may be heterogeneous in their responsiveness to steroid hormones. We tested the hypothesis that testosterone modulates a select population of POMC neurons within the arcuate nucleus of the adult male rat by comparing POMC mRNA signal levels throughout the arcuate nucleus of intact, castrated, and castrated testosterone-replaced adult males. Adult male rats were castrated and implanted (sc) with a Silastic capsule (30 mm) that was either empty (n = 6) or filled with crystalline testosterone (n = 5). Control sham-operated animals (n = 6) were left intact. In each animal the arcuate nucleus was divided into four equal rostral-caudal areas within which we measured POMC mRNA content in individual cells. We report that the effects of castration and testosterone replacement are observed in POMC neurons located in the most rostral region of the arcuate nucleus. After castration, POMC mRNA content was reduced in cells of the most rostral arcuate area (intact, 152 +/- 5 grains/cell; castrate, 119 +/- 2 grains/cell; P less than 0.0005), and replacement with physiological levels of testosterone prevented the decline in POMC mRNA levels so that they remained equivalent to that of the intact animal (castrated testosterone-replaced, 153 +/- 6 grains/cell). There was no significant difference in POMC mRNA signal between intact and castrated testosterone-replaced animals in the most rostral area. POMC neurons in the more caudal aspect of the arcuate (75% of the nucleus) were unaffected by the treatments; alternatively, it is possible that a real change in POMC message content in a subpopulation of cells was obscured by larger numbers of nonresponding cells within the same tissue sections. Based on these observations we conclude that there is a heterogeneous population of POMC neurons in the arcuate nucleus and that testosterone regulates POMC gene expression in a select group of these cells located in the rostral portion of the arcuate nucleus.     

N-acetyltransferase activity in the Harderian glands of the Syrian hamster, Mesocricetus auratus, is regulated by androgens and by hormones of the pituitary-thyroid axis

This study tested the hypothesis that activity of the enzyme N-acetyltransferase (NAT) in the Harderian gland of the Syrian hamster is regulated both by androgens and by hormones of the pituitary-thyroid axis. To test the effects of castration and hypothyroidism, intact or castrated male hamsters were given either tap water or methimazole in their drinking water for 3 weeks. Methimazole suppresses iodination of thyroglobulin, thereby decreasing circulating levels of thyroid hormones and increasing TSH levels. Hypothyroidism or castration caused elevated or depressed Harderian gland NAT activities respectively, compared with euthyroid controls. When castration and hypothyroidism were combined, the animals exhibited high NAT activity compared with castrated euthyroid males. To test the effects of castration and hyperthyroidism, male hamsters were given daily injections of thyroxine (T4) or diluent and were either castrated or left intact for 4 weeks. Intact animals given T4 had depressed Harderian NAT activity; serum thyroid hormone levels were elevated and TSH levels were depressed compared with those of intact controls. Castrated animals had depressed NAT activity below that of intact controls; serum thyroid hormone levels were normal but TSH levels were depressed. Castrated animals given T4 injections had NAT activity similar to that of euthyroid castrated hamsters; thyroid hormone levels were elevated but TSH levels were similar to those seen in euthyroid castrated hamsters. In another experiment, both T4 and tri-iodothyronine (T3) were equally effective in decreasing NAT activity in intact males. To determine the effects of the removal of pituitary influences, male hamsters were hypophysectomized. NAT activity in the Harderian glands of these animals was reduced compared with intact controls.(ABSTRACT TRUNCATED AT 250 WORDS)