Regional cellular heterogeneity and DNA synthetic activity in rat ventral prostate during postnatal development.

Previous studies have provided evidence that the rat ventral prostate grows primarily, if not exclusively, at its distal tips. However, as yet there have been no analyses in which individual cells in defined regions of the prostatic ductal system have been resolved and quantified. Moreover, the possibility that the prostate might grow differently at different times of postnatal development has received little attention. Our objectives were to identify and quantify the proliferating epithelial and stromal cells in defined regions of the rat ventral prostate during its postnatal development. To this end, 3H-thymidine was administered in vivo to rats of ages 10-60 days. A dissection technique was then used by which the distal, intermediate, and proximal segments of the prostatic ductal system were physically isolated from each other without removing the stromal tissue. Longitudinal sections of these segments were examined for cellular composition and DNA synthetic activity. Regional heterogeneity with respect to cell composition and cell proliferation was seen. In rats of all ages, DNA synthetic activity was seen in epithelial and stromal cells throughout the prostate, rather than only in the distal segment. At Days 10 and 20, significantly higher percentages of epithelial and stromal cells were labeled in the distal than in the proximal segments; but at Days 45 and 60, the percentages of labeled epithelial and stromal cells in the distal, intermediate, and proximal segments were similar. Thus, in all segments, and at all ages, substantial labeling was seen throughout the prostate. These data suggest that the prostate grows in both length and width throughout postnatal development, reminiscent of the growth of a tree.     

Effects of aging on growth factors gene and protein expression in the dorsal and ventral lobes of rat prostate

We hypothesize that various growth factors and their receptors gene and protein are modulated in dorsal and ventral lobes of aging prostate. To test this hypothesis, TGFbeta1, TGFbeta2 TGFbeta3, TGFbetaR-I, TGFbetaR-II, TGFalpha, EGF, EGFR, KGF and KGFR gene and protein expression were analyzed in dorsal and ventral lobes of aging rat prostates (1, 3, 6, 9, 12, 18, 24, and 28/30 months). KGF gene expression was very weak or absent in 1, 3, and 6 month old rat dorsal and ventral lobes of prostate whereas it re-expressed in 9, 12, 18, 24 and 30 month old rat prostate. All growth factors and their receptors expect KGF and EGFR were mainly localized in epithelium of ventral and dorsal lobes of aging rat prostates. EGF, TGFalpha, TGFbeta1, and TGFbetaR-I protein expression was lacking in stroma of dorsal and ventral lobes of 1, 3, 6, 9, 12/18 months old rat prostates. However, EGF, TGFbeta1 and TGFbetaR-I proteins re-expressed in stroma of 24 and 28 months old rat prostates. KGF protein expression was lacking in epithelium of dorsal and ventral lobes of all aging rat prostates. This is the first report to demonstrate differential gene and protein expression of growth factors in dorsal and ventral lobes is associated with aging rat prostate, suggesting their role in pathogenesis of prostatic diseases with aging.     

Glyceraldehyde-3-phosphate dehydrogenase expression during apoptosis and proliferation of rat ventral prostate.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme known to play a critical role in neuronal apoptosis. We undertook the current studies to determine whether GAPDH also plays a role in prostate epithelial cell apoptosis in response to androgen deprivation. To do so, we analyzed GAPDH staining by immunohistochemistry during castration-induced involution and androgen-induced regeneration of rat ventral prostate. We found that GAPDH was undetectable in secretory epithelial cells at baseline and that staining did not increase in the epithelium during the period of peak apoptosis from 1 to 3 days after castration. However, GAPDH levels did increase within nuclei of some basal epithelial cells 5 days after castration and within the cytoplasm of all secretory epithelial cells 7 days after castration. GAPDH was also abundant within the cytoplasm of secretory epithelial cells during the period of maximal cell proliferation from 2 to 3 days after androgen replacement and was clearly apparent within nuclei of some epithelial cells 4 days after androgen replacement. Our studies suggest that GAPDH plays multiple roles during prostate epithelial cell apoptosis and proliferation.     

In-vivo expression of a C-type RNA virus in rat ventral prostate epithelial cells.

During evaluation of a procedure for separating rat ventral prostate epithelial from connective tissue cells, isolated fractions were examined by transmission electron microscopy. Characteristic C-type RNA viruses were seen budding from or in close proximity to the plasma membranes of isolated epithelial cells. Dissociation of rat prostate cells and concentration of the epithelial fraction facilitated detection of this virus.     

Androgen regulated expression of a spermine binding protein gene in mouse ventral prostate.

A full length cDNA (MP25) encoding the major mouse prostatic secretory glycoprotein (p25), whose expression is androgen dependent, has been cloned and characterised. Steady-state levels of mRNA are decreased approximately 100-fold after 3 days castration but are restored progressively over 4 days with testosterone treatment. The secreted glycoprotein appears to be a spermine binding protein since the nucleotide and predicted amino acid sequence of MP25 shares extensive homology with a spermine binding protein (SBP) found in rat ventral prostate. Genomic clones indicate that there is a single gene for SBP which consists of 4 exons, the first of which is only 11bp in length. The second exon encodes the signal peptide, the third contains a portion of the spermine binding protein unique to the mouse and the largest exon encodes the bulk of the secreted protein.     

Pneumadin in the rat ventral prostate and its hormonal regulation.

Pneumadin (PNM) is a decapeptide originally isolated from mammalian lungs, and exerts a potent antidiuretic action by stimulating arginine-vasopressin release. We have recently developed a sensitive and specific radioimmunoassay (RIA) for rat PNM and detected high concentrations of PNM--not only in the rat lungs, but also in the prostate. Hence, we investigated whether prostate PNM content is regulated by sex hormones. Male adult rats were orchidectomized or sham-operated and given a subcutaneous injection of testosterone or estradiol (40 and 5 mg/kg), respectively. The animals were decapitated one week after surgery, and their ventral prostates were promptly removed and weighed. PNM concentration and localization in the prostate were investigated by RIA and immunocytochemistry (ICC). Orchidectomy resulted in significant decreases in the prostate weight and PNM concentration, and testosterone administration prevented these effects. Estradiol administration to sham-operated rats caused prostate atrophy without changing PNM concentration. ICC localized PNM immunoreactivity (IR) exclusively in the epithelial cells of the ventral prostate. Orchidectomy markedly reduced PNM-IR concentration, while testosterone abolished this effect. Estradiol did not modify PNM-IR concentration in the atrophic prostate of sham-operated rats. We conclude that PNM content of rat prostate is dependent on the presence of adequate levels of circulating testosterone. The possibility that PNM plays a key role in the maintenance of the prostate growth is unlikely since estradiol-induced gland atrophy is not associated with any decrease in PNM concentration. The localization of PNM in the epithelial cells could suggest that this peptide may be involved in the regulation of some testosterone-dependent secretory functions of the rat prostate.     

Aging in the rat prostate. Reduction in detectable ventral prostate androgen receptor content.

Aging in the rat is associated with a reduction in the detectable androgen receptor content of the ventral prostate. The reduction in cytoplasmic receptor content did not appear to be attributable to an aging-associated production of a receptor-inactivating factor or to an aging-associated change in the sedimentation properties of the androgen receptor of young and aged animals.Saturation analysis of cytoplasmic extracts prepared from two different breeds of similar albino rats and a genetically distinct strain of inbred brown rats demonstrated quantitative aging-associated reductions in the androgen-receptor content per cell of the ventral prostate. The reduction in receptor content per cell appeared to increase progressively in magnitude with increasing age. The mean value for the cytoplasmic androgen receptor sites per cell for the oldest animals (mean age 884 days) was only 14% of the mean value for the young mature animals (mean age 185 days) of the same breed. The binding affinities of the detectable androgen receptor of the young mature and aged animals were essentially identical. This observation does not eliminate the possibility that the observed reduction results from an aging-associated production of defective receptor. Evaluation of the total DNA content of the ventral prostate did not provide evidence for an aging-associated selective loss of receptor-containing cells. These data in toto were consistent with the interpretation that aging is associated with a mean reduction in the androgen-receptor content per receptor-containing cell.Both cytoplasmic and nuclear androgen retention were evaluated in vivo. These experiments provided qualitative confirmation of the in vitro saturation analyses as there was a highly significant aging-associated reduction in the amount of androgen specifically bound by these prostatic compartments. Total specific androgen retention by the ventral prostate of aging adults was reduced by 55% relative to young mature animals. This result was nearly identical to that obtained for the same breed and age category of animals when evaluated by in vitro saturation analysis.Preliminary in vitro experiments revealed a diminution in the uptake of androgen receptor by purified nuclei from aged animals relative to purified nuclei from young mature animals. The magnitude of the diminution in nuclear acceptor capacity was insufficient to account for the reduction in nuclear retention of androgen determined in vivo. The data were consistent with the interpretation that the cytoplasmic receptor is the major determinant of nuclear androgen retention in the ventral prostate.     

Differential regulation of protooncogene c-myc expression in rat ventral prostate after castration

To evaluate the possibility that the protooncogene c-myc plays a role in ventral prostate, the effects of castration have been investigated at a beginning of a period by Northern blot hybridization and the levels of c-myc mRNA were also compared with mRNA of androgen-regulated genes, C1 and TRPM-2. Levels of c-myc mRNA in ventral prostate increased with maximal stimulation reached at 6 hours (early induction) and 48 hours (late induction) after castration, respectively. The level of C1 mRNA did not change and TRPM-2 was not detected at early induction of c-myc mRNA after castration. The level of early induction of c-myc mRNA after castration was increased in ventral prostate treated with cycloheximide, but it was almost reduced by actinomycin-D pretreatment. Administration of androgen at the time of castration prevented early induction of c-myc mRNA. These results suggest that protooncogene c-myc is differentially regulated in ventral prostate after castration.     

性激素对血红素氧化酶在大鼠前列腺腹侧叶表达的影响

血红素氧化酶(heme oxygenase,HO)是产生内源性一氧化碳(carbon monoxide,CO)的限速酶,最近发现内源性CO在调节平滑肌张力方面起重要作用。而人的良性前列腺增生(benign prostates hyperplasia,BPH)所致的膀胱出口梗阻与前列腺平滑肌张力有密切关系,但还不清楚内源性HO/CO系统是否介导了前列腺平滑肌的活动。为了观察性激素对大鼠前列腺腹侧叶中血红素氧化酶-1(heme oxygenase-1,HO-1)和血红素氧化酶-2(heme oxygenase-2,HO-2)基因表达的影响,我们采用睾丸切除术建立雄性SD大鼠去势模型,用RT-PCR方法观察HO-1和HO-2的转录水平,应用免疫组织化学结合图像分析技术,观察去势、外源性雄激素和雌激素对前列腺腹侧叶中HO—1和HO-2蛋白水平的影响。结果表明,HO-1和HO-2在正常大鼠前列腺腹侧叶中都有表达,腺上皮细胞和纤维平滑肌间质呈现HO-1的免疫活性,HO-2的免疫染色仅在腺上皮细胞内检测到;去势组HO-1的mRNA和蛋白表达水平显著低于正常对照组(P<0.01):外源性给予雄激素组和雌激素组的HO-1表达水平明显增高(P<0.01),且雌激素主要增加前列腺纤维平滑肌间质的HO-1表达:HO-2在各组间的表达无明显差异(P>0.05)。这些结果提示,性激素对HO-1有诱导作用,但对HO-2无明显的影响,因此推测一氧化碳-血红素氧化酶(CO—HO)     

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.     

Nuclear non-histone proteins from rat ventral prostate cell under going hypertrophy of hyperplasia.

The distribution of nuclear non-histone proteins and their labelling with [¹⁴C]amino acids or [³²P]O₄ in rat ventral prostate cells undergoing hypertrophy (cell growth) or hyperplasia (cell division) were compared by SDS polyacrylamide gel electrophoresis. Marked quantitative changes in these proteins accompanied castration and replacement of hormone, but no absolute qualitative differences in their staining patterns were established. Preparations from cells undergoing hyperplasia were augmented in high molecular weight proteins and contained fewer proteins of <20000 D. The increased amounts of nuclear non-histone proteins from hormone-treated castrated rats were highly labelled by [¹⁴C]amino acids, but the molecular weights of radioactive proteins from cells undergoing hypertrophy were less diverse than those from replicating cells, Phosphorylation of nuclear non-histone proteins from short-term and long-term castrates, treated with testosterone propionate, was 170 and 60% greater than their controls. Proteins from 20–45 × 10³ D were actively phosphorylated. Nuclear extracts from dividing cells contained additional radioactive high molecular weight proteins and fewer phosphorylated lower molecular weight components. The distribution of phosphorylated proteins and newly synthesized proteins was dissimilar. Quantitative and possible qualitative differences in staining of nuclear proteins isolated in N-ethylmalleamideurea-phosphate buffer from normal or hormone-treated castrated rats were accentuated when they were separated by charge at pH 2.8. In replicating cells, a more generalized synthesis of acidic nuclear proteins from all molecular weight classes occurred, which were not as highly phosphorylated as less heterogeneous nuclear proteins from cells undergoing hypertrophy. Examination of the immediate and the subsequent events following androgen-induced cellular hypertrophy or hyperplasia in the ventral prostate permits their comparison in the same tissue.