scholarly journals A reconstituted cell-free system for the specific transfer of steroid–receptor complexes into nuclear chromatin isolated from rat ventral prostate gland

1971 ◽  
Vol 125 (1) ◽  
pp. 285-295 ◽  
Author(s):  
W. I. P. Mainwaring ◽  
Brenda M. Peterken
Keyword(s):  
Prostate Gland ◽  
Ventral Prostate ◽  
Free System ◽  
Tissue Specific ◽  
Receptor Complexes ◽  
Rat Ventral Prostate ◽  
Reconstituted System ◽  
Androgenic Steroids

1. A system has been developed for the specific transfer of [3H]dihydrotestosterone–receptor complexes into prostatic chromatin in vitro. 2. Under optimum conditions the overall transfer of [3H]dihydrotestosterone into purified chromatin in this reconstituted system is entirely consistent with the results obtained in whole tissue both in vivo and in vitro. 3. The transfer of [3H]dihydrotestosterone into chromatin is tissue-specific and maximal into chromatin isolated from androgen-dependent tissues. 4. The tissue specificity is maintained at two levels: first, in the presence of specific cytoplasmic androgen-receptor proteins; secondly, by the nature and composition of the chromatin itself. 5. Evidence is presented that androgenic steroids in vivo may maintain the tissue-specific nature of chromatin in androgen-dependent tissues by the selective induction of nuclear protein synthesis. 6. The relevance of these findings to the mechanism of action of androgenic steroids is discussed.

scholarly journals Studies on the solubilized ribonucleic acid polymerase from rat ventral prostate gland

1971 ◽  
Vol 123 (4) ◽  
pp. 619-628 ◽  
Author(s):  
W. I. P. Mainwaring ◽  
F. R. Mangan ◽  
B. M. Peterken
Keyword(s):  
Time Course ◽  
Prostate Gland ◽  
Exchange Chromatography ◽  
Ventral Prostate ◽  
Enzyme Preparations ◽  
Rat Ventral Prostate ◽  
Rapid Stimulation ◽  
Androgenic Steroids

1. By using ultrasonic treatment in media of high ionic strength, the RNA polymerase activities associated with prostatic nuclei and nucleoli can be completely solubilized. Such enzyme preparations are entirely dependent on the provision of added DNA for full activity. 2. The solubilized enzymes from the nucleolar and extranucleolar regions can be separated by ion-exchange chromatography. 3. Based on differences in the optimum DNA templates, pH optima and the effects of ammonium sulphate on the activities in vitro, Mn2+- and Mg2+-specific enzymes are associated with both the nucleolar and extranucleolar regions of prostatic nuclei. 4. Androgenic hormones administered in vivo have a particularly pronounced effect on the activity of Mg2+-dependent enzyme associated with the isolated prostatic nucleolus. 5. Time-course experiments in vivo show that androgens induce a rapid stimulation of the solubilized Mg2+-dependent nucleolar enzyme before a pronounced activation of nucleolar chromatin can be measured. 6. The implications of these findings to the mechanism of action of androgenic steroids are discussed.

scholarly journals Studies on the form and synthesis of messenger ribonucleic acid in the rat ventral prostate gland, including its tissue-specific stimulation by androgens

1974 ◽  
Vol 137 (3) ◽  
pp. 513-524 ◽  
Author(s):  
W. Ian P. Mainwaring ◽  
Peter A. Wilce ◽  
Allan E. Smith
Keyword(s):  
Prostate Gland ◽  
Sodium Dodecyl ◽  
Ventral Prostate ◽  
Experimental Conditions ◽  
Free System ◽  
Cell Free System ◽  
Messenger Ribonucleic Acid ◽  
Selective Incorporation

1. When prostate polyribosomes are labelled with radioactive precursors in vivo and subsequently dissociated with sodium dodecyl sulphate, a heterogeneous 6–15S RNA species may be identified that possesses all of the distinctive properties of mRNA. 2. Apart from the selective incorporation of 5′-fluoro-orotic acid into this 6–15S RNA component, it is bound by nitrocellulose filters under experimental conditions where only poly(A)-rich species of RNA are specifically retained. Most importantly, however, only the 6–15S RNA fraction is capable of promoting the incorporation of amino acids into peptide linkage in an mRNA-depleted cell-free system derived from ascites-tumour cells. 3. With the development of a simpler method for labelling the total RNA fraction of the prostate gland in vitro, the poly(A)-enriched RNA fraction may be readily isolated by adsorption and elution from oligo(dT)-cellulose. The synthesis of the poly(A)-enriched 6–15S RNA fraction is stringently controlled by androgens in a highly tissue- and steroid-specific manner. 4. From an analysis of the proteins synthesized in the ascites cell-free system in the presence of the poly(A)-rich RNA fraction, it appears that protein synthesis in the prostate gland is stimulated in a rather general way, even during the earliest phases of the androgenic response. This conclusion may require modification when more specific means of analysis are available than those used in the present investigation. 5. The implications of these findings to the mechanism of action of androgens are discussed.

scholarly journals Testosterone action in the rat ventral prostate. The effects of diethylstilboestrol and cyproterone acetate on the metabolism of [3H]testosterone and the retention of labelled metabolites by rat ventral prostate in vivo and in vitro

1971 ◽  
Vol 125 (1) ◽  
pp. 81-91 ◽  
Author(s):  
J. E. Belham ◽  
G. E. Neal
Keyword(s):  
Cyproterone Acetate ◽  
Prostatic Carcinoma ◽  
Prostate Gland ◽  
Target Organ ◽  
Ventral Prostate ◽  
Nuclear Retention ◽  
Rat Ventral Prostate ◽  
Androgenic Effect

Recent reports have indicated that the prior metabolism of testosterone by the secondary sexual tissues may be necessary for its androgenic effect. The effects of two anti-androgens, diethylstilboestrol and cyproterone acetate (17α-acetoxy-6-chloro-1,2α-methylenepregna-4,6-diene-3,20-dione) used in the chemotherapy of human prostatic carcinoma, have been examined on both the metabolism of testosterone and the retention of its metabolites by the rat ventral prostate gland. Cyproterone acetate was found to inhibit the retention of labelled metabolites of [3H]-testosterone by prostatic nuclei, both in vivo and in vitro. This inhibition appeared to be competitive. In contrast with its effect on nuclear retention of metabolites of testosterone, cyproterone acetate had no significant effect on the metabolism of [3H]testosterone by rat ventral prostate tissue. Diethylstilboestrol similarly had little effect on the metabolism of [3H]testosterone by prostatic tissue, although it did appear partially to inhibit its initial metabolism in all the incubation systems used. Diethylstilboestrol inhibited the nuclear retention of dihydrotestosterone when both [3H]testosterone and diethylstilboestrol were injected intraperitoneally in vivo, but had no effect on dihydrotestosterone retention when both testosterone and diethylstilboestrol were supplied directly to the prostate either in vivo or in vitro. It was concluded that if diethylstilboestrol has an anti-androgenic effect at the level of the target organ as distinct from its effect on androgen production by the testes, then it is probably due to a mechanism differing from that of cyproterone acetate.

scholarly journals Initiation and elongation of polyribonucleotide chains on rat ventral-prostate chromatin transcribed by homologous ribonucleic acid polymerase B.

1977 ◽  
Vol 166 (2) ◽  
pp. 189-198 ◽  
Author(s):  
P Thomas ◽  
P Davies ◽  
K Griffiths
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Control Of Gene Expression ◽  
Rat Ventral Prostate ◽  
Associated Proteins ◽  
Ribonucleoside Triphosphate ◽  
Androgenic Steroids ◽  
Transcriptional Process

The characteristics of initiation of RNA synthesis and the elongation of RNA chains on rat ventral-prostate chromatin by RNA polymerase B were investigated by two methods. 1. Initiation was carried out under low-salt conditions with three ribonucleoside triphosphates, and elongation was begun in the absence of reinitiation by the addition of the fourth ribonucleoside triphosphate and increasing the salt concentration. 2. Stable initiation complexes were formed by preincubation of enzyme with template at 37 degrees C, elongation was started by the addition of all four ribonucleoside triphosphates and reinitiation or spurious RNA synthesis was prevented by rifamycin AF/013. The latter method gave more reliable results. The dependence of those parameters on the androgenic status of the animal was studied. During the first 24h after castration, elongation was mainly affected, whereas after 72h a smaller number of initiation sites for RNA polymerase B on chromatin was evident. Considerable diurnal variations in the various parameters were observed. Changes in the relative concentrations of the chromatin-associated proteins were also observed after castration. In the rat ventral-prostate gland androgenic steroids may not only influence one stage of the transcriptional process, but may affect many factors involved in the control of gene expression.

THE BINDING OF [1,2-3H]TESTOSTERONE WITHIN NUCLEI OF THE RAT PROSTATE

1969 ◽  
Vol 44 (3) ◽  
pp. 323-333 ◽  
Author(s):  
W. I. P. MAINWARING
Keyword(s):  
Molecular Weight ◽  
Equilibrium Dialysis ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Rat Tissues ◽  
Receptor Complex ◽  
Rat Prostate ◽  
Binding Component

SUMMARY The specificity of the binding of [1,2-3H]testosterone to nuclei of various rat tissues in vivo has been studied. A significant amount of radioactivity was retained in the nuclei of androgen-dependent tissues only, particularly the ventral prostate gland. The bound radioactivity was only partially recovered as [1,2-3H]testosterone; the remainder was identified as [3H]5α-dihydrotestosterone. Efforts were made to characterize the binding component, or 'receptor', in prostatic nuclei. On digestion of nuclei labelled in vivo with [1,2-3H]testosterone, with enzymes of narrow substrate specificity, only trypsin released tritium, suggesting that the receptor is a protein. On the basis of subfractionation studies of labelled nuclei, the receptor is an acidic protein. The androgen—receptor complex could be effectively extracted from the prostatic nuclei in 1 m-NaCl and from the results of fractionations on a calibrated agarose column, the complex has a molecular weight 100,000–120,000. The specificity of the binding of steroids to such 1 m-NaCl extracts in vitro was investigated by the equilibrium dialysis procedure. Under these conditions, the specificity of the binding of [1,2-3H]testosterone demonstrated in vivo could not be simulated. The receptor is probably part of the chromatin complex but its precise intranuclear localization cannot be determined by biochemical procedures alone.

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

2001 ◽  
Vol 26 (3) ◽  
pp. 175-184 ◽  
Author(s):  
D Marcantonio ◽  
LE Chalifour ◽  
MA Alaoui-Jamali And H T Huynh ◽  
MA Alaoui-Jamali ◽  
MA Alaoui-Jamali ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Mrna Expression ◽  
Muscle Cells ◽  
Ventral Prostate ◽  
Mrna Levels ◽  
Rat Ventral Prostate ◽  
Steroid Sensitive

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.

scholarly journals Specific changes in the messenger ribonucleic acid content of the rat ventral prostate gland after androgenic stimulation. Evidence from the synthesis of aldolase messenger ribonucleic acid

1974 ◽  
Vol 144 (2) ◽  
pp. 413-426 ◽  
Author(s):  
W I P Mainwaring ◽  
F R Mangan ◽  
R A Irving ◽  
D A Jones
Keyword(s):  
Ribonucleic Acid ◽  
De Novo ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Mrna Synthesis ◽  
Hormonal Stimulation ◽  
Messenger Ribonucleic Acid ◽  
Stimulation Of

1. Aldolase was selected as a suitable marker for following the androgenic regulation of mRNA synthesis in the prostate gland. 2. Antibodies raised in rabbits against crystalline prostate aldolase were used to monitor the synthesis of this androgen-induced enzyme after hormonal stimulation of castrated animals, by using procedures in vivo and in vitro for the translation of prostate poly(A)-rich mRNA. 3. After androgenic stimulation in vivo the poly(A)-rich mRNA was isolated from the prostate gland and other tissues of castrated rats, and added to a protein-synthesizing system in vitro derived from Krebs II ascites-tumour cells. By using this approach it was found that androgens regulate the synthesis of aldolase mRNA in a highly tissue-specific manner. Stimulation of aldolase mRNA synthesis reached a maximum after 8h of androgenic treatment and then declined. 4. The androgenic control of aldolase mRNA synthesis was also investigated in vivo. After treatment of castrated animals with various steroids in vivo [35S]methionine was injected directly into the prostate gland, and labelled aldolase was selectively precipitated from isolated polyribosomes with anti-aldolase serum. The regulation of aldolase mRNA synthesis in the prostate gland was stringently steroid-specific and could only be evoked by androgens. After a single injection of testosterone, aldolase synthesis reached a maximum after 16h of hormonal stimulation and then declined. 5. Although androgens exert significant control over transcriptional processes in the prostate gland, and appear to regulate the synthesis of aldolase mRNA de novo, the possibility exists for additional means of control at the translational level of aldolase synthesis. The results are discussed in the context of the overall mechanism of action of androgens.

scholarly journals The control of the form and function of the ribosomes in androgen-dependent tissues by testosterone

1973 ◽  
Vol 134 (3) ◽  
pp. 795-805 ◽  
Author(s):  
W. I. P. Mainwaring ◽  
P. A. Wilce
Keyword(s):  
Protein Synthesis ◽  
Prostate Gland ◽  
Concomitant Administration ◽  
Ventral Prostate ◽  
Hormonal Stimulation ◽  
Pronounced Increase ◽  
Form And Function ◽  
Mandatory Requirement

1. The ribosome content of the rat ventral prostate gland is controlled by the concentrations of circulating androgens and the polyribosomal complement of the total population of ribosomes is acutely dependent on androgenic stimulation. After the administration of testosterone to castrated rats in vivo, there is a pronounced increase in the amounts of heavy (150–240S) polyribosomes. 2. These results are consistent with a pronounced increase in the mRNA and rRNA content of the prostate gland after the administration of testosterone in vivo. 3. From studies conducted both in vitro, the heavy prostate polyribosomes formed after androgenic stimulation are particularly active in protein synthesis. 4. The androgen-stimulated increase in the formation of prostate polyribosomes has a mandatory requirement for sustained RNA and protein synthesis. 5. Since the androgen-mediated increase in prostate polyribosomes may also be suppressed by the concomitant administration of certain anti-androgenic steroids in vivo, the response in polyribosome formation is probably initiated by the binding of a metabolite of testosterone, 5α-dihydrotestosterone, in the prostate gland. 6. The relevance of these findings to the pronounced increase in protein synthesis in androgen-dependent tissues after hormonal stimulation is discussed.

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