Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate

Binding sites of [3H]testosterone and [3H]dihydrotestosterone in the rat fetal urogenital sinus and postnatal prostate and vagina grown in vitro were examined by steroid autoradiography. Distinct nuclear incorporation of both androgens appeared between 14.5 and 16.5 days of gestation in rat fetuses. Nuclear labelling in the sinus was restricted to the mesenchyme surrounding the epithelium which showed no nuclear labelling. A similar distribution of labelled cells was observed in male and female sinuses up to 18.5 days of gestation. By 20.5 days of gestation, the labelling in the ventral mesenchyme of female urogenital sinuses became less intense but persisted in the mesenchyme of the dorsal sinus wall from which the vagina is formed. In the postnatal prostate, the epithelium showed nuclear [3H]testosterone labelling at 10 days coinciding with the onset of its functional differentiation. Epithelial labelling became more intensive at 4 weeks post partum while that of the mesenchyme declined. The results suggest two phases of androgen action: formation of the prostatic buds mediated by the androgen-activated mesenchyme of the fetal urogenital sinus and the differentiation of the postnatal prostatic epithelium directly stimulated by androgens.     

Change of mosaic pattern by androgens during prostatic bud formation in XTfm/X+ heterozygous female mice

The testicular feminization (Tfm) gene, which is characterized by a deficiency in androgen receptors, is located on the X-chromosome. Using steroid autoradiography, the mosaicism of the Tfm gene has been demonstrated in the androgen target tissues of XTfm/X+ heterozygous female mouse fetuses and the effects of androgens on the mosaic pattern analysed. In the mesenchyme of urogenital sinuses of wild-type female fetuses (X+/X+), more than 95% of the cells were androgen-receptor positive (labelled with [3H]testosterone) while in that of heterozygous fetuses (XTfm/X+), only half of the cells were receptor positive (Tfm gene inactive), and receptor-positive cells and -negative cells formed small irregular patches. When the heterozygous sinuses were cultured in vitro in the presence of androgens, the sinuses underwent male sexual development and formed epithelial buds (prostate gland rudiments) projecting into the surrounding mesenchyme. Autoradiographic analysis revealed that the mosaicism of the mesenchyme disappeared around the developing epithelial buds: almost all the mesenchymal cells in close vicinity to the buds were receptor positive while in the outer layers receptor-positive and -negative cells coexisted. The proportion of receptor-positive cells was greatly increased in the mesenchyme beneath the non-budding area of the sinus epithelium. This androgen-induced increase was observed before the onset of bud formation. The results obtained in the thymidine incorporation experiments suggest that the increase of receptor-positive cells beneath the sinus epithelium might be explained by the migratory behaviour of the androgen-incorporating cells rather than by their selective proliferation.     

Peripheral androgen levels in the male brush-tail possum (Trichosurus vulpecula)

Radioimmunoassays were established for the measurement of total androgens and the specific measurement of testosterone and 5 alpha-dihydrotestosterone in peripheral plasma of the brush-tail possum. Androgen concentrations were measured in blood collected from indwelling jugular cannulae to determine whether the normal pattern of androgen secretion in this species was episodic and to attempt to relate total androgen and the pattern of testosterone secretion to the changes previously reported in prostatic, but not epididymal, weight in the breeding season. Blood was collected from restrained animals at varying time-intervals during daylight hours and darkness. Despite an apparent good adaptation to the sampling procedure there was generally a progressive decline in plasma androgen level during the collection period. This was true for animals bled during or out of the breeding season. There was no significant seasonal effect on the androgen concentration in the initial blood sample. When less restraint was used, two of three animals showed fluctuations in androgen levels over the 7-h sampling period. Testosterone levels in blood obtained by cardiac puncture were four- to nine-fold higher than those of 5 alpha-dihydrotestosterone but levels of these androgens in samples obtained during the breeding season were not significantly different from those obtained out of season. The results do not argue for a pulsatile release of testosterone in the possum but do demonstrate a marked capacity for changes in the peripheral androgen concentration. There was a poor correlation between testosterone and 5 alpha-dihydrotestostosterone levels and prostatic weight.     

Distribution and concentrations of androgens in epithelial and stromal compartments of the human benign hypertrophic prostate

Prostate tissues removed from patients with benign prostatic hypertrophy were separated into epithelia and stromal components and the concentrations of testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol, 4-androstene-3,17-dione, 5 alpha-androstanedione and androsterone in these two fractions were determined by radioimmunoassays after the purification of solvent steroid extracts by Lipidex-5000 column chromatography. On a 'per cell' basis (i.e. relative to DNA), testosterone was equally distributed between the two components, while the other androgens measured were more abundant in the stroma. The observation that 5 alpha-reduced androgens (especially 5 alpha-dihydrotestosterone) were more concentrated in the stroma, and that significant correlations between concentrations of metabolically related androgens were more common in the stroma than in the epithelium, indicate that the stroma is an important site of androgen metabolism in benign prostatic hypertrophic tissues. The present data also support the suggestion that 5 alpha-dihydrotestosterone produced in the prostatic stroma may be transferred to the epithelium by way of sex hormone binding globulin in the extracellular spaces of the prostate.     

The dynamics of the steroidogenic response of perifused Xenopus ovarian explants to gonadotropins

Steroid release before and during maturation was studied in Xenopus ovarian follicles under various conditions of stimulation of LH in a perifusion system. Acute stimulation by 15 micrograms of LH induces a 10-fold increase in the androgen (testosterone and androstenedione) level which reaches a maximum 4 hr later and then slowly decreases until the 25th hour. Repeated stimulations every 2 or 4 hr are followed by the same androgen increase during the first 8 or 10 hr and then by a slow decrease in the secretion despite new LH injections. A significant increase in progesterone secretion is seen only after at least two stimulations (8 hr). Estradiol secretion slowly increases to a moderate level during the first 5 hr and then remains stable whatever the stimulation. During continuous stimulation (LH 0.5 microgram/ml) androgen levels reach an initial maximum after 4 hr and then fluctuate with a 2-hr period. Addition of theophyllin to the medium enhances these fluctuations. After 12 hr when the progesterone has increased, androgen secretion diminishes to reach a basal level without fluctuations. Germinal vesicle breakdown occurs only in follicles that have been appropriately stimulated to secrete androgens and progesterone during the required time.     

Endocrine effects of high-dose ketoconazole therapy in advanced prostatic cancer

The endocrine effects of ketoconazole (400 mg orally every 8 h) were studied in 9 previously untreated patients with advanced prostatic cancer. Five of these patients were followed for 12 months. A rapid fall in the serum concentration of testosterone was noted in all patients studied. Minimal values were observed on day 4 of treatment but thereafter serum testosterone increased slowly. The effect of the drug on unbound testosterone was relatively more important, since sex hormone binding globulin increased markedly during treatment. An increase in progesterone and LH was observed in all patients. This suggests that ketoconazole limits the conversion of C21-precursors into androgens. This block is compensated in part by activation of the hypothalamo-hypophyseal feedback system. Urinary 17-ketosteroids were decreased but 17-hydroxysteroids were unaffected by the treatment. In 5 patients followed monthly over a period of 12 months the mean testosterone concentration ranged from 69 ng/100 ml in one patient to 428 ng/100 ml in another. An excellent inverse correlation could be demonstrated between the mean serum concentration of testosterone and the mean concentration of ketoconazole. The change of serum dehydroepiandrosterone sulphate also correlated inversely with the mean ketoconazole level. Increased concentrations of oestradiol were noted in 2 patients with slight gynaecomastia. It is concluded that long-term suppression of androgen production can be realized by high-dose ketoconazole treatment and that the degree of suppression is proportional to the serum levels of the drug.     

Prostate diseases--role of sex steroids and their inhibitors

The normal prostate as well as prostatic diseases are influenced by androgens. The exact reason for an altered and uncontrolled response to androgens, whether benign as in benign prostate hyperplasia (BPH) or malignant as in the case of prostate cancer (PC), is not known in detail. Nevertheless, restriction of androgen receptor activation by reduction of available androgens is of great clinical value in both diseases. In BPH the inhibition of the conversion of testosterone into 5α-dihydrotestosterone (DHT) by 5α-reductase (5AR) is highly efficient and used in general practice, while the situation in PC is more complex. Specific inhibition of 5AR does not provide as efficient relief of symptoms as general androgen deprivation therapy (ADT), and the use of 5ARI for PC prevention is still under debate. Further, the altered steroid metabolism in castration resistant prostate cancer (CRPC) together with the complex paracrine signalling between different androgen responsive cell types, make the development of more specific drugs targeting androgen receptor signalling both more relevant and challenging.     

Production of testosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and androsterone by dispersed testicular interstitial cells and whole testes in vitro.

The production of 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), androsterone and testosterone by whole rat testes and testicular interstitial cells dispersed with collagenase was studied in vitro. Luteinizing hormone stimulated the production of each of the androgens by cells prepared from 31- to 34-day-old rats. Half maximum stimulation of the production of each androgen occurred with approximately 3.5 ng NIH-LH-B9/ml medium. Androstanediol was the predominant product then androsterone and then testosterone. Luteinizing hormone stimulated the production of testerone, but not androstanediol or androsterone by dispersed interstitial cells from 200-day-old rats. The time-course of production and the effect of the concentration of cells on the production of these androgens suggested that in dispersed testicular interstitial cells from immature animals androstanediol and androsterone are formed, at least partially, by the metabolism of testosterone. In these experiments LH-stimulated testosterone production increased during incubation for 15--60 min and then remained constant up to 180 min. The concentrations of androstanediol and androsterone increased in a linear manner during incubation for 60--180 min. Varying the number of cells incubated yielded a positive correlation between cell concentration and the ratio 5 alpha-reduced androgen : testosterone produced. Luteinizing hormone stimulated production of each androgen by whole tests obtained from rats at 30--175 days of age. The serum concentration of testosterone in these rats increased abruptly at 50 days of age. Significant changes in androgen production in vitro also observed at this age included: (1) increased production of the three steroids when incubated in either the presence or absence of LH and (2) testosterone production, either in the presence or absence of LH, which represented a greater percentage of the total production of the three androgens.     

Norepinephrine amplifies human chorionic gonadotropin-stimulated androgen biosynthesis by ovarian theca-interstitial cells

Ovarian theca-interstitial cells, when cultured in serum-free medium, secreted androgens in response to hCG stimulation. This production was dependent on time (maximum production attained after 96 h) and dose (half-maximal effective dose of hCG, 9 ng/ml). When the sympathomimetics norepinephrine, epinephrine, and isoproterenol were added to the medium, androgen production in response to hCG was enhanced by 100-300%. The ability of the catecholamines to stimulate androgen production was dependent on the continuous presence of hCG. Treatment with catecholamines alone did not induce theca-interstitial cells to produce androgens. Catecholamine stimulation of steroid hormone metabolism was selective for intermediates in the delta 4-pathway, with greatest increases occurring in the production of androstenedione and testosterone. It was found that the effect of the catecholamines on androgen production was dependent on both beta 1-and beta 2-adrenergic receptors. The acquisition of catecholamine responsiveness was specific to hCG; if theca-interstitial cells were induced to differentiate with either prostaglandin E2 or cholera toxin, then isoproterenol did not enhance androgen synthesis. The catecholamine-induced increases in androgen production were not due to a granulosa cell contribution of steroid. The interstitial cells are the only steroid-producing cells in the ovary that are directly innervated by norepinephrine-containing fibers of the sympathetic nervous system. Our finding of catecholamine-augmented androgen production provides a direct link between the autonomic nervous system and regulation of ovarian steroid synthesis.     

Androgenic regulation of rapidly synthesized RNA in the rat ventral prostate.

The influence of castration on the incorporation of nucleotide precursors into RNA of isolated prostatic tissue has been investigated. Castration brought about an apparent increase of incorporation by increasing the specific activity of the uridine nucleotide pool and not by an enhancement in synthesis of rapidly labelled RNA. An actual decrease in this reaction was shown by dividing the radioactivity incorporated into RNA by the specific activity of the uridine nucleotide. Administration of testosterone brought about enhanced synthesis of RNA in the prostates of rats castrated for 3 days as early as 4 h after injection, reaching maximum effect at 8 h. No significant difference was found between the turnover rates of rapidly labelled RNA from castrated, androgen-treated castrated and intact rats. The increased prostatic permeability to nucleosides as an early action of androgens in this organ is discussed.     

Androgen receptor expression in the developing rat prostate is not altered by castration, flutamide, or suppression of the adrenal axis

Mesenchymal epithelial interactions are believed to be important to the growth and development of the neonatal prostate. Prior studies in the rat ventral prostate, using autoradiography and tritiated dihydrotestosterone, indicate that androgen receptors are present in the prostatic stroma on day 3 and are detected in the epithelium by the tenth postnatal day. These findings suggested that androgen stimulation of the prostatic mesenchyme is a crucial step in the growth and development of the prostate. We have examined this developmental program directly using polyclonal antibodies that recognize specific epitopes of the androgen receptor to examine the pattern of androgen receptor expression in intact and neonatally castrate animals. In keeping with previous studies, androgen receptors are present in the prostate stroma at birth and subsequently appear in the prostatic epithelium by the 10th postnatal day. Development of androgen receptor expression in the epithelium was not changed when the animals were castrated at birth, castrated and blocked by flutamide, or castrated and given hydrocortisone to suppress the production of adrenal androgens. These findings suggest that the appearance of androgen receptors in the prostatic epithelium is programmed by androgens before birth or that factors other than testicular or adrenal androgens control the development of epithelial androgen receptors.     

Stimulation of renal deoxyribonucleic acid synthesis by a pituitary-derived renotropin and its inhibition by testosterone and thyroxine

Kidney slices were obtained from castrated-hypophysectomized male rats treated with a single injection of several different gonadotropin preparations (two ovine LH fractions, one bovine LH fraction, and one hCG fraction) or vehicle, then incubated in a buffer containing [3H]thymidine. Only one of the above, an ovine LH preparation, increased [3H]thymidine incorporation into renal DNA, with a peak occurring 8-10 h after injection and therefore termed renotropin. However, in hypophysectomized rats with intact testes, such an effect was not observed. Furthermore, while testosterone propionate alone did not alter basal incorporation in castrated-hypophysectomized rats, it abolished the incorporation that was stimulated by renotropin. These results suggest that androgens, whether of testicular origin or exogenously administered, suppress the increased incorporation of [3H]thymidine stimulated by renotropin. This antagonistic effect of androgen was also observed with T4, but to a lesser degree. Our findings confirm the presence of renotropin, which could not be attributed to other known pituitary hormones, and suggest that there is a complex interaction between this factor and two other renal growth-promoting hormones, testosterone and T4.     

Persistent intraprostatic androgen concentrations after medical castration in healthy men

CONTEXT: The impact of serum androgen manipulation on prostate tissue hormone levels in normal men is unknown. Studies of men with prostate cancer have suggested that prostatic androgens are preserved in the setting of castration. Tissue androgens might stimulate prostate growth, producing adverse clinical consequences. OBJECTIVE: The objective of the study was to determine the effect of serum androgen manipulation on intraprostatic androgens in normal men. DESIGN: Thirteen male volunteers ages 35-55 yr (prostate-specific antigen < 2.0 ng/ml; normal transrectal ultrasound) were randomly assigned to: 1) a long-acting GnRH-antagonist, acyline, every 2 wk; 2) acyline plus testosterone (T) gel (10 mg/d); or 3) placebo for 28 d. Serum hormones were assessed weekly. Prostate biopsies were obtained on d 28. Extracted androgens were measured by RIA, and immunohistochemistry for androgen-regulated proteins was performed. RESULTS: The mean decrease in serum T was 94%, whereas prostatic T and dihydrotestosterone levels were 70 and 80% lower, respectively, in subjects receiving acyline alone compared with controls (P < 0.05). Despite this decrease in prostate androgens, there were no detectable differences in prostate epithelial proliferation, apoptosis, prostate-specific antigen, and androgen receptor expression. CONCLUSION: In this small study of healthy subjects, despite a 94% decrease in serum T with medical castration, intraprostatic T and dihydrotestosterone levels remained 20-30% of control values, and prostate cell proliferation, apoptosis, and androgen-regulated protein expression were unaffected. Our data highlight the importance of assessing tissue hormone levels. The source of persistent prostate androgens associated with medical castration and their potential role in supporting prostate metabolism deserves further study.     

The influence of androgens on protein synthesis by cultured rat epididymal tubules.

Rat epididymal tubules maintained in organ culture for 3 days respond to the addition of androgens to the culture media (testosterone and dihydrotestosterone 1 x 10-minus 5 m and 1 x 10-minus 7 m) with an increased incorporation of amino acids into acid-insoluble material. Significant androgenic stimulation is observed only 24 h after addition of hormone, while an inhibitory effect is found at earlier periods. The stimulation seems to be specifically produced by androgens; it is blocked by cyproterone acetate and is not elicited by oestradiol-17beta or corticosterone. The process appears to involve RNA synthesis since actinomycin D suppresses the stimulatory effect of androgen. Evidence suggests that cAMP production is not a primordial step in the response to androgen since dibutyryl cAMP did not mimick the androgenic effect, theophylline did not potentiate the response and alpha,beta-methylene ATP, which competitively inhibits adenyl cyclase, failed to alter the androgenic effect. Radioactive testosterone and dihydrotestosterone added to the culture media showed a preferential intranuclear localization as well as extensive metabolism. DHT was found to be the principal intranuclear steroid.     

Characterization of a nuclear receptor for testosterone in seminiferous tubules of mature rat testes.

A specific androgen receptor could be demonstrated in the nuclear and cytoplasmic fractions of testicular tissue of mature hypophysectomized rats, either in vivo after injection of testosterone or in vitro after incubation of testis tissue with testosterone. Using agar-gel electrophoresis this receptor could be distinguished from the testicular transport-like protein for androgens (androgen binding protein = ABP). After in vivo administration of testosterone the steroid bound to the receptor in mature rat testis was mainly unmetabolized testosterone. After dissection of testis tissue the larger part of the receptor was shown to be present in the seminiferous tubules. The amount of exogenous testosterone that could be bound per mg of protein in the nuclear extract increased gradually during 20 days after hypophysectomy. Some characteristics of the receptor in the nuclear extract and of ABP were compared : the receptor was more sensitive to temperature increases than ABP; the steroid dissociated more slowly from the receptor than from ABP; cyproterone acetate showed almost no effect on the binding of dihydrotestosterone to ABP, but did compete for the receptor binding sites in the nuclear extract.     

Androgens and dominance: Sex-specific patterns in a highly social fish (Neolamprologus pulcher)

In most vertebrates, aggression and dominance are tightly linked to circulating testosterone. Fish, however, have two androgens (testosterone, T and 11-ketotestosterone, 11KT) that influence aggression and dominance. To date, few studies have compared the relationship between androgen levels and the outcome of aggressive contests in both females and males of the same species. To investigate sex differences in androgens we staged size-matched, limited-resource (territory) contests with 14 female-female and 10 male-male pairs of the highly social cichlid Neolamprologus pulcher. We then examined androgen levels in recently established dominants, who won the contest and subsequently acquired a territory (for 3 h), and subordinates, who lost and did not acquire a territory. Newly dominant females had higher plasma T but similar 11KT levels to newly subordinate females. In contrast, newly dominant males had higher 11KT but similar T levels to subordinate males. The ratio of 11KT to T, which demonstrates physiological importance of T conversion to 11KT, was positively correlated with submissive behavior in female winners, and correlated weakly with aggressive behavior in male winners (p = 0.05). These findings provide support for the hypothesis that different androgens play equivalent roles in female versus male dominance establishment, and suggest that relative levels of 11KT and T are implicated in female dominance behavior and perhaps behavior of both sexes.     

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.     

Age-dependent loss of sensitivity of female urogenital sinus to androgenic conditions as a function of the epithelia-stromal interaction in mice.

The ability of the female urogenital sinus to respond to androgens in forming prostate was determined by growing 13- to 18-day old embryonic female urogenital sinuses and vaginas from 1- to 30-day old mice as grafts to male hosts. All embryonic unrogenital sinuses as well as vaginas from 1-day old mice were responsive to androgens and formed prostate, whereas vaginas from mice 5- or more days old never formed prostate. To determine which tissue, the epithelium or stroma, accounts for the age-dependent loss in responsiveness of the vagina to androgens in forming prostate, recombinations composed of epithelium and stroma from 16-day old embryonic urogenital sinuses and vaginas from 1- to 20-day old mice were grown as grafts to male hosts. The developmental response of these recombinants demonstrated that the age-dependent loss in responsiveness of the intact vagina to androgens results from an age-dependent loss in the ability of vaginal stroma to participate in prostatic morphogenesis. These data emphasize the importance of stromal factors during prostatic morphogenesis and the relationship of temporal factors to developmental properties of urogenital stroma.     

Urinary androgens and cortisol metabolites in field-sampled bonobos (Pan paniscus)

Urinary metabolites of androgens and cortisol were measured in free-living male and female bonobos. Sex differences and correlations between adrenal and gonadal steroid excretion were investigated. The immunoreactive concentrations of androgens were measured with two different androgen assays. One assay used a testosterone (T) antibody raised with a 17beta-hydroxy group, and the other employed an antibody raised against a reduced form, 5alpha-androstane-17alpha-ol-3-one-CM (17alpha) with cross reactivity for epitestosterone and 5alpha-androstanedione. Both assays have been used in bonobo and chimpanzee studies where non-invasive techniques were employed. The levels of 17alpha-androgen metabolites were 1.7- and 3-fold higher than those of T-metabolites in males and females. The two androgen assay results correlated in males but not females. There was a sex difference in the T-metabolites measured. Male levels were significantly higher. Levels of 17alpha in the two sexes were similar. Cortisol metabolite levels (CORT) were similar between the sexes. The T-metabolites were significantly correlated with CORT in males but not in females. In females, the 17alpha-androgen metabolites correlated with CORT. This suggests that either androgen secretion or metabolism differs between the sexes. A parsimonious interpretation of the androgen assay cortisol/androgen correlation differences would be that larger components of dehydroepiandrosterone (DHEA), androstenedione or epitestosterone from the adrenal androgens were being excreted and measured in the females. The CORT/T metabolite interactions in males may reflect male-specific social or metabolic endocrine conditions.     

Combination of an antiandrogen and a 5 alpha-reductase inhibitor: a further step towards total androgen blockade?

While the elimination of androgens of testicular origin can be easily achieved by orchiectomy or medical castration with LHRH agonists, the action of adrenal androgen precursors which are converted into the active androgen 5 alpha-dihydrotestosterone (DHT) in the prostatic tissue itself can be partially neutralized by antiandrogens which compete with DHT for binding to the androgen receptor. In order to increase the efficiency of androgen blockade, we have used 4-MA, an inhibitor of 5 alpha-reductase, the enzyme which converts testosterone into DHT, to reduce intracellular DHT concentrations and thus facilitate the action of the antiandrogen Flutamide. The present data show that the inhibitory effects of 4-MA (17 beta, N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) and of the antiandrogen Flutamide are additive on prostatic growth and on androgen-sensitive prostatic binding protein mRNA levels in the rat, thus clearly suggesting that such a combination could provide the basis for a further improvement in the therapy of prostate cancer.