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The in vivo and in vitro metabolism of (3H)-5alpha-androstane-3lapha,17beta-diol by the male rat anterior pituitary was studied. A rapid and intensive conversion of 5alpha-androstane-3alpha, 17beta-diol into 5alpha-dihydrotestosterone was demonstrated, since following a 30 min. incubation time, 73% of the recovered radioactivity were constituted by 5alpha-dihydrotestosterone. Studies on the subcellular distribution of steroids showed that 5alpha-dihydrotestosterone was the main steroid recovered except from the 105,000 X g pellet. From in vivo and in vitro experiments it was concluded that the transformation of 5alpha-dihydrotestosterone into 5alpha-androstane-3alpha, 17beta-diol was a reversible process, and that this last steroid could exert its biological action mainly via 5alpha-dihydrotestosterone.

Pretreatment of synaptosomal membranes with a diazo-coupling reagent and the presence of Cl- ions were used to differentiate high- and low-affinity populations of postsynaptic gamma-aminobutyric acid (GABAA) receptors. The super-low-affinity GABAA receptors were characterized by the enhancing effect of GABA on [3H]diazepam binding. The GABA antagonists 2-(3-carboxypropyl)-3-amino-4-methyl-6-phenylpyridazinium chloride (SR 95103) and 3-alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R 5135) shifted and suppressed the dose-response curve of GABA on diazepam binding. SR 95103 displaced the lower affinity [3H]GABA binding with higher potency. Dissociation of the binding of the antagonist 2-(3-carboxypropyl)-3-amino-6-p-methoxyphenylpyridazinium bromide ([3H]SR 95531) was polyphasic. Displacing potencies of SR 95531 and GABA were examined on the major (85%) rapid and minor slower phases of dissociation separated kinetically. The slower phase corresponded to higher affinity binding of SR 95531 which was displaced by GABA with about 10 times less potency. Photoaffinity labeling with muscimol decreased the number of [3H]muscimol binding sites by 27%. It decreased the displacing potency of GABA by 72%, but not that of bicuculline methiodide. These findings can be explained by a preferential binding of antagonists to hydrophobic accessory sites around low-affinity GABAA receptors.

The epididymis of adult rats metabolizes 3H 5alpha-androstane-3alpah,17beta-diol (3alpha-diol) by experiments in vitro. After incubation of tissue slices at 37 degrees C for 2 hours, 2% of the radioactivity was found in the water-soluble fraction whereas 98% was found to be ether soluble (free steroids). Further investigation of the free steroids showed the following to be present: 3alpha-diol 39.9%, DHT (17beta-hydroxy-5alpha-androstan-3-one) 33.7%, androsterone (3alpha-hydroxy-5alpha-androstan-17-one) 9.2%, 3beta-diol (5alpha-androstane-3beta,17beta-diol) 2.6%, 5alpha-A-dione (5alpha-androstan-3,17-dione) 1.1%, delta 16-3alpha-ol (5alpha-androst-16-en-3alpha-ol) 1.0%, delta16-3beta-ol (5alpha-androst-16-en-3beta-ol) 2.6%, delta 16-3-one (5alpha-androst-16-en-3-one) 2.9%, and polar compounds 3.3%. When segments of the epididymis (caput and cauda) were incubated in the same way, qualitatively similar metabolites were formed but a greater amount of 3alpha-diol was metabolized by the cauda epididymis. This increase was mainly accounted for by an increased formation of delta 16 compounds (14.3% in cauda, 4.3% in caput). This is most probably due to the presence of larger numbers of mature spermatozoa, which, as we have previously shown, form delta16 steroids from 3alpha-diol and DHT (5).

Enzymatic transformations of androgenic steroids in their target tissues lead to molecules which are androgenically more or less active than testosterone. Among such enzymes, and depending with organs, hydroxylases selectively act upon several androgenic steroids. In testes, the 7 alpha-hydroxylation of biosynthesized testosterone and androstenedione gives molecules implied in the regulation of male hormone production. In prostate, epididymis, sebaceous glands and pituitary, 6 alpha- and 7 alpha-hydroxylations only occur with 5 alpha-reduced and 3 beta-hydroxylated derivatives of 5 alpha-dihydrotestosterone. The cells preferentially dispose of such hydroxylated metabolites, but their formation is decreased by estradiol. The position of hydroxylations in the steroid hormone metabolism of each tissue is shown, and their implication in hormone-dependent pathologic growths are discussed.

Incubation of 4-14C-5 alpha-androstane-3 beta, 17 beta-diol with human normal and hyperplastic prostate minces led to the NADPH-dependent production of a major polar radiometabolite which was identified as 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol and proved the presence of a 3 beta-hydroxysteroid-7 alpha-hydroxylase in prostate. Such evidences may bring new leads for the study of the mechanisms of androgenic steroid action in the prostate gland.

A specific receptor protein has been demonstrated for 5alpha-androstane-3alpha, 17alpha-diol in cytoplasmic extracts of normal and hyperplastic canine prostates. The receptor molecule, with a sedimentation coefficient of 4-5S, has been identified by the use of sucrose gradient centrifugation of tissue fractions which had been labelled in vitro with tritiated 5alpha-androstane-3alpha, 17alpha-diol. The receptor showed a relatively high affinity for this compound whereas binding could not be demonstrated with other labelled C19 steroids. In addition binding of tritiated 5alpha-androstane-3alpha, 17alpha-diol was not affected by the presence of 50-fold excesses of other C19 steroids.

The relative competitive activity of some androstane derivatives was determined by 50% inhibition of [3H]estradiol binding to an unusual estrogen-binding protein (UEBP) of male rat liver. It was shown that: i) the bulk of energy of the steroid-protein complex is derived from hydrophobic interactions; ii) the authentic ability to form specific complexes with UEBP at androgene concentrations close to physiological ones, is determined by 17 beta-hydroxyl and is enhanced by the 3 alpha- or 2 alpha-oxy-group; iii) 3- and 17-keto groups inhibit androgene interaction with UEBP; iiii) the cis-conjunction of rings A and B in the androgen molecule does not block steroid binding to the protein. These data specify significantly the mechanism of androgene interaction with UEBP and shed additional light on the physiological role of this protein.

The activities of hydroxysteroid dehydrogenases of 5-ane and 5-ene steroids were examined in interstitial tissue from testes of rats at different ages. The enzyme reactions were localized in the Leydig cell cytoplasm of isolated cells and in frozen tissue slices. Relative reaction velocites of the NAD-linked hydroxysteroid dehydrogenases were obtained spectrophotometrically with 17 steroid substrates using the 12,000 X g supernatant of isolated interstitial cells from 28-29 day old rats; the rate of 3(alpha,beta) dehydrogenation of 5-ane-3beta steroids was markedly (10 to 20X) higher than that of 5-ene-3beta steroids and 5-ane-3alpha steroids. The hydroxysteroid dehydrogenase activities of testes from 124 rats between the ages of 15 and 138 days were determined using as substrates, 3beta-hydroxy-5beta-androstan-17-one, 3beta-hydroxy-5alpha-androstan-17-one, 3beta,17beta-dihydroxy-5alpha-androstane, dehydroepiandrosterone and pregnenolone. Between the ages of 15 and 32 or 34 days the gonads grow in size more rapidly than the body and the 5-ane-3beta-hydroxysteroid dehydrogenase activities show very marked increases; changes in the 5-ene-3beta-hydroxysteroid dehydrogenases are much less pronounced, so that at 34 days the activity of 3beta-hydroxy-5beta-androstan-17-one dehydrogenase is approximately 20 X that of dehydroepiandrosterone dehydrogenase. After 34 days, the 5-ane-3beta-hydroxysteroid dehydrogenase activities decline. It is suggested that during sexual maturation the testicular biosynthesis of active 5-ane androgens may proceed via 5-ane precursors with the help of age-dependent 5-ane-3beta-hydroxysteroid dehydrogenases.

The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.

Yeasts can advantageously be utilized for the production of the 17beta-hydroxy-derivative, from 3-methoxy-8,14-seco-1,3,5(10),9(11)-estratetraene-14,17-dione (14,17-dione) while 14alpha-hydroxy and 14alpha,17beta-dihydroxy-derivatives are also formed. The biochemical properties of yeasts' enzymes responsible for the formation of the two monohydroxy-derivatives have been studied in detail. In the cell-free extract of Saccharomyces the presence of two hydroxysteroid oxidoreductases could be detected. The first enzyme forms 3beta,17beta-dihydroxy-derivative from 5alpha-androstane-317-dione. This enzyme is responsible for the formation of 17beta-hydroxy-derivative from 14,17-dione. The second enzyme forms 3alpha-hydroxy-derivative from 5beta-androstanedione as well as 14alpha-hydroxy-derivative from its 14,17-dione. The cofactor of both enzymes is pyridine nucleotide. The two enzymes possessing different properties can selectively be inhibited.

A 16-year-old female patient with an adrenal tumour was studied. Clinically she had progressive hirsutism, showed high urinary 17-oxosteroid excretion with normal plasma cortisol. Plasma C19-steroids, both unconjugated (including testosterone) and sulphate-conjugated, were greatly elevated. On surgical exploration an adrenal tumour was histologically an adenoma. On ultrastructural analysis the cells in all zones of the adjoining adrenal were considered normal. Although the tumour cells had the general appearance of a steroid-secreting cell their structure diverged from the cells of every subzone of the cortex. This was the case particularly with mitochondria and lipid inclusions. The only endogenous unconjugated steroids detected in the adjoining cortex were corticosterone and cortisol while in tumour tissue these were present in lesser amounts. The tumour tissue contained large amounts of C19-steroids, 11beta-hydroxy-androstenedione being quantitatively most significant. On the basis of the steroid profile an impaired defect of 21-hydroxylation in tumour cells leading steroid synthesis from corticosteroidogenesis to the C19 pathway is proposed.

Kinetics of testosterone, dihydrotestosterone (DHT) and 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) were studied in 7 elderly healthy men (ages 61 to 80 years) with benign prostatic hyperplasia (BPH). Clearance rates were determined by the constant infusion technique with labeled testosterone and DHT. Metabolic clearance rate (MCR), conversion ratio (CR), the transfer constants (rho) and production rates (PB) were calculated. Plasma androgens were measured by specific radioimmunoassay. Plasma testosterone was 516 +/- 314 (SD) ng/dl, plasma DHT was 74.6 +/- 19.6 (SD) ng/dl and plasma 3alpha-diol was 16.4 +/- 4.1 (SD) ng/dl. An elevated DHT level in elderly men with BPH wasconfirmed. MCRT was 620 +/- 65 (SD) liter/day and MCRDHT was 393 +/- 50 (SD) liter/day. Both MCRT and MCRDHT in elderly men were significantly lower than in young men. PBT was 3.2 +/- 2.1 (SD) mg/day and PBDHT was 291 +/- 87 (SD)migrogram/day. PBDHT was the same in elderly and young men. DHT production is maintained in elderly men despite reduction of testosterone production. From the data, it was claculated that in contrast to young men where greater than 80% of blood DHT is from secreted testosterone, over 50% in elderly men is derived from secretion or production of DHT by the testis or even more likely the prostate.

The presence of a specific receptor for 5alpha-androstan-3beta, 17beta-diol (3beta-diol) in the pituitary cytosol from prepubertal male rats was demonstrated. Its characteristics were: Ka = 5.2.10(7) M-1 KD = 1.9 X 10(-8) M, number of specific binding = 8.7 10(-14) moles per mg of proteins. Its sedimentation constant was 3 S. Competition assays showed that only 3beta-diol itself and estrogens were able to compete for the binding sites for 3beta-diol. Androgens, including 3alpha-diol, were inefficient. This receptor was found only in pituitary cytosol, it was missing from hypothalamic or cortical cytosols. This special localization seemed to foreshadow a specific role for 3beta-diol in the anterior hypophysis.

Metabolism of tritium-labeled testosterone, delta4-androstane-3, 17-dion, dihydrotestosterone. 5alpha-androstane-3alpha, 17beta-diod and 5alpha-androstane-3beta, 17beta-dion by the isolated hypophyses and epiphyses of male and female 30-day rats was studied. The glands were incubated in Eagle's medium containing the mentioned androgens with the concentration of 1-2.10(-8) M for 90 min at 37 degrees C. As revealed, restoration of testosterone and delta4 androstane-3, 17-dion by 5alpha-reductase occurred in the epiphyses and hypophyses; under conditions of incubation androgen hydroxylation was realized only in the 3alpha- and 17beta-positions. Organ and sexual specificity of the androgen metabolism in the hypophyses and the epiphyses was quantitative in character: the metabolism level of all the androgens was greater in female rats than in the male both in the hypophysis and in the epiphyses; the activity of 5-reductase was lower in the epiphyses of animals of the both sexes than in the hypophyses. It appeared that in the process of biotransformation androgens in the organs under study served as precursors of the polar unidentified metabolites poorly retained by the tissues and eliminated from the organs into the incubation medium during the incubation.

The effects of dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and testosterone on the growth of the androgen-dependent Shionogi SC-115 tumour in mice have been compared and the metabolites in the tumour arising from each steroid have been identified. After the transfer of SC-115 tumour cells to castrated male mice, treatment of the recipients with dihydrotestosterone produced a striking proliferative response that enabled earlier tumour detection and led to a higher tumour incidence than obtained with testosterone. At short intervals after the intravenous injection of 200muCi of [1,2-(3)H]testosterone the amounts of radioactivity in tumour, muscle and seminal vesicles were almost equal. The metabolism of [1,2-(3)H]testosterone in tumour and muscle was slight in comparison with the extensive metabolism in seminal vesciles. Whereas up to 7% of the total neutral steroid recovered from whole tumour tissue and isolated nuclei was in the form of [1,2-(3)H]dihydrotestosterone, the amount of this compound in the corresponding preparations from seminal vesciles was several times greater. When the metabolism of [1,2-(3)H]dihydrotestosterone in tumour tissue was studied, it was found that more than 60% of the total neutral steroid in both cytoplasm and nuclei consisted of [1,2-(3)H]dihydrotestosterone. Thus much higher intracellular concentrations of dihydrotestosterone occurred with the administration of this steroid than with testosterone. Tumour cell proliferation was suppressed by oestradiol and the amount of androgen in nuclei was significantly decreased by high doses of this hormone.