Maturational changes in steroidogenic enzyme activities metabolizing testosterone and dihydrotestosterone in two populations of testicular interstitial cells

The present study examined changes in steroidogenic enzyme activities which metabolize testosterone or dihydrotestosterone between days 21-73 of maturation in Band 2 and Band 3 cells isolated by centrifugation of rat testicular interstitial cells on metrizamide density gradients. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities increased progressively in Band 2 and Band 3 cells between days 21-35 of maturation, then both enzyme activities declined to reach low levels in adult Band 2 and Band 3 cells. The significantly higher activities of both enzymes in Band 3, which contains a greater concentration of Leydig cells at each age, suggest their localization in Leydig cells. 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities increased in both Band 2 and Band 3 cells between days 21-50 of maturation and remained elevated; however, dihydrotestosterone was metabolized primarily to 5 alpha-androstane-3 beta,17 beta-diol in Band 2 cells, while 5 alpha-androstane-3 beta,17 beta-diol was the major metabolite of dihydrotestosterone in Band 3 cells. These studies suggest that testosterone accumulation during sexual maturation can be influenced by changing patterns of 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities which metabolize testosterone, and of 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities which metabolize dihydrotestosterone in both Band 2 and Band 3 cells.     

Androgen 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in ventral prostate epithelial and stromal cells from immature and mature rats

To study androgen-mediated differentiation in the rat ventral prostate, we separated the two principal cell types (epithelial and stromal) derived from prostates of immature and mature rats on two continuous Percoll gradients. Cells were immediately placed in culture medium. Testosterone metabolism by the two prostatic cell types was evaluated using [3H]testosterone and quantifying the formation of 5 alpha-[3H]dihydrotestosterone (5 alpha-DHT) and 5 alpha-[3H]androstane-(3 alpha or 3 beta), 17 beta-diols. In epithelial cells from both immature and mature rat prostates the major testosterone metabolites were 5 alpha-DHT and 5 alpha-androstane-3 alpha, 17 beta-diol. Stromal cells metabolized less testosterone than did the epithelial cells. Differences in the relative levels of the various metabolites were observed for the two age groups. To examine in more detail the changes in testosterone metabolism observed in vitro both types of cells and unfractionated cells from immature and mature rat prostates were assayed for testosterone 5 alpha-reductase (using testosterone as substrate) and 3 alpha-hydroxysteroid dehydrogenase (using 5 alpha-DHT as substrate) activities (expressed as pmol substrate reduced/min per 10(6) cells). In immature rats both 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities were localized in the epithelial cell fraction (17 and 52 respectively); stromal cells showed lower 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activity (4 and 4). Relative to epithelial cells from immature rats epithelial cells from mature rats showed a decrease in 5 alpha-reductase (7) and an increase in 3 alpha-hydroxysteroid dehydrogenase (160) activity while stromal 5 alpha-reductase showed little change (3) and 3 alpha-hydroxysteroid dehydrogenase increased to 22.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of dehydroisoandrosterone and androstenedione in human pulmonary endothelial cells in culture

The capacity of endothelial cells from pulmonary arteries and veins to convert dehydroisoandrosterone (3 beta-hydroxy-5-androsten-17-one) and androstenedione to potent, biologically active steroids was investigated. The metabolites of [3H]dehydroisoandrosterone produced in pulmonary artery endothelial cells were androstenedione and 5-androstene-3 beta, 17 beta-diol. The metabolites isolated from incubation of pulmonary arterial cells with [3H]androstenedione were testosterone, 5 alpha-androstane-3,17-dione, 5 alpha-dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), isoandrosterone (3 beta-hydroxy-5 alpha-androstan-17-one), and androsterone. The products of [3H]androstenedione metabolism in human pulmonary venous cells were the same as those formed in arterial cells, and in addition, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol were formed. The rates of metabolite formation from [3H]androstenedione in pulmonary arterial and venous endothelial cells were linear with incubation time up to 3 h. These findings suggest that the pulmonary endothelium is an important site for the metabolism of dehydroisoandrosterone and androstenedione in the human lung. Endothelial cells produce the same metabolites as human lung tissue, with the exception of hydroxylated steroids.     

The metabolism of testosterone in the central nervous system (1). Analysis of testosterone metabolites in the anterior pituitary and hypothalamus using gas chromatography-mass spectrometry (GC-MS), and subcellular localization of testosterone converting enzyme

The aim of this study was to identify accurately the structure of testosterone metabolites in the anterior pituitary and hypothalamus for the investigation of the mechanism of androgen action in the central nervous system. Tissue homogenate and cellular fraction of male rat anterior pituitary and hypothalamus were incubated with testosterone-4-14C and testosterone-19-CD3 (14C/D3 = 1) in the presence of NADH and NADPH. The incubation media were extracted, and they were separated using thin layer chromatography (TLC). Using autoradiogram of TLC, four main radioactive fractions were found on the TLC. The TFA or TMS derivatives of every fraction were analyzed using GC-MS. The main metabolites in the anterior pituitary were identified as 5 alpha-androstan-17-ol 3-one; androst-4-ene-3, 17-dione, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, androst-4-ene-3 alpha, 17 beta-diol and androst-4-ene-3 beta, 17 beta-diol. The result in the hypothalamus was the same as that in the pituitary. The subcellular localization of metabolites in the anterior pituitary was as follows: 5 alpha-androstan-17-ol-3-one, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol were found in microsome; 5 alpha-androstane-3 alpha, 17 beta-diol and androst-4-ene-3 alpha, 17 beta-diol were found in soluble fraction. The result in the hypothalamus was the same as that in the pituitary.     

Testosterone metabolites do not participate in the control of hypothalamic LH-releasing hormone

To determine whether the ability of testosterone to increase intrahypothalamic LH-releasing hormone (LHRH) in orchidectomized rats might be explained by the conversion of the hormone into either its 5 alpha-reduced or oestrogenic metabolites, testosterone, 5 alpha-androstan-17 beta-ol-3-one (DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) (2 mg/rat per day for 6 days) and oestradiol (0.1, 0.5, 1.0 and 5.0 micrograms/rat per day for 6 days) were injected into castrated male rats. After 6 days the rats were killed and serum LH levels and intrahypothalamic LHRH stores measured using specific radioimmunoassay procedures. Testosterone and its 5 alpha-reduced metabolites were used in either the free alcohol or the propionate form (dipropionates in the case of the diols); oestradiol was used as oestradiol-17 beta or in the benzoate form. Treatment with testosterone, DHT, 3 alpha-diol and 3 beta-diol resulted in a significant decrease in serum LH levels; all the 5 alpha-reduced testosterone derivatives were more effective than testosterone in this respect. Testosterone and DHT propionates suppressed LH release following orchidectomy totally; 3 alpha-diol and 3 beta-diol dipropionates were less effective. Testosterone increased intrahypothalamic LHRH stores, this effect being much higher after testosterone propionate, i.e. when intrahypothalamic LHRH stores were restored to pre-castration levels. None of the 5 alpha-reduced steroids was capable of modifying the low intrahypothalamic levels of LHRH found following orchidectomy; only 3 alpha-diol dipropionate exhibited some activity, but this was much lower than that of testosterone propionate. Oestradiol-17 beta was totally ineffective in decreasing serum LH in orchidectomized animals; in contrast, oestradiol benzoate progressively decreased serum LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of C19 steroids in vitro by the submaxillary salivary gland of mature and immature domestic pigs

Homogenates of the submaxillary glands of immature and mature pigs were incubated with 3H-labelled C19 steroids which have been shown previously to be metabolized in vitro by the submaxillary gland of mature boars. Dehydroepiandrosterone was metabolized largely to androstenedione, 5 alpha-androstane-3,17-dione and androsterone, and to small amounts of testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstanediols. Testosterone yielded predominantly 5 alpha-androstane-3 alpha, 17 beta-diol with smaller amounts of other 5 alpha-reduced products, i.e. 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione and androsterone; 5 alpha-dihydrotestosterone and the two epimeric 5 alpha-androstane-3 alpha/3 beta, 17 beta-diols were interconverted. These and earlier results show that the porcine submaxillary gland has the capacity in vitro to metabolize selected C19 steroids in a way which is not related to either sexual maturity or sex of the animal; in this respect the findings support certain aspects of previous histochemical studies.     

Effect of photoperiod and gonadectomy on testosterone metabolism in vitro by the prostate of the golden hamster

Testosterone metabolism was studied in vitro in the prostate of intact and castrated golden hamsters maintained either in short days (8 h light: 16 h darkness, 8L: 16D) or in long days (14L: 10D). Testosterone was found to be converted into 17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-DHT), 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3, 17-dione and androstenedione. The mean conversion of testosterone to 5 alpha-DHT was higher in prostates from animals maintained in long days than in short days (P less than 0.0025) while that to androstenedione was higher in short days (P less than 0.0005); no significant changes in the formation of the other three metabolites were noted. Castration of animals maintained in short days resulted in a significant (P less than 0.05) decrease in the mean conversion to all four metabolites. In contrast, castration of animals kept in a long-day regime caused a significant (P less than 0.01) decrease in the mean formation of 5 alpha-DHT but a significant (P less than 0.05) increase in the mean formation of 5 alpha-androstane-3 alpha, 17 beta-diol.     

Steroid metabolism in fish. II. Testosterone metabolites in the bile of the marine winter flounder Pseudopleuronectes americanus and the freshwater Atlantic Salmon Salmo salar

Intraarterial injection of [3H]testosterone in the flounder Pseudopleuronectes americanus and the landlocked salmon Salmo salar resulted in the concentration of radioactive catabolites in the gall bladder bile. The catabolites were quantitatively conjugated with glucuronic acid. In flounder bile, three steroids, testosterone, 5 beta-dihydrotestosterone and 5 beta-androstane-3 alpha,17 beta-diol accounted for most of the radioactivity. Quantitatively the concentration of 5 beta-dihydrotestosterone usually exceeded that of the other two compounds. In salmon bile, 5 alpha-dihydrotestosterone was also present as a minor catabolite and the dihydrotestosterones accounted for approximately one-half the total radioactivity. In both species there was evidence for trace amounts of 5 beta-androstane-3 beta,17 beta-diol, 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol but no evidence for 5 beta- or 5 alpha-androstanolones, 4-androstene-3 alpha,17 beta-diol, or its 3 beta-epimer. There were no obvious sex-related differences in the composition of the biliary testosterone metabolites.     

Plasma concentrations of ovarian steroids in the freshwater European silver eel (Anguilla anguilla L.): effects of hypophysectomy and transfer to sea water

Intact and hypophysectomized freshwater (FW) silver eels were transferred to tanks of FW or artificial sea water (SW; salinity = 0.60 osmol/l) which were simultaneously renewed twice a week. Fish were killed 2 months after transfer and plasma was assayed for ovarian steroids. In all fish, 5 alpha-androstane-3 beta,17 beta-diol was present, while 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol were undetectable. In intact FW eels, plasma levels of testosterone, 5 alpha-androstane-3 beta,17 beta-diol and oestradiol-17 beta were approximately 0.15 nmol/l. In intact SW eels, no change in plasma levels of testosterone and 5 alpha-androstane-3 beta,17 beta-diol was found, whereas the concentration of oestradiol-17 beta was increased significantly (P less than 0.01), indicating stimulation of aromatase activity. In hypophysectomized compared with intact FW fish, plasma levels of testosterone and 5 alpha-androstane-3 beta,17 beta-diol were decreased (P less than 0.05) and there was a slight but significant (P less than 0.01) augmentation of the plasma concentration of oestradiol-17 beta which may have involved the removal of pituitary-dependent inhibition of aromatase activity, possibly by 5 alpha-reduced compounds. In hypophysectomized compared with intact SW fish, plasma levels of testosterone, 5 alpha-androstane-3 beta,17 beta-diol and oestradiol-17 beta were decreased (P less than 0.05); in the case of oestradiol-17 beta, this may have reflected the diminished ovarian synthesis of testosterone, its precursor. The plasma level of oestradiol-17 beta was, however, higher in SW than in FW fish, even in hypophysectomized eels. This suggests that extra-pituitary mechanisms mediate, at least partly, the effects of transfer to SW on aromatase activity.     

Structure and steroidogenic enzymes of the seminal vesicles of the urohaze-goby (Glossogobius olivaceus)

The in vitro steroid metabolism in the seminal vesicles of the brackish water goby (urohaze-goby, Glossogobius olivaceus) was studied using males in the breeding season. The moderate activity of delta 5-3 beta-hydroxysteroid dehydrogenase was histochemically detected only in the epithelial cells of the organ, though these cells have the characteristics of secretory cells ultrastructurally. Cell-free homogenates (800 g supernatant fluid) of the whole tissue were aerobically incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, or testosterone in the presence of NAD+ or NADPH. Pregnenolone and dehydroepiandrosterone were converted to progesterone and androstenedione, respectively. Progesterone was transformed to 5 alpha-pregnane-3,20-dione (main product) and 17 alpha-hydroxyprogesterone. 17 alpha-Hydroxyprogesterone was metabolized into androstenedione (main product) and 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione. From androstenedione, 5 alpha-androstane-3,17-dione (main product) and epiandrosterone were obtained. Testosterone was transformed to 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione, and androstenedione. These results indicate that the steroid metabolic patterns in the seminal vesicles of G. olivaceus are closely resembled to those in the testes.     

Metabolism of [14C]testosterone by human foetal and adult brain tissue

The metabolism of [14C]testosterone in vitro by various areas of the human foetal brain has been studied and compared with that of adult brain. The predominant metabolites were 5alpha-dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol, and also androstenedione, and all areas of the foetal brain showed similar activity. In the foetal pituitary gland, the activity of 5alpha-reductase was less prominent than that of 17beta-hydroxysteroid-dehydrogenase. Small quantities of oestradiol-17beta were produced from testosterone by the hypothalamus, temporal lobe and amygdala only, and no aromatization could be detected in the pituitary gland. 5alpha-Reductase activity was much lower in adult brain tissues and no oestradiol was identified in adult temporal lobe tissue.     

Role of testosterone and its metabolites in the differentiation of the mammary gland in rats.

The capacity of various androgens to virilize the differentiating mammary gland in the female rat fetus has been determined. Testosterone, 5alpha-androstane-3alpha, 17beta-diol (3alpha-diol), and dihydrotestosterone (DHT) virilize the anlagen of the mammary gland by suppressing nipple formation but 5alpha-androstane-3beta, 17beta-diol, androsterone, and dehydroepiandrosterone sulfate do not affect female mammary differentiation. However, unlike the genitalia and wolffian ducts of the female rat fetus in which the masculinizing potency of DHT and 3alpha-diol is greater than that of testosterone, testosterone is more potent than its metabolites DHT and 3alpha-diol, in virilizing the mammary gland. The results suggest that testosterone is the fetal androgen mediating masculine development of the mammary gland.     

Metabolism of androgens in vitro by human foetal skin.

Fresh scalp, genital, chest and axillary skin from human foetuses of 12-41 weeks' maturity was incubated in Krebs improved Ringer I medium with (7alpha-3h)dehydroepiandrosterone, (7alpha-3H)testosterone and (7alpha-3H)androstenedione. The metabolites identified were androstenedione, 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, 5-androstene-3beta, 17beta-diol and testosterone. The results provide evidence for the presence of 3beta-hydroxysteroid dehydrogenase, delta4-5 isomerase, 17beta-hydroxysteroid dehydrogenase, delta4-3-oxosteroid-5alpha reductase and 3alpha-hydroxysteroid dehydrogenase in human foetal skin. There were quantitative differences in the various enzyme activities between different body sites and skin specimens of different gestational age. 5alpha-Reductase activity was particularly high in genital skin. 3beta-Hydroxysteroid dehydrogenase delta4-5 isomerase activity was low in skin from a 12-week foetus, but high in skin specimens from 28-, 38- and 41-week foetuses. 17beta-Hydroxysteroid dehydrogenase activity was already high in the skin of the 12-week foetus and remained so in the older foetuses. These results were correlated with the development of the foetal sebaceous glands, and were in general agreement with a parallel enzyme histochemical study. The role of androgen metabolism in human foetal skin is discussed.     

Uptake and conversion of progesterone and testosterone to 5alpha-reduced products by enriched gonadotropic and chromophobic rat anterior pituitary cell fractions.

Monodispersed cells from anterior pituitaries of male rats were prepared by Pronasedissociation and incubated with [3H]progesterone or [3H]testosterone. The cells were then separated into enriched fractions consisting of gonadotropic, somatotropic or chromophobic cells by velocity sedimentation at unit gravity for 4 h. The uptake of [3H]progesterone and [3H]testosterone by the gonadotropic enriched cell fraction was 1.8 to 3.2 times greater than the somatotropic and chromophobic enriched cell fractions. The gonadotropic and chromophobic enriched cell fractions metabolized [3H]progesterone and [3H]testosterone appreciably. The principal metabolites were identified and quantitated by reverse isotopic dilution. After incubation with [3H]progesterone, the principal metabolite was [3H]5alpha-dihydroprogesterone which ranged from 11.5% for the gonadotropic cells to 7.6% for the chromophobic cells. Smaller amounts of 3alpha-hydroxy-5alpha-pregnan-20-one (3.7 to 4.8%) and 20alpha-dihydroprogesterone (2.1 to 4.3%) were also identified. After incubation with [3H]testosterone, the principal metabolite was 5alpha-dihydrotestosterone which ranged from 12.6% for the gonadotropic cells to 10.3% for the chromophobic cells. Smaller amounts of 5alpha-androstane-3alpha, 17beta-diol (4.1 to 5.5%) and androstenedione (1.8 to 3.0%) were identified. After incubation with [3H]progesterone or [3H]testosterone the same metabolites were also identified in the somatotropic cell fraction but were thought to be present because of contamination with gonadotropic cells. Dissociated pituitary cells from orchidectomized rats had a 2-fold increase in the uptake of [3H]testosterone and a greater metabolism of [3H]testosterone to 5alpha-dihydrotestosterone as compared to pituitary cells from intact rats (12.6 vs 25.7%).     

Relationship between the structures and steroidogenic functions of the testes of the urohaze-goby (Glossogobius olivaceus)

The testis of the brackishwater goby (Glossogobius olivaceus, the urohaze-goby in this text) consists of two main components, the glandular and the seminiferous tissue. After manual separation of the two tissues, in vitro steroidogenesis in each tissue was examined using testes from mature males in the breeding season. Cell-free homogenates (800g supernatant fluid) of each tissue were aerobically incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, or 5 alpha-pregnane-3,20-dione in the presence of NAD+ or NADPH. (1) Glandular tissue: Pregnenolone and dehydroepiandrosterone were converted to progesterone and androstenedione, respectively, in the presence of NAD+. In the presence of NADPH, the following metabolism of steroids was established. Progesterone was transformed to 5 alpha-pregnane-3,20-dione (main product), 17 alpha-hydroxyprogesterone, 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, and androstenedione. 17 alpha-Hydroxyprogesterone was metabolized into 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione (main product), 3 beta, 17 alpha-dihydroxy-5 alpha-pregnan-20-one, androstenedione, and 5 alpha-androstane-3,17-dione. From androstenedione, 5 alpha-androstane-3,17-dione (main product) and epiandrosterone were obtained. Testosterone was transformed to 5 alpha-dihydrotestosterone (main product), 5 alpha-androstane-3 beta, 17 beta-diol, epiandrosterone, and 5 alpha-androstane-3,17-dione. 5 alpha-Pregnane-3,20-dione was metabolized into 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, 5 alpha-androstane-3,17-dione, epiandrosterone, and 5 alpha-dihydrotestosterone. (2) Seminiferous tissue: Almost all of the above metabolites were obtained, but the yield was much smaller, especially for 5 alpha-reduced metabolites, compared with that for glandular tissue. From these results, it is concluded that steroidogenesis in the testis of G. olivaceus is characterized by the predominant activity of 5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase and that these are localized mainly in glandular tissue, together with delta 5-3 beta-hydroxysteroid dehydrogenase + delta 5-delta 4 isomerase, 17 alpha-hydroxylase, and C-17-C-20 lyase.     

In vivo uptake and metabolism of 3-h-5alpha-androstane-3alpha,17beta-diol and of 3-h-5alpha-androstane-3beta,17beta-diol by human prostatic hypertrophy.

Tritiated 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-diol) respectively were administered to patients with benign prostatic hypertrophy (bph) undergoing prostatectomy. In prostate and skeletal muscle homogenates and in plasma the total radioactivity content as well as the formation of metabolites were measured. Histological examination of each ectomized prostate was performed to evaluate the cellular composition of the tissue. After 3alpha-diol injection, a higher uptake of radioactivity in the prostate was obtained than after 3beta-diol. Within 30 min the 3alpha-isomer was very efficiently converted to 5alpha-DHT, while most of the 3beta-isomer remained unchanged. There was, however, also after administration of the 3beta-diol a substantial biconversion to 5alpha-DHT as has been confirmed by recrystallization to constant specific radioactivity. Only after 3beta-diol epiandrosterone was detected in small but significant amounts. 3alpha-diol administration resulted in distinct concentrations of 3beta-diol, whereas the conversion of 3beta-diol to the 3alpha-isomer was insignificant. When comparing the histological composition of the prostatic tissue with the accumulation of radioactivity and the formation of metabolites only a weak correlation between glandular structure and radioactivity uptake after 3alpha-diol administration could be revealed.     

Sexual differences in the Japanese quail: behavior, morphology, and intracellular metabolism of testosterone

Three experiments were carried out to study whether differences in the intracellular metabolism of testosterone (T) can explain sexually differential responses to T in Japanese quail. In the first experiment, a series of dose-response curves in which length of Silastic testosterone implants was related to effects on several behavioral and physiological variables was established. In Experiment 2, adult males and females were assigned to six experimental groups: intact males and females (I-males and I-females), castrated males and females implanted subcutaneously with 40-mm Silastic implants of T (T-males and T-females), and castrated males and females without hormone treatment (CX-males and CX-females). No CX-bird (male or female) and no I-female exhibited male sexual behavior. However, I-males and T-males regularly copulated during the behavioral tests. No crowing was ever heard in CX-animals and I-females. T-females crowed less than T-males and their crowing sounded weaker than those of males. The cloacal glands of T-females were less developed than those of males. Radioimmunoassay of T and 5 alpha-DHT showed that T-males and T-females have similar plasma levels of androgens. No striking differences were observed in the way testosterone is metabolized by the pituitary gland and central nervous tissues of males and females. By contrast, the production of 5 alpha-dihydrotestosterone (5 alpha-DHT) and 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha, 3 alpha-diol) was higher in the cloacal glands of males than in those of females. These sex differences were not detected between T-males and T-females. In experiment 3, the cloacal gland of males produced more 5 alpha-reduced metabolites than those of females. The pituitary gland of females also produced more 5 beta-androstane-3 alpha, 17 beta-diol (5 beta, 3 alpha-diol). In syringeal muscles, the production of 5 beta-dihydrotestosterone (5 beta-DHT) and 5 beta, 3 alpha-diol was higher in females compared to males.     

The determination of 5alpha-androstane-3alpha, 17beta-diol in human plasma by radioimmunoassay

Antibodies have been raised in rabbits against 3alpha, 17beta-dihydroxy-5alpha-androstane-6-0-carboxymethyloxime coupled with Cohn's fraction IV-4. The antiserum exhibited significant cross reactions with 5beta-androstane-3alph1, 17beta-diol, 5alpha-dihydrotestosterone, and testosterone. No cross reactions were observed with 5alpha-androstane-3beta,17beta-diol and 5-androstene-3beta,17beta-diol. The methodological criteria for the measurement of 5alpha-androstane-3alpha, 17beta-diol in human plasma were as follows: The specificity was ensured by separating the cross reacting steroids by thin layer chromatography. The intraassay and interassay coefficients of variation were found to be 6.2 and 10.2%, respectively. The sensitivity was 30 pg. The recovery of different amounts of 5alpha-androstane-3alpha,17beta-diol added to human plasma (80, 120, and 200 pg) yielded 91.3, 92.5, and 93.5%, respectively. The following concentrations of 5alpha-androstane-3alpha,17beta-diol have been determined in human plasma (mean +/- SD, ng/dl): Normal males: 18.98 +/- 5.9; normal females: 2.65 +/- 0.27; females with idiopathic hirsutism: 11.9 +/- 6.4; prepubertal children: not detectable.     

Identification of two 5alpha-reduced pregnanes as major metabolites of progesterone in immature rat ovaries (1000 x g supernatant) in vitro.

Incubation of the 1000 x g supernatant obtained from 23-day-old rat ovarian homogenate with labeled progesterone resulted in the production of 3 major metabolites; 5alpha-androstane-3alpha,17beta-diol, and two 5alpha-reduced pregnanes that were identified as 3alpha-hydroxy-5alpha-pregnan-20-one and 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one. The 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one has not been hitherto isolated from mammalian ovaries. The steroids were identified by their mobilities on thin-layer and gas-liquid chromatography, by mass spectroscopy, derivative formation and by recrystallization to constant specific activity. In another experiment, incubation of the 1000 x g supernatant from 23-day-old rat ovaries with 3alpha-hydroxy-5alpha-pregnan-20-one as substrate resulted in the production of 5alpha-androstane-3alpha,17beta-diol. It is suggested that 5alpha-androstane-3alpha,17beta-diol is produced in immature rat ovaries by a pathway in which the identified 5alpha-reduced pregnanes serve as intermediates.     

Is hirsutism an evolving syndrome?

The possibility that hirsutism is an evolving syndrome rather than a static condition involving only one gland has been considered. To assess this proposal 60 untreated hirsute patients aged 12-32 years were divided into five groups according to the duration of the hirsutism (less than 1, 1-2, 2-3, 3-5 and greater than 5 years). Peripheral plasma concentrations of LH and FSH, androstenedione, dehydroepiandrosterone sulphate, testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, cortisol, oestradiol-17 beta and oestrone were determined by radioimmunoassay. When the values obtained were compared with those from normal menstruating women, the results showed that in group I there was a significant increase only in the mean plasma 5 alpha-androstane-3 alpha, 17 beta-diol concentration. The mean concentration of this steroid was also raised in all other groups. In groups II and III mean basal levels of plasma dehydroepiandrosterone sulphate were also significantly increased and showed a marked increase after ACTH stimulation (1 mg tetracosactide acetate, i.m.) as did the concentrations of androstenedione and 17 alpha-hydroxyprogesterone. Finally, in groups IV and V, a significant increase in mean plasma concentrations of LH, androstenedione, oestrone and testosterone was found in the basal condition. The clinical picture also became gradually more severe from group I to group V. These data suggest that hirsutism could be an evolving syndrome progressively involving peripheral androgen metabolism, the adrenal gland and finally the ovary possibly through alterations of hypothalamic-pituitary function.