Age dependent changes in androgen metabolism in the rat prostate

Oxidation and reduction of androstenedione, testosterone, dihydrotestosterone (DHT), 5 alpha-androstan-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol (3 alpha- and 3 beta-A'diol) were measured in homogenates from the ventral prostate (VP), dorsal prostate (DP), lateral prostate (LP), the coagulating gland (CG) and seminal vesicles (SV) in intact rats of different ages from young mature (3-6 months) to senescent rats (20-30 months). Some very old intact rats (30-32 months) were treated with testosterone in order to rule out the effect of this hormone on androgen metabolism. The enzymatic activities for young mature rats were significantly altered by increasing age, both with regard to differences between the various organs as well as differences in cofactor requirement. With increasing age, the specific activity of most enzymes gradually decreased. With testosterone as substrate, 5 alpha-reductase activity was significantly reduced in the old rats in all tissues studied and was undetectable in the oldest animals in the VP and the SV. On the other hand, 5 alpha-reductase could not be recorded in any tissue in any tissue in old rats when androstenedione was the substrate. 3 alpha-Hydroxysteroid oxidoreductase (3 alpha-HSOR) in the VP was the only enzyme which did not decrease in activity by increasing age. In the other lobes this enzyme activity decreased similar to 3 beta-hydroxysteroid oxidoreductase (3 beta-HSOR) and the 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) activity. Administration of testosterone to old rats increased the specific activity of most of the enzymes studied.     

Occurrence of hydroxysteroid oxidoreductases in liver of turtles

1. Hydroxysteroid oxidoreductases have been partially purified from the cytosol fraction (105,000 g supernatant) of liver from a fresh-water turtle (Podocnemis expansa) and a sea-water turtle (Chelonia mydas mydas) by precipitation with ammonium sulphate (AS, 10-80% saturation). 2. The following enzymes were detected (substrates in brackets): 3 alpha-hydroxysteroid oxidoreductase (androsterone), 3 beta-hydroxysteroid oxidoreductase (DHEA) and 17 beta-hydroxysteroid oxidoreductase (testosterone, oestradiol-17 beta). NAD as well as NADP were effective as cofactors. 3. In fresh-water turtle, highest activities of the 3 alpha-enzyme were measured in the 20% AS fraction (cofactor NAD), of the 3 beta-enzyme in the 60% AS fraction (cofactor NAD) and of the 17 beta-enzyme in the 40% AS fraction (cofactor NADP). 4. In sea-water turtle, highest activities were observed for all three enzymes in the 60% AS fraction. 5. Generally, enzyme activities were higher in sea-water turtles than in fresh-water turtles. The most active enzyme in both turtles was found to be the 3 alpha-hydroxysteroid oxidoreductase, followed by the 17 beta- and the 3 beta-hydroxysteroid oxidoreductases.     

Dutasteride affects progesterone metabolizing enzyme activity/expression in human breast cell lines resulting in suppression of cell proliferation and detachment

Recent evidence indicates that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that breast carcinoma and tumorigenic breast cell lines have higher 5alpha-reductase and lower 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO activities and mRNA expression levels than normal tissue and non-tumorigenic cell lines. The 5alpha-reduced progesterone metabolites such as 5alpha-dihydroprogesterone (5alphaP) promote both mitogenic and metastatic activity in breast cell lines in culture, whereas the 4-pregnene metabolites, 4-pregnen-3alpha-ol-20-one (3alphaHP) and 4-pregnen-20alpha-ol-3-one (20alphaHP) have the opposite (anti-cancer-like) effects. The 5alpha-reductase inhibitor dutasteride has been shown to inhibit 5alpha-reduction of testosterone to 5alpha-dihydrotestosterone in prostate tissue, resulting in decreased prostate volume. The aim of this study was to determine if dutasteride is an effective inhibitor of progesterone 5alpha-reduction in human breast cell lines and if such inhibition reduces mammary cell proliferation and detachment. The effect of dutasteride on progesterone metabolizing enzyme activities and mRNA expression were examined in tumorigenic MCF-7 and non-tumorigenic MCF-10A human breast cell lines. Dutasteride (10(-6)M) inhibited progesterone conversion to 5alpha-pregnanes by >95% and increased 4-pregnene production. The results indicated that effects of dutasteride on the progesterone metabolizing enzymes are due to direct inhibition of 5alpha-reductase activity and to altered levels of expression of 5alpha-reductase and HSO mRNAs. Treatment of cells with progesterone without medium change for 72 h resulted in significant conversion to 5alpha-pregnanes and increases in cell proliferation and detachment. The increases in proliferation and detachment were blocked by dutasteride and were reinstated by concomitant treatment with 5alphaP, providing proof-of-principle that the effects were due not to progesterone but to the 5alpha-reduced metabolites. This study provides the first evidence that dutasteride is a potent progesterone 5alpha-reductase inhibitor and that such inhibition may be beneficial in breast cancer.     

Conversion of androstenedione to testosterone and other androgens in guinea-pig alveolar macrophages

Alveolar macrophages obtained by bronchoalveolar lavage of lungs of male and female guinea pigs were incubated with tritium-labelled androstenedione to evaluate the steroid metabolizing enzymes in these cells. The radiolabeled metabolites were isolated and thereafter characterized as testosterone, 5 alpha-androstanedione, 5 alpha-dihydrotestosterone, androsterone, isoandrosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol. Thus, the following androstenedione metabolizing enzymes are present in guinea-pig alveolar macrophages: 17 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase, 3 beta-hydroxysteroid dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase. The predominant androstenedione metabolizing enzyme activity present in alveolar macrophages was 17 beta-hydroxysteroid dehydrogenase. The rate of testosterone formation increased with incubation time up to 4 h, and with macrophage number up to 1.6 X 10(7) cells per ml. Androstenedione metabolism was similar in alveolar macrophages obtained both from male and female guinea pigs. These results suggest that alveolar macrophages may be a site of peripheral transformation of blood-borne androstenedione to biologically potent androgens in vivo and, therefore, these cells may contribute to the plasma levels of testosterone in the guinea pig.     

Metabolism of androgens in the seminal vesicles and the different lobes of the prostate in young mature rats

Oxidation and reduction of 4-androstene-3,17-dione (androstenedione), 17 beta-hydroxy-4-androsten-3-one (testosterone), 17 beta-hydroxy-5 alpha-androstan-3-one (DHT), 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-A'diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-A'diol) were measured in homogenates from ventral (VP), dorsal (DP) and lateral prostate (LP), the coagulating gland (CG) and seminal vesicle (SV) of the intact sexually mature rat using NAD(H) or NADP(H) as cofactors. The specific activity of the various enzymes varied significantly between the different organs. 5 alpha-Reductase activity was highest in the DP and the CG, and undetectable in the LP. 17 beta-Hydroxysteroid oxidoreductase (17 beta-HSOR) activity was mainly confined to the LP. 3 alpha-Hydroxysteroid oxidoreductase (3 alpha-HSOR) activity was also highest in the LP. In the VP the highest 3 alpha-HSOR activity was recorded using NAD(H) as cofactor. In the other organs, similar or higher enzymatic activities were measured using NADP(H) as added cofactor. 3 beta-Hydroxysteroid oxidoreductase (3 beta-HSOR) activity was high in the LP and low or undetectable in the other tissues. Our results indicate that isoenzymes of 3 alpha-HSOR, 3 beta-HSOR and 17 beta-HSOR are present in the accessory sex organs of the rat.     

Metabolism of dehydroisoandrosterone and androstenedione by human A-549 alveolar type II epithelial-like cells

The A-549 cell line was initiated from an explant of human lung carcinoma tissue. The biochemical characteristics of these cells are similar to those of normal alveolar type II epithelial cells. To gain some insight into the steroid-metabolizing capabilities of A-549 cells, the metabolism of tritium-labeled dehydroisoandrosterone and androstenedione by these cells was studied. The metabolism of dehydroisoandrosterone led to the exclusive formation of 5-androstene-3 beta,17 beta-diol. The major product of androstenedione metabolism was testosterone; and, 5 alpha-reduced steroids also were formed, viz. 5 alpha-androstane-3,17-dione, androsterone, isoandrosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol. Estrogens, viz., estrone and estradiol-17 beta, were not products of androstenedione metabolism by A-549 cells. The rates of metabolite formation from either dehydroisoandrosterone or androstenedione were linear as a function of incubation time up to 3 h, and with cell number up to 1 X 10(6) cells/ml. The apparent Km of 17 beta-hydroxysteroid oxidoreductase for dehydroisoandrosterone was 11 microM, and that for androstenedione was 13 microM. The predominant formation of 5-androstene-3 beta,17 beta-diol from dehydroisoandrosterone, and testosterone from androstenedione is a likely indication that the principal C19-steroid-metabolizing enzyme in A-549 cells is 17 beta-hydroxysteroid oxidoreductase; the other steroid-metabolizing enzymes expressed in these cells are 5 alpha-reductase, 3 beta-hydroxysteroid oxidoreductase and 3 alpha-hydroxysteroid oxidoreductase. The findings of this study demonstrate that A-549 cells express steroid-metabolizing enzymatic activities that are qualitatively similar to those found in other human pneumonocytes and human lung tissue, except for 3 beta-hydroxysteroid oxidoreductase-5----4-isomerase activity, which is not expressed in these cells with dehydroisoandrosterone as the substrate.     

Reductive metabolism of 5 alpha-dihydrotestosterone by rat ventral and dorsolateral prostate: kinetic parameters of the enzymes

In male sex accessory organs the active androgen 5 alpha-dihydrotestosterone (DHT) is metabolized to 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) by the reductase activities of 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR; EC 1.1.1.50) and 3 beta-hydroxysteroid oxidoreductase (3 beta-HSOR; EC 1.1.1.51). After separation of radiosubstrate and products by HPLC, these enzymes activities in subcellular preparations of rat ventral and dorsolateral prostate were determined from the conversion of [3H]DHT to the radiometabolites 3 alpha-diol and 3 beta-diol and 3 beta-triols (5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol plus 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol). Whereas both enzymes were found in the dorsolateral prostate, 3 beta-HSOR reductase activity was near the limit of detection in ventral prostate. Unlike the equal distribution of 3 alpha-HSOR reductase between the microsomal and cytosol fractions of the ventral prostate, both 3 alpha- and 3 beta-HSOR reductase activities of the dorsolateral prostate are mainly confined to its cytosol fraction. Km and Vmax of the 3 alpha- and 3 beta-HSOR reductases in dorsolateral prostate cytosol were 1.8 microM, 24.6 pmol.mg-1 min-1 and 25.4 microM, 45.7 pmol.mg-1 min-1, respectively. We surmise from these and earlier studies that 3 beta-HSOR reductase is the rate-limiting prostatic enzyme in the catabolic disposition of intracellular DHT.     

No inhibitory effects of gestrinone and medroxyprogesterone acetate on the estrogen production by ovaries of hypophysectomized rats stimulated by gonadotropins.

The in vivo effects of gestrinone (R2323) and medroxyprogesterone acetate (MPA) on the estrogen production by rat ovaries were investigated. Hypophysectomized immature female rats treated with 2.5 or 5 IU of pregnant mare serum gonadotropin (PMS) were daily given vehicle only, gestrinone (0.5 mg/kg body weight) or MPA (10 mg/kg body weight), and the activities of 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, 17, 20-lyase, 17 beta-hydroxysteroid dehydrogenase and aromatase in ovaries of these rats were measured. Gestrinone suppressed the 3 beta-hydroxysteroid dehydrogenase activity and increased activities of 17 alpha-hydroxylase, 17, 20-lyase and aromatase in ovaries stimulated by 5 IU of PMS, while MPA suppressed activities of 17 alpha-hydroxylase and aromatase in these ovaries. On the other hand, the aromatase activity in ovaries stimulated by 2.5 IU of PMS was suppressed by gestrinone and increased by MPA, and neither gestrinone nor MPA affected the production of aromatizable androgens from progesterone by these ovaries. Thus, gestrinone and MPA administrated in vivo showed divergent influences on steroidogenic enzyme activities in ovaries, but they did not affect the serum concentration of estradiol-17 beta. The present results suggest that neither gestrinone nor MPA reduced estrogen production by the rat ovary under the gonadotropin stimulation although they influenced some process of its steroidogenesis.     

3 Beta-hydroxysteroid dehydrogenase-isomerase activity in bovine adrenocortical cells in culture: lack of response to ACTH treatment

Primary cultures of bovine adrenocortical cells (BAC) were used to determine whether the adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase complex (3 beta-HSD), like the 17 alpha-hydroxylase (17-OHase), responded to ACTH treatment with an increase in activity. Both enzymes influence the steroidogenic path leading to 17 alpha-hydroxyprogesterone formation and thus could affect adrenal androgen biosynthesis. 3 beta-HSD Activity in postmitochondrial supernatant fluid, homogenates or cell monolayers remained unchanged after cells had been maintained in 1 microM ACTH up to 48 h. Since ACTH exposure led to a marked increase in 17-OHase activity over the same time period, it is concluded that, under the conditions used, the 3 beta-HSD-isomerase complex in BAC is nonresponsive to tropic hormone treatment.     

Metabolism of testosterone and dihydrotestosterone in cultured rat hepatoma cells.

Steroid metabolism in hepatoma tissue culture (HTC) cells derived from a male rat was investigated. Steroids in ethanol were incubated with the cells for various lengths of time. Volume of ethanol never exceeded 1% of incubation volume. Thin-layer and paper chromatography were used. Incubation was with tritiated steroids. It was demonstrated that testosterone as well as dihydrotestosterone is transformed. The main enzyme activities detected were 5alpha-reduction and 3alpha-, 3beta, and 17beta-hydroxysteroid dehydrogenation. The pattern of metabolism was reproducible and varied with time, substrate concentration, and number of cells incubated. Some steroids interfered with androgen metabolism. 17beta-estradiol, 17-epitestosterone, and progesterone competed for the 17beta-hydroxyprogesterone dehydrogenase. it is concluded that 3beta and 17beta reduction in the HTC cells may be catalyzed by the same enzyme which might differ considerably from the 3beta-hydroxysteroid dehydrogenase assayed in intact liver cells. A hepatoma derived from a female rat also produced considerable amounts of 3beta-derivatives of testosterone.     

Steroid 5 alpha-reductase activity in endothelial cells from human umbilical cord vessels

The metabolism of radiolabeled progesterone and androstenedione was evaluated in endothelial cells from human umbilical cord vein and arteries maintained in culture. The predominant metabolite of progesterone was 5 alpha-pregnane-3,20-dione and that of androstenedione was 5 alpha-androstane-3,17-dione. Thus, the major pathway of progesterone and androstenedione metabolism within these cells is via steroid 5 alpha-reductase. The rate of formation of 5 alpha-pregnane-3,20-dione from progesterone by venous endothelial cells was linear with incubation time up to 4 h and with cell number up to 1.6 X 10(6) cells/ml. The apparent Km of 5 alpha-reductase for progesterone was 0.4 microM; and, the Vmax was 55 pmol 5 alpha-pregnane-3,20-dione formed/mg protein X h. The rate of 5 alpha-androstane-3,17-dione formation from androstenedione also was linear with incubation time up to 4 h. In addition to 5 alpha-androstane-3,17-dione, the metabolism of androstenedione by either venous or arterial cells resulted in the formation of various minor metabolites, including testosterone and 5 alpha-reduced steroids, viz. 5 alpha-dihydrotestosterone, androsterone, isoandrosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, and 5 alpha-androstane-3 beta, 17 beta-diol. Estrogens (i.e. estradiol-17 beta and estrone) were not detected as products of androstenedione metabolism. The formation of these metabolites are indicative that the steroid-metabolizing enzymes present in endothelial cells are: 5 alpha-reductase, 17 beta-hydroxysteroid oxidoreductase, 3 alpha-hydroxysteroid oxidoreductase, and 3 beta-hydroxysteroid oxidoreductase.     

In vitro metabolism of testosterone on hepatic tissue of chicken (Gallus domesticus)

Among the subcellular fractions of chicken liver homogenates, the microsomal and cytosol fractions were most active in metabolism of testosterone with mutually different enzymological features. On the other hand, the nuclear and mitochondrial fractions had far lower activity of metabolizing the steroid. Metabolism by the cytosol fraction: the following steroids were identified as the metabolites of testosterone. 5 beta-Dihydrotestosterone (17 beta-hydroxy-5 beta-androstan-3-one), 5 beta-androstane-3 alpha,17 beta-diol and its 3 beta-epimer, 3 alpha-hydroxy-5 beta-androstan-17-one and its 3 beta-epimer and 5 beta-androstanedione. Metabolism by the microsomal fraction: from testosterone under aerobic condition, androstenedione was obtained as the major metabolite, besides the minor polar metabolites, production of which diminished when incubated in the atmosphere of carbon monoxide. From the results, testosterone was accepted to be firstly converted by the cytosol fraction into 5 beta-dihydrotestosterone which was then reduced to 5 beta-androstane-3 alpha,17 beta-diol and its 3 beta-epimer. These diols were further converted partially to 3 alpha -and 3 beta-hydroxy-5 beta-androstan-17-ones. These pathways were supported by the results of our incubation study with 5 beta-dihydrotestosterone and 5 beta-androstanedione as substrates. By the microsomes, testosterone was aerobically and anaerobically transformed to androstenedione as the major metabolite. Throughout our incubation experiments, no 5 alpha-reduction of a delta 4-3-oxo-steroid was detected in the chicken liver.     

Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes: potent inhibition by imidazole-type antimycotic drugs and lack of inhibition by steroid 5 alpha-reductase inhibitors.

5 alpha-Dihydrotestosterone, the principal androgen mediating prostate growth and function in the rat, is formed from testosterone by steroid 5 alpha-reductase. The inactivation of 5 alpha-dihydrotestosterone involves reversible reduction to 5 alpha-androstane-3 beta,17 beta-diol by 3 beta-hydroxysteroid oxidoreductase followed by 6 alpha-, 7 alpha-, or 7 beta-hydroxylation. 5 alpha-Androstane-3 beta,17 beta-diol hydroxylation represents the ultimate inactivation step of dihydrotestosterone in rat prostate and is apparently catalyzed by a single, high-affinity (Km approximately 0.5 microM) microsomal cytochrome P450 enzyme. The present studies were designed to determine if 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes is inhibited by agents that are known inhibitors of androgen-metabolizing enzymes. Inhibitors of steroid 5 alpha-reductase (4-azasteroid analogs; 10 microM) or inhibitors of 3 beta-hydroxysteroid oxidoreductase (trilostane, azastene, and cyanoketone; 10 microM) had no appreciable effect on the 6 alpha-, 7 alpha-, or 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (10 microM) by rat prostate microsomes. Imidazole-type antimycotic drugs (ketoconazole, clotrimazole, and miconazole; 0.1-10 microM) all markedly inhibited 5 alpha-androstane-3 beta,17 beta-diol hydroxylation in a concentration-dependent manner, whereas triazole-type antimycotic drugs (fluconazole and itraconazole; 0.1-10 microM) had no inhibitory effect. The rank order of inhibitory potency of the imidazole-type antimycotic drugs was miconazole greater than clotrimazole greater than ketoconazole. In the case of clotrimazole, the inhibition was shown to be competitive in nature, with a Ki of 0.03 microM. The imidazole-type antimycotic drugs inhibited all three pathways of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation to the same extent, which provides further evidence that, in rat prostate microsomes, a single cytochrome P450 enzyme catalyzes the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol. These studies demonstrate that certain imidazole-type compounds are potent, competitive inhibitors of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes, which is consistent with the effect of these antimycotic drugs on cytochrome P450 enzymes involved in the metabolism of other androgens and steroids.     

Costs and consequences of cellular compartmentalization and substrate competition among human enzymes involved in androgen and estrogen synthesis

The impact of compartmental expression of steroidogenic enzymes and of changes in flux through delta5 and delta4 metabolism on sex steroid synthesis was investigated by rebuilding pathways using recombinant enzyme expression by infection of insect cells with recombinant baculovirus constructs. Human cytochromes 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase (P450arom), always coexpressed with their redox partner NADPH-P450 oxidoreductase (CPR) and 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3betaHSD; types 1 or 2), were compartmentally expressed in different cell populations or coexpressed together with pregnenolone (100 nM) as substrate. Estrone was compared among cell compartments expressing different enzyme combinations or in cells coexpressing all enzymes (experiment 1). Additionally, P450c17, 3betaHSD, and CPR were all coexpressed, and androstenedione was measured in cells with different 3betaHSD expression levels or activity using an inhibitor, trilostane (experiment 2). Steroids were measured by immunoassay and mass spectrometry. In experiment 1, partitioning of P450c17, P450arom, and 3betaHSD markedly decreased estrone synthesis in comparison to cells coexpressing enzymes in different combinations. However, partitioning P450arom with 3betaHSD from P450c17 in different cell populations resulted in more estrone than either of the other two-cell compartment models. In experiment 2 (cells coexpressing P450c17, 3betaHSD, and CPR), androstenedione secretion was (paradoxically) higher at lower levels of 3betaHSD, and partial inhibition of 3betaHSD by trilostane also increased androstenedione when 3betaHSD expression was high. We conclude 1) that tissue or cell-specific, partitioned expression of sex steroid synthesizing enzymes limits rather than maximizes estrogen synthesis and 2) that limiting metabolism by 3betaHSD can paradoxically promote androgen synthesis when 3betaHSD expression is high by promoting delta5-steroid flux.     

In vitro metabolism of testosterone by cultured Sertoli cells and the effect of FSH.

Cultures of Sertoli cells isolated from testes of 18-and 36-day-old Long Evans rats were used to investigate their capacity to metabolize testosterone and the effect of FSH on such metabolism. Three different approaches were used: 1) investigation of the metabolism of radiolabeled testosterone under saturating substrate conditions; 2) study of the metabolism of radiolabeled testosterone utilizing trace amounts of high specific activity substrates; 3) the utilization of radioimmunoassay for measurement of estradiol-17 beta. The following steroids were isolated and identified by recrystallization to constant specific acitvity from the control and FSH-treated cultures; testosterone (unconverted substrate), androstenedione, dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha, 17 beta-diol. Radioimmunoassay data suggests that the Sertoli cells produce an estradiol-17 beta-like compound from unlabeled testosterone and that this production is stimulated by FSH. However, the radioactive metabolite from all our studies that behaved chromatographically like estradiol--17 beta failed to crystallize to constant specific activity, while in each experiment, authentic radiolabeled estradiol-17 beta added as recovery tracer did. The data demonstrate that : 1) cultures of Sertoli cells from immature rats have 5 alpha-reductase, 3 alpha- and 17 beta-hydroxysteroid oxidoreductase activities; 2) these enzymes may be affected by FSH; 3) based on radiolabeled metabolic techniques, Sertoli cells were unable to biotransform testosterone to estradiol-17 beta even in the presence of FSH.     

Role of 5 alpha-reduction in progesterone's ability to release FSH in estrogen-primed ovariectomized rats.

In ovariectomized estrogen-primed rats, progesterone as well as 5 alpha-dihydroprogesterone (5 alpha-DHP) are capable of inducing the release of gonadotropins. This study examined the need of 5 alpha-reduction as a prerequisite for the action of progesterone. The 5 alpha-reductase inhibitor, N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide was injected at a 1 or 2 mg dose/rat 2 h prior to an injection of 0.4 or 0.8 mg progesterone/kg body weight at 0900 h to immature ovariectomized, estrogen-primed rats and serum was analyzed for LH and FSH at 1500 h. Pituitary and hypothalamic 5 alpha-reductase activity was measured at the time of progesterone administration and at the time of the surge by incubating tissue homogenates with [3H]progesterone. Substrate, ([3H]progesterone) and product ([3H]5 alpha-DHP), were separated by reverse phase HPLC. The pituitary 5 alpha-reductase activity was not blocked at 1500 h. However, both pituitary and hypothalamic 5 alpha-reductase was blocked at the time of progesterone administration. No effect was seen by acute administration of the 5 alpha-reductase inhibitor upon either the 0.4 or 0.8 mg progesterone/kg-induced release of LH and FSH. There was, however, a specific, significant inhibition of progesterone-induced FSH but not LH release when the 5 alpha-reductase inhibition was sustained throughout the afternoon of the gonadotropin surge. These results indicate a biologically significant role for the irreversible 5 alpha-reduction of progesterone in the modulation of the release of FSH.     

Isolation of multiple forms of indanol dehydrogenase associated with 17 beta-hydroxysteroid dehydrogenase activity from male rabbit liver

Seven multiforms of indanol dehydrogenase were isolated in a highly purified state from male rabbit liver cytosol. The enzymes were monomeric proteins with similar molecular weights of 30,000-37,000 but with distinct electrophoretic mobilities. All the enzymes oxidized alicyclic alcohols including benzene dihydrodiol and hydroxysteroids at different optimal pH, but showed clear differences in cofactor specificity, steroid specificity, and reversibility of the reaction. Two NADP+-dependent enzymes exhibited both 17 beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes and 3 alpha-hydroxysteroid dehydrogenase activity for 5 beta-androstan-3 alpha-ol-17-one. Three of the other enzymes with dual cofactor specificity catalyzed predominantly 5 beta-androstane-3 alpha,17 beta-diol dehydrogenation. The reverse reaction rates of these five enzymes were low, whereas the other two enzymes, which had 3 alpha-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes or 3(17)beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes, highly reduced 3-ketosteroids and nonsteroidal aromatic carbonyl compounds with NADPH as a cofactor. All the enzymes exhibited Km values lower for the hydroxysteroids than for the alicyclic alcohols. The results of kinetic analyses with a mixture of 1-indanol and hydroxysteroids, pH and heat stability, and inhibitor sensitivity suggested strongly that, in the seven enzymes, both alicyclic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities reside on a single enzyme protein. On the basis of these data, we suggest that indanol dehydrogenase exists in multiple forms in rabbit liver cytosol and may function in in vivo androgen metabolism.     

In vitro inhibition by ketoconazole of human testicular steroid oxidoreductases

An oral antimycotic agent, ketoconazole has been demonstrated to be an inhibitor of cytochrome P-450-dependent monooxygenases. To investigate its effect on steroid oxidoreductases, in vitro studies were carried out using subcellular fractions of human testes. Ketoconazole competitively inhibited activities of 3 beta-hydroxy-5-ene-steroid oxidoreductase/isomerase and NADH-linked 20 alpha-hydroxysteroid oxidoreductase for steroid substrate and the Ki values were 2.9 and 0.9 microM, respectively. In contrast, ketoconazole inhibited neither 17 beta-hydroxysteroid oxidoreductase nor NADPH-linked 20 alpha-hydroxysteroid oxidoreductase, indicating that the two 20 alpha-hydroxysteroid oxidoreductases are distinct. Further, ketoconazole inhibited non-competitively the above enzyme activities for the corresponding cofactors of NAD and NADH. From the binding mode of ketoconazole to cytochrome P-450 and the present findings, it appears likely that the agent binds to a site which is different from that of steroids or pyridine nucleotides.     

In vitro steroidogenesis in testes of three infants, two with ambiguous external genitalia and one with true precocious puberty: Evidence for the presence of active 17 beta-hydroxysteroid oxidoreductase in immature human testes

The present report investigated steroidogenesis in vitro in testis tissues obtained from two boys aged 8 months and 4 years with ambiguous external genitalia and male vagina, and a 4-year-old body with true precocious puberty. Histologically, testes of the former two boys are still immature and the testis of the last one contains differentiated Sertoli cells and primary spermatocytes, but no mature Leydig cells are recognized in any of them. In each testis, 17 beta-hydroxysteroid oxidoreductase is active for androstenedione in the presence of an excess amount of NADPH, while delta 5-3 beta-hydroxysteroid dehydrogenase and delta 4-steroid 5 alpha-reductase activities are limited. 17 alpha-Hydroxylase and C17--20 lyase are significantly active in each testis and are enhanced in the testis of the boy with precocious puberty. Although the testis tissue used in the present study may not be biologically normal and the number of cases investigated is still limited, the above results indicate that active 17 beta-hydroxysteroid oxidoreductase is present in immature human testes and that delta 5-3 beta-hydroxysteroid dehydrogenase may become active in the human testis at the advanced stage of the development of testicular function during the puberty.     

Direct effects of lithium chloride on the activities of delta 5-3 beta and 17 beta-hydroxysteroid dehydrogenase in the testis and Bidder's organ of the adult toad (Bufo melanostictus)--in vitro study

Steroidogenic key enzymes, i.e. delta 5-3 beta and 17 beta-hydroxysteroid dehydrogenase (delta 5-3 beta and 17 beta-HSD) activities, in the testis and Bidder's organ of the toad were inhibited and ascorbic acid synthesis in these organs was decreased by a wide range of lithium concentration in in vitro study. A significant inhibition was noted at a concentration of 2.0 mM, which is easily achieved in the blood during the treatment of manic patients by lithium chloride. This experiment reflected that lithium exerts a direct inhibitory effect on hydroxysteroid dehydrogenase activities in the testis and Bidder's organ--a rudimentary ovary in Bufo.