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.     

Multiple steroid metabolic pathways in ZR-75-1 human breast cancer cells

In order to characterize the main enzymatic systems involved in androgen and estrogen formation as well as metabolism in ZR-75-1 human breast cancer cells, incubation of intact cells was performed for 12 or 24 h at 37 degrees C with tritiated estradiol (E2), estrone (E1), androst-5-ene-3 beta, 17 beta-diol (5-ene-diol), dehydroepiandrosterone (DHEA), testosterone (T), androstenedione (4-ene-dione), dihydrotestosterone (DHT) or androsterone (ADT). The extra- and intracellular steroids were extracted, separated into free steroids, sulfates and non-polar derivatives (FAE) and identified by HPLC coupled to a Berthold radioactivity monitor. Following incubation with E2, 5-ene-diol or T, E1, DHEA and 4-ene-dione were the main products, respectively, thus indicating high levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). When 4-ene-dione was used, on the other hand, a high level of transformation into 5 alpha-androstane-3,17-dione (A-dione), Epi-ADT and ADT was found, thus indicating the presence of high levels of 5 alpha-reductase as well as 3 alpha- and 3 beta-hydroxysteroid dehydrogenase. Moreover, some T was formed, due to oxidation by 17 beta-HSD. No estrogen was detected with the androgen precursors T or 4-ene-dione, thus indicating the absence of significant aromatase activity. Moreover, significant amounts of sulfates and non-polar derivatives were found with all the above-mentioned substrates. The present study shows that ZR-75-1 human breast cancer cells possess most of the enzymatic systems involved in androgen and estrogen formation and metabolism, thus offering an excellent model for studies of the control of sex steroid formation and action in breast cancer tissue.     

Androsterone 3beta-substituted derivatives as inhibitors of type 3 17beta-hydroxysteroid dehydrogenase

Androsterone derivatives substituted at position 3 were synthesized starting from dihydrotestosterone in a short sequence of reactions. They proved to be potent inhibitors (IC50 = 57-147 nM) of type 3 17beta-hydroxysteroid dehydrogenase, a key enzyme of steroidogenesis, which catalyzes the transformation of androstenedione to steroid active androgen testosterone.     

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.     

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.     

4-Azasteroidal 5 alpha-reductase inhibitors without affinity for the androgen receptor

In efforts to develop potent 5 alpha-reductase inhibitors without affinity for the androgen receptor, synthetic 3-oxo-5 alpha-steroids were tested for their ability to inhibit 5 alpha-reductase, using [14C]testosterone as the substrate, and for their ability to inhibit the binding of [3H]5 alpha-dihydrotestosterone to the androgen receptor of rat prostate cytosol. 2',3' alpha-Tetrahydrofuran-2'-spiro-17-(5 alpha-androstan-3-one) is not an inhibitor of 5 alpha-reductase and has a high affinity for the androgen receptor; substitution of the -CH2- at the 4-position with N-H resulted in a good inhibitor of 5 alpha-reductase. The 4-N-CH3 derivative is even more active, whereas the N-CH2-CH3 derivative is inactive. These 4-aza derivatives have much lower affinity for the androgen receptor than the parent compound. The 4-N-H derivatives of several 3-oxo-5 alpha-steroids were found to be 20-100% as potent as their corresponding 4-N-CH3 analogs as inhibitors of 5 alpha-reductase, whereas their androgen receptor affinities were at least 40-fold lower than their 4-N-CH3 analogs. Their 5 beta-isomers did not inhibit either 5 alpha-reductase or the androgen receptor binding of [3H]5 alpha-dihydrotestosterone. Two of these 4-N-H steroids, 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one and 17 beta-N, N-diisopropylcarbamoyl-4-aza-5 alpha-androstan-3-one, are potent 5 alpha-reductase inhibitors with Ki values equal to 29.2 +/- 1.7 and 12.6 +/- 0.8 nM, respectively, but have little affinity for the androgen receptor. The inhibition of 5 alpha-reductase by both compounds is competitive with testosterone. When [3H]testosterone was incubated with minced rat prostate in the presence of either of these two 4-azasteroids, the nuclear concentration of 5 alpha-dihydrotestosterone decreased and that of testosterone increased. The total nuclear uptake of testosterone plus 5 alpha-dihydrotestosterone was not significantly affected. These 4-azasteroids should be useful for investigating the importance of 5 alpha-reductase in androgen action in vivo.     

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.     

Crystal structures of the multispecific 17beta-hydroxysteroid dehydrogenase type 5: critical androgen regulation in human peripheral tissues

Human type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD5;AKR1C3) plays a major role in the metabolism of androgens in peripheral tissues. In prostate basal cells, this enzyme is involved in the transformation of dehydroepiandrosterone into dihydrotestosterone, the most potent androgen. It is thus a potential target for prostate cancer therapy because it is understood that the testosterone formation by this enzyme is an important factor, particularly in patients who have undergone surgical or medical castration. Here we report the first structure of a human type 5 17beta-HSD in two ternary complexes, in which we found that the androstenedione molecule has a different binding position from that of testosterone. The two testosterone-binding orientations in the substrate-binding site demonstrate the structural basis of the alternative binding and multispecificity of the enzyme. Phe306 and Trp227 are the key residues involved in ligand recognition as well as product release. A safety belt in the cofactor-binding site enhances nicotinamide adenine dinucleotide phosphate binding and accounts for its high affinity as demonstrated by kinetic studies. These structures have provided a dynamic view of the enzyme reaction converting androstenedione to testosterone as well as valuable information for the development of potent enzyme inhibitors.     

Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat during oestrogen administration.

This investigation was undertaken to elucidate the amount of oestradiol and duration of its administration necessary to cause complete feminization of the activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase in male rat liver. With the exception of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase, 5 microgram oestradiol/d for 8 days and less was sufficient to cause complete feminization. The order of oestrogen sensitivity was cytoplasmic 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 3 beta-hydroxysteroid dehydrogenase greater than microsomal 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 5 alpha-reductase greater than cytoplasmic 17 beta-hydroxysteroid dehydrogenase. Although the changes occurring after oestradiol administration are qualitatively the same as after testectomy, they occur more rapidly. This rules out the possibility that oestradiol exerts its effect via androgen deprivation. Diethylstilboestrol administration causes the same changes in cytoplasmic 17 beta- and microsomal 3 beta-hydroxysteroid dehydrogenase activity as oestradiol, although the dosage must be increased 100 fold. The effect of diethylstilboestrol on 5 alpha-reductase activity changes with the dose applied. Doses up to 100 microgram/d partially feminize the activity, but at higher doses the enzyme activity is repressed.     

Androstenedione metabolism in human lung fibroblasts

Human lung fibroblasts in culture metabolized [3H]androstenedione to a number of different compounds, including testosterone, 5 alpha-androstanedione, androsterone, 5 alpha-dihydrotestosterone, isoandrosterone, and 5 alpha-androstane-3 alpha,-17 beta-diol. The major products were 5 alpha-androstanedione and testosterone. Estrone, estradiol-17 beta and 5 beta-reduced steroids were not formed. The production rates of testosterone and 5 alpha-androstanedione from [3H]androstenedione by lung fibroblasts were studied both as a function of incubation time and substrate concentration. The rates of formation of testosterone and 5 alpha-androstanedione remained linear with time up to 4 h. The apparent Km of human lung fibroblast 5 alpha-reductase was 1 microM, and that of 17 beta-hydroxysteroid oxidoreductase was 11 microM. The findings of this study suggest that mesenchyma may contribute to the metabolism of androstenedione in human lung tissue.     

Comparison of the effect of cortisol on aromatase activity and androgen metabolism in two human fibroblast cell lines derived from the same individual

The effect of preincubation with cortisol on estrogen and androgen metabolism was investigated in human fibroblast monolayers grown from biopsies of genital and non-genital skin of the same person. The activity in the cells of aromatase, 5 alpha-reductase, 17 beta-hydroxysteroid oxidoreductase and 3 alpha-hydroxysteroid oxidoreductase was investigated by isolating estrone, estradiol, estriol, dihydrotestosterone, androstanedione, androsterone, 3 alpha-androstanediol, testosterone and androstenedione after incubation of the cells with [14C]testosterone or [14C]androstenedione. For experiments with 14C-labeled substrate the cells were incubated in medium, charcoal stripped of steroids without Phenol Red. Preincubation from 6 to 36 h with cortisol in concentrations of 10(-8) - 10(-6) M showed maximal stimulation of aromatase activity after 12 h preincubation with cortisol in concentrations of 0.5-1.0 x 10(-6) M in both cell lines. When preincubation with cortisol was omitted no estrogen synthesis was detected. The formation of androgen was not altered after preincubation with cortisol. Pronounced differences were found in estrogen and in androgen metabolism in the two cell lines suggesting a local regulation of the hormonal environment. The aromatase activity, which is low in many tissues could be stimulated by cortisol without altering the androgen metabolism was found to be a suitable system for investigations of the cellular interconversion of androgens and estrogens and for investigations of the in vitro regulation of the enzymes involved.     

The hypophysis in the regulation of androgen and oestrogen dependent enzyme activities of steroid hormone metabolism in rat liver cytosol.

In order to determine whether the gonadal and hypophyseal modes of regulation recently reported for the microsomal enzymes of hepatic steroid metabolism are also valid for cytoplasmic enzymes, three enzymes whose activities exhibit sex differences (male:female activity ratio shown in brackets), 5beta-reductase(1.7:1), 20alpha-hydroxysteroid dehydrogenase(5 : 1) and 17beta-hydroxysteroid dehydrogenase (4:1), as well as one enzyme whose activity shows no sex difference, 3beta-hydroxy-delta5-steroid dehydrogenase, were investigated after various interferences with the endocrine balance (gonadectomy, hypophysectomy, combination of both operations, administration of testosterone or oestradiol). From the results of this and a previous study the following statements can be made about the endocrine control of hepatic enzyme activities. Those enzymes whose activities show sex differences are either androgen or oestrogen dependent; the sex hormone acts in either an inductive or repressive manner. 1) Criteria for androgen dependency are the feminization of enzyme activity after testectomy or inhibition of testicular function by administration of oestradiol; masculinization of the enzyme activity after administration of testosterone to male or female castrates. Using these criteria the following enzymes investigated in this laboratory fall into this category: all microsomal enzymes which show sex differences in their activity (3alpha-, 3beta-, delta4-3beta, 20-hydroxysteroid dehydrogenase; cortisone alpha-reductase; steroid hydroxylases and 16alpha-hydroxylase) as well as the cytoplasmic 20alpha-hydroxysteroid dehydrogenase. Apart from the single exception of 20alpha-hydroxy-steroid dehydrogenase the presence of the hypophysis is obligatory for the androgen to be effective. The hypophysis does not only work in a permissive manner, but participates in establishing the sex specific activity levels in a manner which is antagonistic to the androgen action. 2) Criteria for oestrogen dependency are that the female animal reacts to gonadectomy, as well as to the inhibition of ovarian function after testosterone administration, by a masculinization of the enzyme activities. After administration of oestradiol, but not gonadectomy, the male animal exhibits typical female activity. Using these criteria the cytoplasmic 5beta-reductase and 17beta-hydroxysteroid dehydrogenase are oestrogen dependent. The repressive oestrogen effect observed on 17beta-hydroxysteroid dehydrogenase is antagonistic to hypophyseal action, whereas in the case of 5beta-reductase it is synergistic. 3) The activities of cytoplasmic 3beta-hydroxy-delta5-steroid dehydrogenase and microsomal 7alpha-hydroxylase show no sex differences and are not influenced by any interference with the endocrine balance.     

Comparative study on the metabolism of the androgen precursor androstenedione in two gastropod species: in vitro alterations by TBT and TPT

A comparative study was performed to assess the metabolism of the androgen precursor androstenedione (AD) in two gastropod species from the Muricidae family: Bolinus brandaris and Hexaplex trunculus. AD was mainly converted to 5alpha-dihydrotestosterone by microsomal fractions isolated from Bolinus brandaris, whereas it was primarily metabolized to testosterone by Hexaplex trunculus. Sex differences in the metabolism of AD were only detected in Bolinus brandaris and attributed to higher 5alpha-reductase activity in males. Thereafter, the effect of the organotin compounds, tributyltin (TBT) and triphenyltin (TPT), on the metabolism of AD was investigated. A significant interference was only detected in females, and differences between the modes of action of the two compounds were observed: TPT was a strong inhibitor of 5alpha-reductase activity in B. brandaris at a concentration as low as 100 nM whereas only TBT (10 microM) altered the metabolism of AD in H. trunculus by increasing the activity 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Thus, this work shows that the metabolism of the androgen precursor AD strongly differs among gastropod species, both in terms of activity and metabolic profile, and further demonstrates the ability of TBT and TPT to interfere with key enzymatic pathways involved in androgen synthesis.     

Sex steroid hormone metabolism and prostate cancer

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.     

The effects of diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and (4R)-5,10-SECO-19-norpregna-4,5-diene-3,10,20-trione (SECO) on androgen biosynthesis in the rat testis and epididymis

We have investigated the effects of two 4-ene-steroid 5 alpha-reductase inhibitors, diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and (4R)-5,10-seco-19-norpregna-4, 5-diene-3,10,20-trione (SECO), on testicular and epididymal androgen biosynthesis. Kinetic analyses revealed that both compounds inhibited epididymal DHT biosynthesis. 4-MA was a competitive inhibitor of epididymal nuclear and microsomal 4-ene-steroid 5 alpha-reductases (3-oxo-5 alpha-steroid: NADP 4-ene-oxidoreductase EC 1.3.1.22) with Kiapp values of 12.8 and 15.1 nmol/l compared to the respective Kmapp values of 185 and 240 nmol/l. Values for the Vmaxapp were always within 70-130% of the control. SECO at 1.0 mumol/l, also inhibited epididymal nuclear and microsomal 4-ene-steroid-5 alpha-reductases, causing respectively 2.9 and 5.2-fold increases in Kmapp. The Vmaxapp values were unchanged. However, SECO concentrations of 5 and 25 mumol/l abolished 4-ene-steroid 5 alpha-reductase activity at all testosterone concentrations. To examine the specificity of these compounds, we investigated their effects on the enzymes that convert pregnenolone to testosterone. Rat testis microsomes converted pregnenolone to testosterone via the 4-ene-3-oxo pathway, with the major metabolites being progesterone, 17-hydroxyprogesterone, 4-androstenedione and testosterone; some 17-hydroxypregnenolone was also formed. Very small amounts of dehydroepiandrosterone (DHA) and 5-androstenediol were detected. SECO, at a concentration that completely inhibited epididymal 4-ene-steroid 5 alpha-reductase activity, did not alter the metabolic profile of pregnenolone metabolism. However, 4-MA prevented the appearance of 4-ene steroids, and large quantities of 17-hydroxypregnenolone and DHA accumulated, suggesting that inhibition of the 3 beta-hydroxysteroid: NAD(P)+ oxidoreductase (EC 1.1.1.51) and 3-oxosteroid 5-ene-4-ene-isomerase (EC 5.3.3.1) [3 beta-hydroxysteroid dehydrogenase-isomerase] was occurring. Optimal conditions for the microsomal conversion of DHA to 4-androstenedione were determined; kinetic analyses of the 3 beta-hydroxysteroid dehydrogenase-isomerase activity revealed that 4-MA inhibited this reaction non-competitively, reducing Vmaxapp values to 25% of the control. The Kiapp determined from the intercept replot, was 121 nmol/l, and the Kmapp was always between 90 and 130% of the control value. It is concluded that SECO is more specific than 4-MA in its effects on androgen biosynthesis in the testis and epididymis and that both these drugs should provide useful tools in assessments of the relative contributions of 5 alpha-reduced androgens to androgen dependent processes.     

Metabolism of [4-14C]testosterone and precursors by homogenates of rat testes after chronic LHRH-treatment

Adult male rats were injected daily for 8 days with an LHRH agonist. Twenty-four hours after the last injection testes-homogenates were incubated in the presence of a 4-14C-labeled steroid, either progesterone, 17 alpha-hydroxyprogesterone, dehydroepiandrosterone, androstenedione or testosterone. The activity of several enzymes involved in the androgen biosynthetic pathway was inferred from the amount of metabolites produced under these conditions. After LHRH-treatment a significant increase in the 17,20-lyase activity was observed without any significant change in the activity of 17 alpha-hydroxylase, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase and 17 beta-hydroxysteroid dehydrogenase. The results of the experiments indicate that the decreased testosterone secretion observed in rats after chronic LHRH-administration is not due to an inhibition of the enzyme-systems studied.     

Effects of repeated androgen treatments on metabolism and nuclear binding of androgen in the infant murine submandibular gland

1. Androgen responsiveness of esteropeptidase of the murine submandibular gland developed rapidly in normal males compared with in normal females and castrated males. 2. Repeated treatments of infant mice of both sexes with testosterone (T), 5 alpha-dihydrotestosterone (DHT) or 5 alpha-androstane-3 alpha, 17 beta-diol increased androgen responsiveness of this enzyme, but did not affect those of 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDase; androgen metabolizing enzymes) of the gland. 3. Exchange assay of nuclear androgen receptor using 3H-DHT showed that in both sexes, amounts of binding in animals pretreated with T were higher than those in animals pretreated with sesame oil. 4. These results suggest that there is parallelism between the androgen responses and amounts of nuclear androgen binding, not androgen responses of 5 alpha-reductase and 3 alpha-HSDase.     

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.     

Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat following gonadectomy.

The activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase of rat liver were determined at different time points after gonadectomy on day 75 of life. Following testectomy the activities in male rats assume female values. However this change is relatively slow, 10--14 days being necessary for significant trends in individual activities to develop, and 40--60 days before the final level of activity is reached. The changes in enzyme activities after ovariectomy are only slight. The change in microsomal 5 alpha-reductase activity following gonadectomy of male rats is biphasic, the activity increasing initially to the normal female level before falling to the intermediate "neonatally androgen-imprinted" level. The reaction of 17 beta-hydroxysteroid dehydrogenase activity to testectomy and ovariectomy indicates that in the course of several years, during which we have investigated the behaviour of this enzyme in Chbb/THOM rats, the regulation of its activity has changed from one of oestrogen dependency to one of androgen dependency.     

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.