Prostatic cytosol binding of 5 alpha-androstane-3 beta, 17 beta-diol and estradiol-17 beta

The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.     

Binding characteristics of [3H]delta(5)-androstene-3beta,17beta-diol to a nuclear protein in the rabbit vagina

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.     

Histo-autoradiographic study of a testosterone target organ, the epididymis of the lizard (Lacerta vivipara), after administration of 3H-17 beta-estradiol]

[6,7 3H] oestradiol-17 beta was injected into castrated male viviparous lizards in order to compare retention of this steroid to that of testosterone during the period of sexual activity. Retention of the isotope in epididymis was greater than in blood, lung and stomach. Radioautographs of epididymis indicated that oestradiol or a metabolite was concentrated in cell nuclei and over discharged secretory granules as was observed with testosterone. Reason of such binding is not yet known.     

Estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase from rabbit liver--II. Mechanisms of enzyme-steroid interaction

Binding of [3H]estradiol, [3H]testosterone and [3H]progesterone to purified NADP-dependent estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with moderate [corrected] affinity (Ka congruent to 10(7) [corrected] M-1 at 4 degrees C) and equal binding capacity. High-rates were shown for both association and dissociation processes. The steroids competitively inhibited the binding of each other to EHSD. At the same time, their relative binding affinities (RBA) were dependent on the nature of [3H]ligand. The results of RBA determinations for 72 steroids and their analogues by inhibition of [3H]progesterone binding to EHSD suggest that androgens and gestagens bind preferentially to the same site on EHSD molecule, while estrogens (at least by their D-ring) bind to another site. The assumption that EHSD molecule has more than one binding site for steroids is corroborated by (i) substrate inhibition revealed for a number of steroids; (ii) the estrogen ability to potentiate 20 alpha-reduction of progesterone; (iii) stimulatory effect of 5 alpha (beta)-androstane-3 alpha (beta), 17 beta-diols on [3H]testosterone and progesterone binding; and (iv) reciprocal effect of NADP on [3H]estradiol and [3H]testosterone binding to EHSD. Significant differences in sensitivity to pH and changes in NaCl concentration upon metabolism and binding of various steroids have been found. At concentrations of 16 mM dithiothreitol potentiated catalytic conversion of some steroids and had no effect on metabolism of others. Both the affinity for steroids and binding capacity of EHSD are found to be cofactor-dependent. It is speculated that EHSD has a complex active center including at least two mutually influencing steroid-binding sites tightly related with cofactor-binding site. The polyfunctionality of EHSD may be due to both the excess of functional protein groups that form individual constellations upon binding of any steroid and also to conformational lability of EHSD molecule implying alternative orientations of steroids at the binding site.     

Serum levels of 5-androstene-3 beta,17 beta-diol sulphate, 5 alpha-androstane-3 alpha, 17beta-diol sulphate and glucuronide, in late onset 21-hydroxylase deficiency

Serum sulphates of 5-androstene-3 beta,17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), as well as 5 alpha-androstane-3 alpha,17 beta-diol glucuronide (3 alpha-DIOL-G) and unconjugated androstenedione (AD) and testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI) and 17 alpha-hydroxyprogester-one (17OHP) were measured by specific radioimmunoassays (RIA) in 14 women with late-onset 21-hydroxylase deficiency (LOCAH), and in normal women (n = 73). The diagnosis of LOCAH was made on the finding of a (17OHP) response level greater than 30 nmol/l following ACTH stimulation, and/or an elevation of urinary metabolites of 17OHP. Mean values for serum concentrations of all steroids measured and the free androgen index (100 X T nmol/l divided by SHBG nmol/l) were significantly elevated, and SHBG levels depressed in patients with LOCAH. These studies show that in LOCAH, in addition to the unconjugated steroids AD and T, the sulphoconjugated steroids DHEA-S, 5-ADIOL-S and 3 alpha-DIOL-S are increased, as is the glucuronide conjugate 3 alpha-DIOL-G and the index of bioavailable testosterone (FAI), and that mean SHBG levels are depressed. These data suggest that as well as AD, 5-ADIOL-S and DHEA-S may act as pro-hormones for more potent steroids (T and 5 alpha-dihydrotestosterone) in peripheral tissues, while 3 alpha-DIOL-S and 3 alpha-DIOL-G may both reflect peripheral androgen metabolism in patients with LOCAH.     

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.     

Effect of testosterone and 17beta-estradiol treatment on sex steroid binding proteins in the male of the green frog Rana esculenta

In this paper we report the effect of gonadectomy and/or long-term sex steroid (testosterone and estradiol-17beta) treatment and prolonged captivity (two months) on testosterone and estradiol-17beta binding proteins (TBP and EBP, respectively) in the plasma of the male of the green frog Rana esculenta. Experiments were carried out during different periods of the reproductive cycle. Gonadectomy and prolonged captivity were carried out in winter, when the spermatogenic activity slowed down and the concentration of circulating androgens was high. Both gonadectomy and prolonged captivity resulted in a significant decrease in TBP binding activity, which could not be restored by the hormonal treatment. On the contrary, when the hormonal treatment was carried out in the early summer, when the spermatogenesis was active but the concentration of circulating androgens was low, a significant increase in TBP binding activity was observed. Neither gonadectomy, nor the prolonged captivity, nor the hormonal treatment affected EBP levels. Our data indicate that TBP apparent changes in response to testosterone and estradiol-17beta treatment varied according to the period of the reproductive cycle, an indication that studies on sex steroid binding proteins regulation should take into consideration the internal endocrine condition before drawing any final conclusion especially in species with a seasonal mode of reproduction.     

Radioautographic study of the effect of estradiol-17 , estrone, estriol, progesterone, testosterone and corticosterone on the in vitro uptake of 2,4,6,7- 3 H estradiol-17 by uterine eosinophils of the rat

The in vitro uptake of 2,4,6,7-tritiated estradiol-17beta in uterine eosinophils of the rat was inhibited by the presence of nonradioactive estradiol-17beta, estrone, and estriol, but not by progesterone, testosterone, or corticosterone. This action is attributed to competition between tritiated estradiol and the various estrogenic compounds for the same binding site. Compounds without any estrogenic activity do not compete. The proposal is made that the eosinophil binding system and the 8S-5S binding system are involved in different mechanisms of estrogen action. The parallelism between the doses of estradiol and estriol needed to promote certain estrogenic early effects in the uterus, and the affinity of these steroids for the eosinophil uptake sites, suggests that uterine eosinophils might be responsible for some of these early effects, such as water imbibition, histamine releasing activity, and estrogen priming effect.     

Inhibition of hepatic 17 beta- and 3 alpha-hydroxysteroid dehydrogenases by antiinflammatory drugs and nonsteroidal estrogens

Antiinflammatory agents and estrogens have been tested as inhibitors of two isozymes of guinea pig liver testosterone 17 beta-dehydrogenase (NADP) 1.1.1.64) and rat liver 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50). Antiinflammatory steroids and estradiols were highly inhibitory to 3 alpha-hydroxysteroid dehydrogenase and one isozyme of testosterone 17 beta-dehydrogenase, respectively, but nonsteroidal antiinflammatory agents and nonsteroidal estrogens such as hexestrol, dienstrol, diethylstilbestrol and zearalenone showed potent inhibitions on all the enzymes. Although the inhibitory potency of indomethacin for one isozymes of testosterone 17 beta-dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase decreased with changing pH from 9.7 to 7.0, that of the nonsteroidal estrogens for all the enzymes was little affected by pH. No additive effect in double inhibitor experiments with indomethacin and the nonsteroidal estrogens was observed, and the compounds were all competitive inhibitors with respect to steroidal substrate. The results suggest that there is a very similar region in substrate binding sites of the enzymes.     

Comparison of the effects of gossypol, estradiol-17 beta and testosterone compensation on male rat reproductive organs

The effects of gossypol acetic acid (GAA) were compared with those induced by estradiol-17 beta (E2), testosterone, and a combination of these steroids. GAA was administered s.c. to adult rats at doses of 25 to 30 mg . kg BW . day for 30 days, while steroids in polydimethylsiloxane tubing of various lengths were implanted s.c. for 30 days or longer. GAA and E2 at the doses used had similar effects: they caused a graded atrophy of sex organs, discriminative degeneration of spermatogenic cells, impairment of Sertoli cells, decrease in serum testosterone, reduction in androgen receptor binding and retardation in body growth. Supplementing GAA and E2 treatments with 14-cm testosterone implants had a counteracting effect on organ weight losses: seminal vesicles recovered above, ventral prostate within and epididymides below control values, whereas the testes did not respond. The organs most refractory to supplementation therapy were those in which GAA and E2 were most effective in depressing androgen receptor binding. Aside from having similar antiandrogenic effects as E2 and other steroids, GAA induced a specific flagellar syndrome which testosterone therapy could not prevent, indicating that this action is hormonally independent.     

Kinetic and stereochemical studies on reaction mechanism of mouse liver 17 beta-hydroxysteroid dehydrogenases

The kinetic mechanism of two major monomeric 17 beta-hydroxysteroid dehydrogenases from mouse liver cytosol was studied at pH 7 in both directions with NADP(H) and three steroid substrates: testosterone, 5 beta-androstane-3 alpha, 17 17 beta-diol, and estradiol-17 beta. In each case the reaction mechanism of the two enzymes was sequential, and inhibition patterns by-products and dead-end inhibitors were consisted with an ordered bi bi mechanism with the coenzyme binding to the free enzyme, although there was difference in affinity and maximum velocity for the steroidal substrates between the two enzymes. Binding studies of the coenzyme and substrate indicate the binding of coenzyme to the free enzyme, in which 1 mol of NADPH binds to 1 mol of each monomeric enzyme. The 4-pro-R-hydrogen atom of NADPH was transferred to the alpha-face of the steroid molecule by the two enzymes.     

Identification, characterization, and nucleotide sequence of the F17-G gene, which determines receptor binding of Escherichia coli F17 fimbriae.

Enterotoxigenic Escherichia coli strains express fimbriae which mediate binding to intestinal mucosal cells. The F17 fimbriae mediate binding to N-acetylglucosamine-containing receptors present on calf intestinal mucosal cells. These fimbriae consist of F17-A subunit peptides. Analysis of the F17 gene cluster indicated that at least the F17-A, F17-C, F17-D, and F17-G genes are indispensable to obtain adhesive F17 fimbriae (unpublished data). Genetic evidence is presented that the F17-G protein, a minor fimbrial component, is required for the binding of the F17 fimbriae to the intestinal villi. The F17-G gene was cloned and sequenced. An open reading frame of 1,032 bp encoding a polypeptide of 344 amino acids, starting with a signal sequence of 22 residues, was localized. The F17-G mutant strain produced F17 fimbriae which were morphologically identical to the fimbriae purified from strains which contained the intact F17 gene cluster. However, this F17-G mutant could no longer adhere to calf villi. The F17-G locus was shown to act in trans: transformation of the F17-G mutant strain, still expressing the genes F17-A, F17-C, and F17-D, with a vector expressing the F17-G gene restored the binding activity of this mutant strain.     

Crystal structures of mouse 17alpha-hydroxysteroid dehydrogenase (apoenzyme and enzyme-NADP(H) binary complex): identification of molecular determinants responsible for the unique 17alpha-reductive activity of this enzyme

Very recently, the mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, has been characterized and identified as the unique enzyme able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epitestosterone (epi-T), the 17alpha-epimer of testosterone. Indeed, the other AKR enzymes that significantly reduce keto groups situated at position C17 of the steroid nucleus, the human type 3 3alpha-HSD (h3alpha-HSD3), the human and mouse type 5 17beta-HSD, and the rabbit 20alpha-HSD, produce only 17beta-hydroxy derivatives, although they possess more than 70% amino acid identity with m17alpha-HSD. Structural comparisons of these highly homologous enzymes thus offer an excellent opportunity of identifying the molecular determinants responsible for their 17alpha/17beta-stereospecificity. Here, we report the crystal structure of the m17alpha-HSD enzyme in its apo-form (1.9 A resolution) as well as those of two different forms of this enzyme in binary complex with NADP(H) (2.9 A and 1.35 A resolution). Interestingly, one of these binary complex structures could represent a conformational intermediate between the apoenzyme and the active binary complex. These structures provide a complete picture of the NADP(H)-enzyme interactions involving the flexible loop B, which can adopt two different conformations upon cofactor binding. Structural comparison with binary complexes of other AKR1C enzymes has also revealed particularities of the interaction between m17alpha-HSD and NADP(H), which explain why it has been possible to crystallize this enzyme in its apo form. Close inspection of the m17alpha-HSD steroid-binding cavity formed upon cofactor binding leads us to hypothesize that the residue at position 24 is of paramount importance for the stereospecificity of the reduction reaction. Mutagenic studies have showed that the m17alpha-HSD(A24Y) mutant exhibited a completely reversed stereospecificity, producing testosterone only from Delta4, whereas the h3alpha-HSD3(Y24A) mutant acquires the capacity to metabolize Delta4 into epi-T.     

Estradiol-17 beta inhibition of androgen uptake, metabolism and binding in epididymis of adult male rats in vivo: a comparison with cyproterone acetate

The effects of estradiol-17 beta on androgen uptake, metabolism and binding were studied in rat epididymis in vivo in comparison with cyproterone acetate. Steroids (250 ug/100 g body weight) were injected 5 min prior to 3H-testosterone in castrate rats. Estradiol-17 beta inhibited 3H-testosterone uptake into epididymal cytosol by 58% as compared to 38% by cyproterone acetate. 3H-Testosterone uptake into epididymal nuclei was inhibited 95% by estradiol-17 beta and 83% by cyproterone acetate. Total bound radioactivity in cytosol fractions was reduced to a greater extent by estradiol-17 beta than cyproterone acetate when either 3H-testosterone or 3H-dihydrotestosterone was injected. Binding of 3H-dihydrotestosterone to nuclear receptors was completely abolished by estradiol-17 beta; whereas approximately 20% binding remained in the nuclear extract after cyproterone acetate treatment. Metabolism of 3H-testosterone in vivo was also altered by estradiol-17 beta, resulting in diminished conversion to 3H-dihydrotestosterone. Cyproterone acetate, on the other hand, did not affect 3H-testosterone metabolism. Estradiol-17 beta and cyproterone acetate inhibited in vitro binding of 3H-dihydrotestosterone to the intracellular cytoplasmic receptor, but not the intraluminal androgen binding protein (ABP). These data suggest that estradiol-17 beta may have a more potent antiandrogenic effect on the epididymis than cyproterone acetate due to inhibition of 5 alpha reduction of testosterone as well as binding to the androgen receptor.     

Dietary green and white teas suppress UDP-glucuronosyltransferase UGT2B17 mediated testosterone glucuronidation

The anabolic steroid testosterone can be used by athletes to enhance athletic performance and muscle growth. UDP-glucuronosyltransferase (UGT2B17) is the key enzyme involved in the glucuronidation of testosterone to testosterone glucuronide, which also serves as a marker for the testosterone/epitestosterone (T/E) ratio used to detect testosterone abuse in sport. Inhibitors of testosterone glucuronidation could have an impact on circulating testosterone levels, thus aiding performance, as well as potentially affecting the urinary T/E ratio and therefore masking testosterone abuse. Previous reports have revealed that non-steroidal, anti-inflammatory drugs, diclofenac and ibuprofen, inhibit the UGT2B17 enzyme. The aim of this study is to analyse dietary tea samples for inhibition of testosterone glucuronidation and, where inhibition is present, to identify the active compounds. Analysis of testosterone glucuronidation was conducted by performing UGT2B17 assays with detection of un-glucuronidated testosterone using high performance liquid chromatography. The results from this study showed that testosterone glucuronidation was inhibited by the green and white tea extracts, along with specific catechin compounds, notably: epicatechin, epigallocatechin gallate (EGCG) and catechin gallate. The IC50 inhibition value for EGCG was determined, using a Dixon plot, to be 64 μM, equalling the most active NSAID inhibitor diclofenac. Thus, common foodstuffs and their constituents, for the first time, have been identified as inhibitors of a key enzyme involved in testosterone glucuronidation. Whilst these common compounds are not substrates of the UGT2B17 enzyme, we showed that they inhibit testosterone glucuronidation which may have implications on current doping control in sport.     

Regulation of the gene for estrogenic 17-ketosteroid reductase lying on chromosome 17cen----q25

17-Ketosteroid reductase (17KSR), also known as 17 beta-hydroxysteroid dehydrogenase, catalyzes the reversible interconversion of estradiol to estrone and of androstenedione to testosterone. Using a recently cloned human placental 17KSR cDNA, we show that the 1.4-kilobase mRNA for this enzyme is detected only in tissues producing estrogens, and a 2.4-kilobase mRNA is detected in some estrogenic tissues and some androgenic tissues. This tissue distribution suggests that the interconversion of androstenedione and testosterone may be mediated by a different enzyme. Southern blotting studies show that the mRNA for this estrogenic 17KSR is encoded by two very similar genes localized to chromosome 17cen----q25 by analysis of DNA from mouse/human somatic hybrid cell lines. 8-Br-cAMP increases the abundance of estrogenic 17KSR mRNA as well as mRNAs for other steroidogenic enzymes in JEG-3 choriocarcinoma cells. By contrast, cAMP decreases estrogenic 17KSR mRNA in primary cultures of human cytotrophoblasts and human granulosa cells, a pattern of tropic regulation that differs from other steroidogenic enzyme mRNAs.     

Properties and regulation of 17β-hydroxysteroid oxidoreductase of OVCAR-3, CAOV-3, and A431 cells: Effects of epidermal growth factor,estradiol, and progesterone

Although there is a growing body of evidence that 17β-hydroxysteroid oxidoteductase plays a role the regulation of steroid levels in epithelial tumors of the endometrium and breast, out knowledge of its role in other gynecologic tumors is limited. In this investigation, the 17β-hydroxysteroid oxidoreductase activity of cell lines derived from two ovarian tumors (OVCAR-3, CAOV-3) and an epidermoid tumor of the vulva (A431) was assayed under conditions which differentiate between 17β-hydroxysteriod oxidoreductase type 1, a cytosolic isoform highly specific for estradiol, and type 2, a membrane bound isoform reactive with both estradiol and testosterone. On the basic of estradiol/testosterone activity ratios, all three cell lines appear to have type 2-like activity, with the specific activity of A431 markedly greater than that of the other cell lines. Estradiol, progesterone, or EGE, alone or in combination, were without effect on the enzymatic activity of OVCAR-3 cells. EGE decreased the activity of CAOV-3 cells slightly. In contrast, EGE stimulated A431 17β-hydroxysteriod oxidoreductase activity 7–8-fold over a 5-day exposure. Estradiol or progesterone, singly or in combination, also did not effect the enzymatic activity of A431 cells. However, progesterone inhibited the increase in activity seen in the presence of EGE. With EGE, estradiol, and progesterone together, the increase in enzymatic activity was comparable to that with EGE alone. The effects of estradiol and progesterone appear to result from steroid actions following binding of EGE to low-affinity receptors on A431 cells. © 1995 Wiley-Liss, Inc.     

Serum 5-androstene-3 beta,17 beta-diol sulphate, sex hormone binding globulin and free androgen index in girls with premature adrenarche

Serum sex hormone binding globulin (SHBG), testosterone (T), DHEA sulphate (DHEA-S), androstenedione (AD) and delta 5-androstene-3 beta,17 beta-diol sulphate (5-ADIOL-S) levels were measured by specific radioimmunoassay in 16 girls presenting with premature adrenarche (PA) and in 14 normal girls. Mean levels of steroids measured were elevated, and SHBG significantly depressed, in the girls with PA, with values (mean +/- SE) for DHEA-S (1.73 +/- 0.17 vs 0.25 +/- 0.06 mumol/l), 5-ADIOL-S (104 +/- 8 vs 31 +/- 4 nmol/l), AD (0.89 +/- 0.06 vs 0.62 +/- 0.04 nmol/l), and T (0.49 +/- 0.03 vs 0.23 +/- 0.06 nmol/l). SHBG levels were 68 +/- 6 vs 108 +/- 5 nmol/l, and the free androgen index [100 x T (nmol/l) divided by SHBG (nmol/l)] was 0.89 +/- 0.17 vs 0.22 +/- 0.01. These studies show that SHBG is depressed in girls with premature adrenarche; with the increased testosterone levels, this results in a markedly elevated free androgen index, a measure of testosterone which is bioavailable to target tissue. This may be compounded by the elevated levels of 5-ADIOL-S in girls with PA since its role may be as a prohormone for more potent androgens (testosterone, 5 alpha-dihydrotestosterone) in target tissues such as pubic skin.     

Androgenic and estrogenic 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase in human ovarian epithelial tumors: evidence for the type 1, 2 and 5 isoforms

17β-Hydroxysteroid dehydrogenase/17-ketosteroid reductases (17HSD/KSR) play a key role in regulating steroid receptor occupancy in normal tissues and tumors. Although 17HSD/KSR activity has been detected in ovarian epithelial tumors, our understanding of which isoforms are present and their potential for steroid metabolism is limited. In this investigation, 17HSD/KSR activity from a series of ovarian epithelial tumors was assayed in cytosol and microsomes under conditions which differentiate between isoforms. Inhibition studies were used to further characterize the steroid specificities of isoforms in the two subcellular fractions. Activity varied widely between tumors of the same histopathologic classification. The highest levels of activity were observed in mucinous tumors. Michaelis constants, maximum velocities, estradiol-17β/testosterone (E₂/T) activity ratios and inhibition patterns were consistent with a predominance of microsomal 17HSD/KSR2 and cytosolic 17HSD/KSR5, isoforms reactive with both E₂ and T, with evidence of estrogenic 17HSD/KSR1 in cytosol from some samples. In tumors where activity and mRNA expression were both characterized, Northern blots, PCR and sequence analysis indicated 17HSD/KSR5 was the predominant isoform. The presence of 17HSD/KSR5, which also has both 3-HSD/KSR and 20HSD/KSR activity, and 17HSD/KSR2 which also has 20-HSD activity, could influence not only estrogen and androgen binding but progesterone receptor occupancy, as well, in receptor-containing tumors.     

Identification of a new Sertoli cell metabolite of testosterone: 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol, by HPLC and GC-MS

Testosterone metabolism by isolated rat Sertoli cells cultured in vitro was investigated using HPLC and GC-MS techniques. Monolayer cultures of Sertoli cells (greater than 90% pure and free of Leydig cells) were incubated for 3-day periods with a stable labeled [2,2,4,6,6-d5]testosterone prepared and used in a 1:1 proportion with unlabeled testosterone as the substrate (5 X 10(-7)M). After incubation, the metabolites were extracted from the media and reacted with oestradiol-antibodies. The antibody-bound components were separated on reverse phase HPLC and the fraction corresponding to oestradiol was analyzed by GC-MS in the form of TMS-ether. One of the metabolites whose mass spectrum contained d0 + d5 species was detected and interpreted to be a triol with a mol. wt of 308. Mass spectra data indicated that this testosterone metabolite is one of the sixteen possible isomers of 3,16,17-trihydroxy androstane. This substance was identified based on the Vm value (27.81) closely resembling that of 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol TMS-ether (Vm reported = 27.78) [1] and when compared directly with synthesized compounds [2-3]. Recently we have demonstrated that similar Sertoli cell preparations contain two 16 alpha-hydroxylases by their ability to convert oestradiol to oestriol [4] and 5 alpha-androstane-3 alpha,17 beta-diol to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol [3], where the former conversion is not affected by FSH, the latter is significantly stimulated by the presence of FSH. Presence of this new product represents the first example of testosterone conversion to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol and confirms our previous observation that 16 alpha-hydroxylation of 5 alpha-reduced androgens can occur in the rat testis.