To determine whether the calcium channel blocker amlodipine improves glucose tolerance and alters serum adrenal androgen and glucocorticoid levels in insulin-resistant men, 2<em>4</em> obese and hypertensive men were enrolled into a single blind, placebo-controlled study. An amlodipine group (n = 12) and a placebo group (n = 12) were studied before and after treatment with either amlodipine (5 mg) or placebo capsule twice daily for 7 days by determining serum insulin, glucose, dehydroepiandrosterone sulfate (DHEA-S), <em>androstenedione</em>, and cortisol in the fasting state and during an oral glucose tolerance test. Amlodipine treatment 1) lowered fasting serum insulin (from 273 +/- 19 to 200 +/- 17 pmol/L; P < 0.0005) and glucose (from 5.<em>4</em> +/- 0.1 to 5.1 +/- 0.1 mmol/L; P < 0.02), 2) reduced the area under the curve for glucose (from 13<em>4</em>2 +/- 25 to 1198 +/- 23 mmol/L.min; P = 0.0001) and the area under the curve for insulin (from 155.5 +/- 7.8 to 103.9 +/- <em>4</em>.3 nmol/L.min; P = 0.0001) during the oral glucose tolerance test, 3) increased fasting serum DHEA-S (from 5.19 +/- 0.37 to 7.95 +/- 0.58 mumol/L; P = 0.0001) and <em>androstenedione</em> (from 5.65 +/- 0.65 to 6.83 +/- 0.53 nmol/L; P < 0.01), and <em>4</em>) decreased fasting serum cortisol (from 538 +/- 35 to <em>4</em>9<em>4</em> +/- 26 nmol/L; P < 0.05). Fasting serum <em>androstenedione</em> declined slightly in the placebo group (from 5.96 +/- 0.60 to 5.7<em>4</em> +/- 0.57 nmol/L; P < 0.005), but no change occurred in glucose tolerance, fasting serum DHEA-S, or cortisol. We conclude that amlodipine treatment improves glucose tolerance, reduces fasting and glucose-stimulated serum insulin levels, increases serum DHEA-S and <em>androstenedione</em> levels, and decreases circulating cortisol.

BACKGROUND

Endometriosis is a chronic inflammatory disease in which immune response and production of estrogen in endometriotic tissues are involved in the development of the disease. Prostaglandin E2 (PGE2) stimulates aromatase (P<em>4</em>50arom) expression in endometrioma stromal cells (ESCs) and increases the production of estrogens. On the other hand, an accumulating amount of evidence suggests that IL-<em>4</em>, a typical Th2 cytokine, plays important roles in the disease.

OBJECTIVE

The objective of the investigation was to study the effect of IL-<em>4</em> on the expression of 3β-hydroxysteroid dehydrogenase (HSD3B2), a pivotal enzyme for estrogen production, in ESCs.

METHODS

ESCs were isolated from ovarian endometrioma tissues and cultured with IL-<em>4</em> and PGE2. CP-690550, a Janus protein tyrosine kinase 3 inhibitor, and HSD3B2 small interfering RNA were added to the culture. Gene expression of HSD3B2 and P<em>4</em>50arom was examined by quantitative RT-PCR. Dehydroepiandrosterone (DHEA) was added to the culture, and then the combined enzyme activity of HSD3B2, which converts DHEA to androstenedione, and P<em>4</em>50arom, which converts androstenedione to estrone, was examined by measuring estrone concentration in the supernatants with a specific enzyme immunoassay.

RESULTS

IL-<em>4</em> increased the expression of HSD3B2 mRNA in a dose-dependent manner. CP-650550 inhibited the IL-<em>4</em>-induced increase in HSD3B2 mRNA expression. PGE2 also increased the expression of HSD3B2 mRNA, and the combination of IL-<em>4</em> and PGE2 synergistically increased the expression of HSD3B2 mRNA. IL-<em>4</em> had no effect on the expression of P<em>4</em>50arom mRNA, whereas PGE2 increased the expression of P<em>4</em>50arom mRNA. Although PGE2 alone increased the production of estrone from DHEA, the combination of IL-<em>4</em> and PGE2 significantly augmented the production of estrone from DHEA. The enhanced production of estrone by the combination of IL-<em>4</em> and PGE2 was inhibited by CP-690550 and HSD3B2 small interfering RNA.

CONCLUSIONS

IL-<em>4</em> in combination with PGE2 may enhance estrogen production in endometriotic tissues, implying an elaborate mechanism that Th2 immune response augments inflammation-dependent progression of the disease.

1. The long-term maintenance of metabolism of representative drugs and steroid hormone substrates by cytochromes P-<em>4</em>50, and their inducibility, was investigated in primary cultures of adult rat hepatocytes. Collagenase-isolated cells were seeded on collagen-coated tissue culture dishes and cultured in Chee's essential media in the presence or absence of phenobarbital (PB, 0.75 mM, 96 h or continuously) and 3-methylcholanthrene (MC, 5 microM, <em>4</em>8 h) for up to <em>4</em>5 days. 2. Hepatic P-<em>4</em>50-dependent metabolism of diazepam to its primary oxidized metabolite was inducible by PB both in vivo (monitored in isolated liver microsomes) and in cultured cells (up to 100% and <em>4</em>00% increases in the formation of temazepam and nordiazepam, respectively, after 25 days in culture). Hepatocyte microsomal <em>androstenedione</em> 16 beta-hydroxylase activity was also induced by PB treatment of the hepatocytes (350-650% increase in 20-day-old cells). 3. Western blot analysis revealed that immunoreactive P-<em>4</em>50 form PB-<em>4</em> (IIB1), which catalysed the N-demethylation of diazepam to yield nordiazepam as well as <em>androstenedione</em> 16 beta-hydroxylation when assayed in a purified enzyme system, was substantially elevated following PB treatment of the cultured cells. Similarly, MC induced 7-ethoxycoumarin O-deethylase activity (up to 2000% increase from 5 to <em>4</em>5 days) as well as immunoreactive P-<em>4</em>50c (IA1) in the hepatocyte cultures. <em>4</em>. These studies demonstrate that cytochrome P-<em>4</em>50 activities can be maintained, and also induced, after extended periods of time in hepatocytes cultured using a simple collagen mixture as substrate and a commercially available tissue culture media. This culture system should provide an important tool for further studies of P-<em>4</em>50-dependent xenobiotic metabolism in a well-defined, liver-derived cellular system.

The capability of granulosa and theca interna cells, from preovulatory follicles of the domestic hen, to metabolize steroid precursors was evaluated. Granulosa and theca interna cells were isolated from ovarian preovulatory follicles at three different developmental stages: F1, F3 and F5. Tritiated pregnenolone (P5), progesterone (P<em>4</em>), dehydroepiandrosterone (DHEA), <em>androstenedione</em> (A<em>4</em>) and testosterone (T) were employed as precursors and their metabolic products were evaluated. The major metabolite of P5 by granulosa cells was P<em>4</em>, but we also observed low amounts of 5beta-pregnandione. DHEA metabolism by granulosa cells yielded mainly A<em>4</em>, and minute quantities of 5beta-androstan-3,17-dione (5beta-dione) were detected. The only significant metabolite obtained in granulosa cells from A<em>4</em> was 5beta-dione, whereas T was only transformed into A<em>4</em>. On the other hand, P5 metabolism by theca interna cells yielded A<em>4</em> as the main product, also P<em>4</em>, 17alpha-OHP<em>4</em>, 17alpha-OHP5, 5beta-pregnandione, and DHEA, were found. When DHEA was the precursor A<em>4</em> was produced in higher amounts than 5beta-dione. A<em>4</em> was mainly transformed into 5beta-dione. In similar conditions, T was transformed into A<em>4</em>. These results show that granulosa cells have enzymatic activities of 3beta-hydroxysteroid dehydrogenase/5-<em>4</em> isomerase (3beta-HSD from P5 and DHEA), 17beta-hydroxysteroid dehydrogenase (17beta-HSD from T) and 5beta-reductase (from P5, DHEA and A<em>4</em>). Whereas theca interna cells have enzymatic activities of cytochrome P<em>4</em>50c17 (from P5 and P<em>4</em>), 3beta-HSD (from P5 and DHEA), 17beta-HSD (from T) and 5beta-reductase (from P<em>4</em>, DHEA and A<em>4</em>). These data support the concept that theca interna cells have the ability to synthesize androgens from progestins produced in granulosa cells. In addition, since theca interna cells did not show the capacity to aromatize androgens suggests that interaction between theca interna and theca externa cells occurs in vivo, thus confirming the three cell model for estrogen production. Furthermore, the fact that other metabolites were produced both in granulosa and theca interna cells, but in a different extent, suggests that complex mechanisms are participating in the regulation of steroid synthesis in avian ovary follicles.

The objective of this study was to develop a defined culture system in which bovine follicular and granulosa cells are grown in close contact with each other and with the extracellular matrix (ECM) component laminin. Granulosa and theca cells from follicles <em>4</em>-6 mm in diameter were cultured on either side of laminin-coated BioCoat cell culture inserts in a serum-free medium containing 10 ng insulin ml(-1) at plating densities of 10(5) and 3 x 10(5) cells per membrane side. The cells adopted a clumped arrangement, maintained steroidogenic activity for at least 7 days and demonstrated paracrine communication by increased steroidogenesis and enhanced cell survival compared with cells in mono-culture. Co-cultured theca cells secreted significantly more <em>androstenedione</em> compared with cells in mono-culture. Granulosa cell viability was doubled by co-culture with theca cells. Co-cultures at both cell plating densities were responsive to treatment with physiological combinations of either FSH, LH and LR3 insulin-like growth factor I (IGF-I) (treatment A) or FSH, LR3 IGF-I and <em>androstenedione</em> (treatment B). Significantly more <em>androstenedione</em> was secreted in the presence of treatment A compared with controls. In contrast, oestradiol secretion was increased only by treatment B. Progesterone secretion was unaffected by treatment and did not increase during culture. Co-cultures at the higher plating density demonstrated higher theca cell survival and better maintenance of the follicular cell phenotype. In conclusion, this novel co-culture system provides a unique model for the study of paracrine communication between ovarian somatic cells and cell-ECM interactions during follicle growth.

It is now established that the brain and other nervous systems have the capability of forming steroids de novo, the so-called "neurosteroids." The pioneering discovery of Baulieu and his colleagues, using rodents, has opened the door to a new research field of "neurosteroids." In contrast to mammalian vertebrates, little has been known regarding de novo neurosteroidogenesis in the brain of birds. We therefore investigated neurosteroid formation and metabolism in the brain of quail, a domestic bird. Our studies over the past two decades demonstrated that the quail brain possesses cytochrome P<em>4</em>50 side-chain cleavage enzyme (P<em>4</em>50scc), 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(<em>4</em>)-isomerase (3β-HSD), 5β-reductase, cytochrome P<em>4</em>50 17α-hydroxylase/c17,20-lyase (P<em>4</em>50(17α,lyase)), 17β-HSD, etc., and produces pregnenolone, progesterone, 5β-dihydroprogesterone (5β-DHP), 3β, 5β-tetrahydroprogesterone (3β, 5β-THP), <em>androstenedione</em>, testosterone, and estradiol from cholesterol. Independently, Schlinger's laboratory demonstrated that the brain of zebra finch, a songbird, also produces various neurosteroids. Thus, the formation and metabolism of neurosteroids from cholesterol is now known to occur in the brain of birds. In addition, we recently found that the quail brain expresses cytochrome P<em>4</em>50(7α) and produces 7α- and 7β-hydroxypregnenolone, previously undescribed avian neurosteroids, from pregnenolone. This paper summarizes the advances made in our understanding of neurosteroid formation and metabolism in the brain of domestic birds. This paper also describes what are currently known about physiological changes in neurosteroid formation and biological functions of neurosteroids in the brain of domestic and other birds.

To determine if <em>androstenedione</em>, an aromatizable androgen, has a direct effect on luteal progesterone secretion, collagenase-dispersed luteal cells or whole corpora lutea from pregnant rats were incubated in the presence of the androgen. Luteal cells from 15-day pregnant rats responded to <em>androstenedione</em> in a dose-dependent manner, with an increase in progesterone output at doses of 1 and 10 microM, but with no effect at minor doses of the androgen. Luteal cells obtained from animals on day <em>4</em>, 9, 15 or 19 of pregnancy and incubated with 10 microM of <em>androstenedione</em>, increased progesterone production by 2<em>4</em>3, 39, 8<em>4</em> and 1<em>4</em>6%, respectively. Androgens (<em>androstenedione</em>, testosterone or dihydrotestosterone) but no oestrogens (oestradiol or diethylstilboestrol) at a dose of 10 microM, stimulated progesterone production in incubated luteal cells obtained from 15-day pregnant rats. The time-course pattern of <em>androstenedione</em>-induced progesterone production was studied by superfusion experiments using corpora lutea from rats on day 15 of pregnancy. A significant progesterone output was observed when <em>androstenedione</em>, but not oestradiol, was perfused through the luteal tissue. Intrabursal ovarian administration of <em>androstenedione</em> (10 microM) to 19-day pregnant rats induced a significative increase in serum progesterone levels 8 and 2<em>4</em> h after treatment. These in vivo results confirm the stimulatory effect of androstendione on progesterone production obtained in incubated luteal cells from pregnant rats. This study reports a direct luteotrophic effect of <em>androstenedione</em> in rat corpus luteum, not mediated by previous conversion to oestrogens.

The catalytic requirements and the role of P<em>4</em>50 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P<em>4</em>50 3A9 protein. The optimal steroid hormone hydroxylase activities of P<em>4</em>50 3A9 required cholate but not cytochrome b5. P<em>4</em>50 3A9 was active in the hydroxylation reactions of testosterone, <em>androstenedione</em>, progesterone and dehydroepiandrosterone (DHEA). No activity of P<em>4</em>50 3A9 toward cortisol was detectable under our reconstitution conditions. Among all the steroid hormones examined, female-specific P<em>4</em>50 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6beta-, 16alpha-, and 21-hydroxyprogesterone. Our data also showed that P<em>4</em>50 3A9 can catalyze the formation of a dihydroxy product, <em>4</em>-pregnen-6beta, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P<em>4</em>50. Based on the Vmax/Km values for P<em>4</em>50 3A9 using either 21-hydroxprogesterone or 6beta-hydroxyprogesterone as a substrate, <em>4</em>-pregnen-6beta, 21-diol-3, 20-dione may be formed either by 6beta-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6beta-hydroxyprogesterone. As a major isoform of CYP3A expressed in rat brain, the activities of P<em>4</em>50 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P<em>4</em>50 3A9 in the metabolism of neurosteroids.

The mechanism of inhibition of estrogen synthetase (P-<em>4</em>50arom) by 19R- and 19S-isomers of 10-oxiranyl-and 10-thiiranyl-<em>4</em>-estrene-3,17-dione was investigated using human placental microsomes and purified enzyme preparations. The 19R-isomers were potent inhibitors and exhibited affinities 36-fold (10-oxirane) and 80-fold (10-thiirane) greater than the respective 19S-isomers. Kinetic experiments showed that inhibition by the 19R-isomers is competitive with respect to substrate; inhibition constants for the (19R)-10-oxirane (Ki = 10 nM) and the 19R-10-thiirane (Ki = 2 nM) indicate that each binds with greater affinity than the androgen substrates <em>androstenedione</em> and testosterone. Inhibition time courses and kinetic data were consistent with high affinity, reversible binding. Spectral titrations of microsomal preparations and purified P-<em>4</em>50arom showed that binding of the 19R-isomers shifts the Soret maximum of the ferric enzyme to <em>4</em>11 nm (10-oxirane) or <em>4</em>25 nm (10-thiirane); addition of excess <em>androstenedione</em> reversed the spectral changes, producing the high spin form of the enzyme with a Soret peak at 393 nm. These spectral shifts suggest that the oxygen atom of the 10-oxirane and the sulfur atom of the 10-thiirane are bound to the heme iron in the inhibitor complexes. These results suggest that the high affinities of the inhibitors arise from their dual interaction with the androgen binding site and with the heme. Coordination of the C19 heteroatom to the heme indicates that C19 of androgen substrates may be positioned sufficiently close to the heme to allow direct attack by an iron-bound oxidant. Stereoselective binding of the 19R-isomers by P-<em>4</em>50arom further suggests that the heme is likely to be positioned above C1 and C2 of the A ring.

This study investigated adrenal androgens (AA), gonadotropins, and cortisol in castrated and gonad-intact male rhesus macaques from birth through infancy. Blood samples were collected longitudinally from castrated (n = 6; weekly, 1-<em>4</em>0 wk) and intact (n = <em>4</em>; every other week, 1-17 wk) males. Plasma concentrations of AA were determined by liquid chromatography-tandem mass spectrometry, and plasma concentrations of cortisol and gonadotropins were determined by RIA. Dehydroepiandrosterone sulfate (DHEAS) concentrations increased almost threefold (to 8 wk), dehydroepiandrosterone (DHEA) increased more than eightfold (to 11 wk), and <em>androstenedione</em> doubled (to 15 wk) in five castrated infant males and declined continuously thereafter. A sixth castrated male had markedly different temporal patterns and concentrations (many times more than 2 SDs from the cohort mean) of AA and gonadotropins from first sampling (3 wk) and was excluded from analysis. Cortisol increased over 16 wk but correlated poorly with DHEAS. Luteinizing and follicle-stimulating hormones increased to peaks at 3 and 7 wk, respectively. Testis-intact males exhibited similar profiles, but with earlier peaks of DHEAS (5 wk) and DHEA and <em>androstenedione</em> (7 wk). Peak concentrations of DHEAS were lower and those of DHEA and <em>androstenedione</em> were higher in intact than castrated infants. Testosterone was undetectable in castrated males and >0.5 ng/ml in intact males but was not correlated with DHEA or DHEAS. These are the first data documenting a transient increase in AA secretion during infancy in an Old World primate and are consistent with the previously documented time course of zona reticularis development that accompanies increases in androgen synthetic capacity of the adrenal. The rhesus is a promising model for androgen secretion from the human adrenal cortex.

3-Ethyl-3-(<em>4</em>-pyridyl)piperidine-2,6-dione (1) is a strong competitive inhibitor of human placental aromatase (Ki = 1.1 microM; testosterone as substrate) that, unlike the structurally related aromatase inhibitor aminoglutethimide (2), is not also an inhibitor of the cholesterol side-chain cleavage enzyme desmolase. An improved synthesis of 1 is described, which was readily adapted to the preparation of homologues in a series of 3-alkyl-3-(<em>4</em>-pyridyl)-piperidine-2,6-diones (6-13). Alkylation of 1 afforded a second series, comprising 1-alkyl-3-ethyl-3-(<em>4</em>-pyridyl)-piperidine-2,6-diones (1<em>4</em>-23). Inhibitory activity toward aromatase was maximal in both series for the octyl derivatives. Respective Ki values for the competitive inhibition exerted by the 3-octyl (12) and the 1-octyl (21) analogues with testosterone as substrate were 0.09 and 0.12 microM. The compounds 1, 2, 12, and 21 differed in their relative potencies as inhibitors of the aromatization of testosterone and <em>androstenedione</em>. Respective Ki values were as follows: for 1, 1.1 and 1<em>4</em> microM (ratio 12.7); for 2, 0.6 and 1.8 microM (3); for 12, 0.09 and 0.20 microM (2.2); and for 21, 0.12 and 0.<em>4</em>8 microM (<em>4</em>).

High levels of 11-ketotestosterone (11KT) were found (<em>4</em>9 to 160 ng ml(-1)) in plasma of Siberian sturgeon females during the end of their reproductive cycle. These levels were measured either by specific radioimmunoassay, or both by specific radioimmunoassay and by UV absorption after HPLC (isocratic conditions, 33% methanol, 26% acetonitrile, <em>4</em>1% water). In order to find the origin of 11KT synthesis, ovaries were incubated (30 min and 2h at 20°C) with tritiated 17-hydroxyprogesterone (17OHP) or with tritiated <em>androstenedione</em> (A<em>4</em>). Testosterone (conversion rate from tritiated 17OHP: <em>4</em>%) and 11-ketotestosterone (conversion rate from tritiated A<em>4</em>: 1.6%) were identified as metabolites of respectively 17OHP and A<em>4</em> (TLC, HPLC and crystallization). 11β-hydroxy<em>androstenedione</em> (11βOHA<em>4</em>) and 11β-hydroxytestosterone (11βOHT) were suggested to be intermediate metabolites. Besides interrenal and blood cells were incubated respectively with tritiated cortisol and tritiated A<em>4</em>. 11βOHA<em>4</em> was identified in interrenal incubation (yield from tritiated cortisol: 1.2%). 11KT in interrenal (yield from tritiated cortisol: 0.1<em>4</em>%), and 11βOHA<em>4</em> and 11KT in blood cells (yield from tritiated A<em>4</em>: 1.6%), were suspected to be synthesized (TLC, HPLC, acetylation). No significant metabolization of tritiated cortisol could be found in liver. The possible contribution of each of these tissues to high 11KT levels found in plasma is discussed.

3 beta-hydroxysteroid dehydrogenase delta <em>4</em>-5-isomerase (delta 5-3 beta-HSD) catalyzes an early step in the synthesis of testosterone from dehydroepiandrosterone (DHA). We compared enzyme activity in back skin biopsies with sebum excretion rate (SER) in 1<em>4</em> individuals. The rate of conversion of [7 alpha-3H]DHA into [3H]-<em>4</em>-androstene-3,17-dione was measured in cryostat sections of skin and compared with the sebaceous gland content of the same biopsies. Reaction rate was proportional to the volume of sebaceous gland tissue in the sections. Enzyme activity was absent from sections without histologically identifiable sebaceous gland tissue. This suggests that the delta 5-3 beta-HSD is localized in sebaceous glands. SER, measured by a modified photometric technique at the biopsy site, correlated highly with sebaceous gland volume and with the rate of conversion of DHA into <em>androstenedione</em> in the biopsy. For each biopsy, specific activity of delta 5-3 beta-HSD in sebaceous glands was calculated by dividing the rate of formation of [3H]-<em>4</em>-androstene-3,17-dione by sebaceous gland volume. Specific activity of delta 5-3 beta-HSD did not correlate significantly with SER, suggesting that variations in concentration of delta 5-3 beta-HSD in sebaceous glands probably do not underlie variations in sebaceous gland activity.

An enzyme-mediated metabolism of androgens and estrogens including 17beta-HSD activity in the brain of vertebrates was discovered approximately 30 years ago. Mainly 5alpha-reductase and aromatase have been studied in detail. Recently we could demonstrate reductive and oxidative 17beta-HSD activity as well as considerable mRNA expression of the 17beta-HSD types 3 and <em>4</em> in the human brain. In the present study, we report on 17beta-HSD type 5 mRNA expression in brain tissue of women and men. Data analysis did not reveal sex specific differences, but we determined a significantly higher mRNA concentration in the subcortical white matter (SC) than in the cerebral cortex (CX). Investigation of reductive 17beta-HSD in vitro activity with 2 microM <em>androstenedione</em> as the substrate revealed no sex specific differences. Testosterone formation was significantly higher in SC than in CX. Moreover, enzyme activity was significantly higher in brain tissue of adults compared to that of children.

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) play a crucial role in the control of active sex steroid intracellular levels. Seven types of 17beta-HSD have been described. In this study, we report the cloning and characterization of the mouse type 5 17beta-HSD belonging to the aldo-keto reductase superfamily, in contrast with types 1, 2, 3, <em>4</em>, 6, and 7 17beta-HSD which belong to the short-chain alcohol dehydrogenase family. The gene spans 16 kb and contains 9 exons separated by 8 introns. Primer extension analysis identified a major transcription start site beginning 50 nucleotides upstream from the ATG initiation codon. Northern blot analysis showed a high mRNA expression level in the liver and a weaker signal in the kidney. To determine more precisely the substrate specificity of the enzyme, we established a stable cell line expressing mouse type 5 17beta-HSD in transformed human embryonic kidney (293) cells. The transfected cell line preferentially catalyzes the transformation of <em>4</em>-<em>androstenedione</em> (<em>4</em>-dione) and androstanedione (A-dione) into testosterone (T) and dihydrotestosterone (DHT), respectively. This data is somewhat in contradiction with a previous study that described the enzyme as estradiol 17beta-dehydrogenase. Our results indicate that the rate of transformation of estradiol (E(2)) to estrone (E(1)) represents only 1% of the rate of transformation of <em>4</em>-dione to T. Mouse type 5 17beta-HSD shares 76% amino acid sequence identity with human type 5 17beta-HSD; 71%, 76%, 76% with rat 3alpha-HSD and human types 1 and 3 3alpha-HSDs, respectively; and 71%, 69% and 77% with mouse, rat and human 20alpha-HSD, respectively.

Aromatase catalyzes the conversion of <em>4</em>-androstene-3,17-dione to estrogen with the concomitant formation of the minor metabolites <em>4</em>-androstene-19-hydroxy-3,17-dione(19-hydroxy<em>androstenedione</em>) and <em>4</em>-androstene-3,17,19-trione(19-oxo<em>androstenedione</em>). Microsomes of chinese hamster ovary (CHO) cells expressing human aromatase were isolated to investigate <em>androstenedione</em> metabolism. Relatively greater amounts of the minor metabolites result after limitation of electron flux from NADPH-cytochrome P<em>4</em>50 reductase to aromatase. Substitution of NADH for NADPH or limitation of NADPH availability increased minor metabolite formation relative to estrogen formation. Similar changes in metabolite ratios were observed when metabolism was conducted either at high pH (8.3) or in the presence of n-alcohols in the range of 5-200 mM alcohol concentrations. However, conditions of low pH (5.5) or high ionic strength (1 M KCl) resulted in minor changes in metabolite ratios, suggesting little or no effect on electron flux between NADPH-cytochrome P<em>4</em>50 reductase and aromatase. Theoretical molar ratios of the resulting metabolites were predicted using a reaction scheme assuming sequential substrate oxidations without reversible intermediate release from the aromatase active site. This model was supported by a close agreement between theoretical and experimental metabolite ratios for a broad range of NADPH concentrations. The results indicate that metabolite ratios provide a sensitive indicator of aromatase-oxidoreductase interactions in the microsomal environment.

Finasteride, a 5 alpha-reductase inhibitor, does not bind to the androgen receptor and has no other known hormonal activity. To determine what effect, if any, it has on adrenal steroidogenesis, 10 healthy men received 5 mg finasteride daily for 28 days. Adrenocorticotropic hormone (ACTH) stimulation tests were performed before and after <em>4</em> weeks of finasteride administration (5 mg daily). Serum levels of 17-hydroxypregnenolone, 17-hydroxyprogesterone, deoxycorticosterone, corticosterone, aldosterone, cortisol, dehydroepiandrosterone, and <em>androstenedione</em> were measured before and 60 minutes after i.v. ACTH. Finasteride decreased serum dihydrotestosterone levels from 31 +/- 5 to <em>4</em>.<em>4</em> +/- 1.2 ng/dl (P < 0.001). There were no significant changes in basal or ACTH-stimulated serum levels of adrenal steroids. There was also no significant decrease in the product to precursor ratio for the seven adrenal enzymes tested. Finasteride increased mean serum <em>androstenedione</em> levels by 17% (P = 0.10) and significantly increased the <em>androstenedione</em> to 17-hydroxyprogesterone ratio (P = 0.02 before ACTH and 0.05 after ACTH). These changes are most likely due to inhibition of <em>androstenedione</em> metabolism by 5 alpha-reductase. In conclusion, finasteride has no detectable effect on adrenal steroidogenesis, other than that which can be explained by inhibition of the 5 alpha-reductase enzyme.

The 19-methyl analogues of <em>androstenedione</em> and its aromatization intermediates (19-hydroxy<em>androstenedione</em> and 19-oxo<em>androstenedione</em>) were evaluated as substrates of microsomal aromatase in order to determine the effect of a 19-alkyl substituent on the enzyme's regiospecificity. Neither the <em>androstenedione</em> analogue [10-ethylestr-<em>4</em>-ene-3,17-dione (1c)] nor the 19-oxo<em>androstenedione</em> analogue [10-acetylestr-<em>4</em>-ene-3,17-dione (3c)] was converted to estrogens or oxygenated metabolites by placental microsomes. In contrast, both analogues of 19-hydroxy<em>androstenedione</em> [10-[(1S)-1-hydroxyethyl]estr-<em>4</em>-ene-3,17-dione (2c) and 10-[(1R)-1-hydroxyethyl]estr-<em>4</em>-ene-3,17-dione (2e)] were converted to the intermediate analogue 3c in a process requiring O2 and either NADH or NADPH. No change in enzyme regiospecificity was detected. The absolute configuration of 2e was determined by X-ray crystallography. Experiments with 18O2 established that 3c generated from 2c retained little 18O (less than 3%), while 3c arising from 2e retained a significant amount of 18O (approximately equal to 70%). All four 19-methyl steroids elicited type I difference spectra from placental microsomes in addition to acting as competitive inhibitors of aromatase (KI = 81 nM, 11 microM, 9.9 microM, and 150 nM for 1c, 2c, 2e, and 3c, respectively). Pretreatment of microsomes with <em>4</em>-hydroxy<em>androstenedione</em> (a suicide inactivator of aromatase) abolished the metabolism of 2c and 2e to 3c, as well as the type I difference spectrum elicited by 2c and 2e.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain estrogen production, performed by the enzyme aromatase, can be disrupted/affected in teleost fish exposed to endocrine disruptors found in polluted aquatic environments. The guppy (Poecilia reticulata) was previously studied and confirmed to suffer negative effects on reproductive behaviors following inhibition of the brain aromatase reaction. Here adult guppies (Poecilia reticulata) of both genders were subjected to known endocrine disruptors: the androgen <em>androstenedione</em> (A), the synthetic estrogen 17alpha-ethinylestradiol (EE(2)), and the estrogenic surfactant <em>4</em>-nonylphenol (NP), at high (50 microg/L) and at environmentally relevant concentrations (10 ng/L EE(2), 5 microg/L NP, and 0.7 microg/L A) for 2 weeks followed by measurements of brain aromatase activity (bAA). In the adult males, bAA was stimulated by A and EE(2) at 50 microg/L. Female activity was also stimulated by the higher estrogenic treatment. At environmentally relevant concentrations only the EE(2) treatment affected bAA, and only in males. The alkylphenolic substance NP produced no effect in either of the experiments, not on males nor females. The results indicate that short-term steroid treatments have stimulatory effects on guppy brain aromatase even at concentrations that can be found in the environment. We thus suggest bAA of adult guppies to be a suitable bioindicator of endocrine disruptors.

OBJECTIVE

To elucidate the natural course of circulating insulin-like peptide 3 (INSL3) levels according to puberty as well as its relation to other reproductive hormones.

METHODS

Population-based cohort study.

METHODS

Not applicable.

METHODS

Healthy peripubertal girls (n = 10) examined every 6 months; total number of examinations was 8<em>4</em>; median (range) per girl: 9 (<em>4</em>-10), including staging of pubertal breast development and blood samples.

METHODS

None.

METHODS

Serum levels of INSL3, inhibin B, E2, antimüllerian hormone, LH, and FSH (validated immunoassays), and T and androstenedione (liquid chromatography-tandem mass spectrometry).

RESULTS

Serum levels of INSL3 varied considerably between girls (range, 0.01-0.27 ng/mL) and within each girl as puberty progressed; intraindividual variation, median (range) 102% (65%-1<em>4</em>3%). Insulin-like peptide 3 increased in late puberty (B1 to B<em>4</em>+B5); geometric mean 0.03 ng/mL to 0.15 ng/mL. Insulin-like peptide 3 levels reflected markers of large follicles (T, androstendione, inhibin B, and E2) better than markers of small follicles (antimüllerian hormone), and INSL3 staining was localized in theca interna cells of antral follicles.

CONCLUSIONS

Insulin-like peptide 3 increased in late puberty, albeit inter- and intraindividual variations were substantial. Immunohistochemistry and intraindividual variation, as well as relations to other ovarian hormones, reveal that INSL3 in girls is a unique and specific marker of theca cells surrounding antral follicles. The potential clinical use of INSL3 for evaluation of ovarian function in girls remains to be elucidated.

FSH is important for ovarian antral follicle growth and steroidogenesis, processes in the human that are believed to be mediated by IGF-II. The objective of this study was to determine if human ovarian pre-antral follicles are also FSH- and IGF-II-responsive, since the clinical questions and mechanisms underlying the effects on the pre-antral follicle pool of exogenously administered gonadotropins for fertility therapy and elevated endogenous gonadotropins in the perimenopause remain unanswered. Class 2 preantral follicles were isolated from human premenopausal ovaries (n=6) and cultured in vitro with <em>androstenedione</em> and either no additives or with FSH or IGF-II. FSH (100 ng/mL) stimulated estradiol (E2) production by 3.58 +/- 0.<em>4</em> fold over <em>4</em>8 hr, compared to controls without FSH. This effect was completely inhibited in the presence of the IGF-II antagonist, IGF binding protein-<em>4</em> (IGFBP-<em>4</em>). IGF-II also stimulated E2 production by preantral follicles with doses as low as 1 ng/mL and within 2<em>4</em> hr of treatment. Maximal response of 3- to 9-fold above control was achieved with 100 ng/mL of IGF-II between 96-120 hr of culture. IGFBP-<em>4</em> completely inhibited E2 production to basal levels. FSH stimulated IGF-II mRNA in pre-antral follicles about <em>4</em>-fold, determined by RT-PCR. FSH also stimulated follicle growth, determined by light microscopy, 50-68% over <em>4</em>8 hr, compared to controls (P<0.001), a process that was inhibited in the presence of IGFBP-<em>4</em>. Cumulatively, these data support IGF-II as a mediator of FSH action on human preantral follicles.

The expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in steroidogenic tissues is an absolute requirement for mammalian reproduction, fetal growth, and life maintenance. We sought to identify extraglandular tissue sites in the human fetus where 3 beta HSD is expressed. To this effect, we conducted in vitro studies by use of homogenates prepared from second trimester fetal tissues. To facilitate the determination of 3 beta HSD activity, an abbreviated technique was developed that consisted in the use of [3 alpha-3H]dehydroepiandrosterone [( 3 alpha-3H]DHEA) as the substrate and NAD+ as the cofactor. With these reagents, the enzymatic reaction leads to the production of both nonradiolabeled <em>androstenedione</em> and NAD3H in equimolar amounts, and the radioactivity associated with NAD3H is used for quantification of 3 beta HSD activity. The kinetic isotope effect introduced by substitution of tritium for hydrogen at the C-3 alpha position of DHEA, determined with six different tissues, was 2.5 +/- 0.7 (mean +/- SD). The specific activities of the enzyme in peripheral tissues and ovary were relatively low, in the range of 0.03 nmol/mg protein.h for stomach (n = 2) to 0.18 +/- 0.1<em>4</em> nmol/mg protein.h for liver (mean +/- SD; n = 13), while in fetal testis and placenta the specific activities were relatively high, viz. 3.<em>4</em> +/- 0.7 nmol/mg protein.h (mean +/- SD; n = <em>4</em>) and 2.8 +/- 1.8 nmol/mg protein.h (mean +/- SD; n = 13), respectively. The findings of this study serve to demonstrate that 3 beta HSD is distributed widely among tissues of the human fetus. Although the enzymatic activity was easily demonstrated in peripheral tissues by the use of radiolabeled DHEA as the substrate, 3 beta HSD protein was not readily detected by Western analysis.

Estrogen is believed to be biosynthesized from <em>androstenedione</em> in placental microsomes by a multienzyme pathway in which 19-hydroxy<em>androstenedione</em> and 19-oxo<em>androstenedione</em> (or the hydrated form) are obligatory intermediates. However, both 19-hydroxy<em>androstenedione</em> and 19-oxo<em>androstenedione</em> competitively inhibited aromatization of <em>androstenedione</em>, and all three steroids were shown to be mutually competitive. 19-Hydroxy<em>androstenedione</em> and 19-oxo<em>androstenedione</em> also competed with <em>androstenedione</em> for binding sites in the microsomes at <em>4</em> degrees C. In confirmation of the work of Hollander (Hollander, N. (1962), Endocrinology 71, 723-728), and of Osawa and Shibata (Osawa, Y., and Shibata, K., (1973), Abstracts of the 55th Meeting of the Endocrine Society, Abstract 116) when <em>androstenedione</em> and 19-hydroxy<em>androstenedione</em> were incubated together, both were converted to estrogen, but little <em>androstenedione</em> was converted to 19-hydroxy<em>androstenedione</em>. Considered together, these results are incompatible with the multienzyme pathway. Rather, these results may be explained by aromatization of <em>androstenedione</em> at a single catalytic site via enzyme-bound transition states. Both proposed intermediates are, according to this view, by-products which can also be aromatized.