The metabolism of (1<em>4</em>)C-labeled testosterone by cultured human fibroblasts and amniotic fluid cells was investigated. Radiolabeled testosterone was incubated with the cultured cells for <em>4</em>8 hr, and the labeled metabolites present in the medium were subsequently identified. The major metabolic products of testosterone formed by cultured fibroblasts were Delta(<em>4</em>)-<em>androstenedione</em>, dihydrotestosterone, androsterone, and androstanediol. The amount of testosterone metabolized through each of two pathways was calculated and used to form a ratio designated the 17beta-hydroxyl/17-ketonic ratio. Fibroblasts from normal male and female children and adult females had high 17beta-hydroxyl/17-ketonic ratios indicating testosterone metabolism occurred primarily through the 17beta-hydroxyl pathway. There was change in the pattern of testosterone metabolism with age in males, i.e., adult males had much lower 17beta-hydroxyl/17-ketonic ratios than did male children. The testosterone metabolism of fibroblast cultures derived from three children with testicular feminization and their mothers was compared to normal age and sexmatched controls. Fibroblasts of children with testicular feminization metabolized testosterone predominantly through the 17-ketonic pathway and manifested a pattern of testosterone metabolism distinctly different from their sex and age matched controls. The mothers of children with testicular feminization could be distinguished from normal females by their much lower 17beta-hydroxyl/17-ketonic ratios. The much lower amounts of dihydrotestosterone and androstanediol produced by fibroblasts from patients with testicular feminization as compared with normals suggests there is a decrease in testosterone 5alpha-reductase activity in these patients. Cultured amniotic fluid cells metabolized testosterone to the same four major metabolites found in fibroblast cultures, but their activity was much lower than that of fibroblasts. Most of the amniotic fluid cell cultures metabolized testosterone largely through the 17beta-hydroxyl pathway as did fibroblasts from normal children.

The effect of indole-3-carbinol (IC), an anticarcinogen present in cruciferous vegetables, to alter the metabolism of <em>4</em>-<em>androstenedione</em> (AD) by female rat liver microsomes was investigated and compared to that of its main gastric conversion product, diindolylmethane (DIM) as well as other specific cytochrome P<em>4</em>50 inducers. DIM was a more potent inducer of the hydroxylase which converts androsterone to its 6 beta-hydroxylated derivative 3 alpha, 6 beta-dihydroxy-5 alpha-androstan-17-one (A) than IC after either oral or intraperitoneal administration and was also a better in vitro inhibitor. Isosafrole (ISF), which like IC and DIM, induces CYP1A2 as well as gestodene, were powerful inhibitors of the in vitro reaction. Naringenin produced only a weak inhibitory effect while 3-methylcholanthrene was inactive. SKF-525A, a prototypic hydroxylase inhibitor, or 17 beta-N,N-diethylcarbamoyl-<em>4</em>-methyl-<em>4</em>-aza-5 alpha-androst-1-ene-3-one which inhibits steroid 5 alpha-reductase, also decreased the formation of A from AD by liver microsomes. The infusion of human growth hormone by osmotic minipump, which feminizes hepatic steroid metabolism, increased the ability of male rat liver microsomes to convert AD to A and to respond to induction by IC. The identity of A, the main polar derivative of AD, induced by IC, DIM and ISF, was tentatively assigned by a combination of GC-MS and results from metabolic studies with intermediates in the pathway leading to its formation. It is proposed that the protective role of indole carbinols against mammary carcinoma due to decreased formation of 16 alpha-hydroxyestrone from estrone may be further enhanced by the diminished availability of AD for aromatization to estrone.

Skin, the largest organ of the human body, synthesizes active sex steroids from adrenal C19 precursor steroids. Normal human breast epidermal keratinocytes in primary culture were used to evaluate the enzymatic activities responsible for the formation and degradation of active androgens and estrogens during keratinocyte differentiation. Enzymatic activities, including 3beta-hydroxysteroid dehydrogenase/Delta5-Delta<em>4</em> isomerase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD), 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) were measured using [3H] steroids as substrates. After 10-60 days in culture, no 3beta-HSD activity was detected, but all other activities were measured, demonstrating the ability of keratinocytes to convert <em>androstenedione</em> (<em>4</em>-DIONE) into the potent androgen dihydrotestosterone (DHT). Furthermore, marked changes in enzymatic activity were observed during cell differentiation: 17beta-HSD was first detected during the third week of culture, the level of activity reaching a peak during the fourth week. This peak was followed by a progressive decrease during keratinization. On the other hand, 5alpha-reductase and 3alpha-HSD activities were first detected during the fourth week of culture. The enzymatic activities involved in the formation and degradation of sex steroids were also characterized in the immortalized human keratinocyte cell line HaCaT. It was then found that HaCaT cells possess a pattern of steroid metabolizing enzymes similar to that of human epidermal keratinocytes in culture. Since glucocorticoids are known to exert potent pharmacological effects on the skin, the effect of dexamethasone (DEX) on cell proliferation and enzymatic activities was determined using HaCaT cells. DEX causes a 55% decrease in HaCaT cell proliferation (IC50: 10nM) whereas DEX caused a three- to five-fold stimulation of oxidative 17beta-HSD activity in intact cells in culture (ED50: 30 nM) and this stimulatory effect was competitively blocked by the glucocorticoid antagonist RU<em>4</em>86. A four-fold increase in type 2 17beta-HSD mRNA levels was also observed as measured by real-time PCR, correlating with the increase in oxidative activity. No effect of DEX on the other enzymatic activities (3beta-HSD, 5alpha-reductase, and 3alpha-HSD) was observed. Since increased levels of inflammatory cytokines have been detected in some skin diseases then these cytokines might play a role in the differentiation of keratinocytes. In this regard, we found that interleukin-<em>4</em> (IL-<em>4</em>) induced the expression of 3beta-HSD in HaCaT cells, thus allowing the cells to produce a different set of sex steroids from adrenal C19 precursors. The present data thus indicate that HaCaT cells are a useful model to further study the regulation of the enzymes involved in the metabolism of sex steroids in keratinocytes.

To confirm that plasma delta <em>4</em> <em>androstenedione</em> (delta <em>4</em>) is the main precursor for 5 alpha-androstane-3 alpha, 17 beta-diol glucuronide (Adiol G) in patients with idiopathic hirsutism (IH), delta <em>4</em> was cutaneously applied to five normal women and five women with IH. Several parameters of androgen metabolism were assayed basally and throughout the studies. Those included plasma delta <em>4</em>, testosterone, and dihydrotestosterone as well as urinary Adiol G and testosterone glucuronide excretion. Under basal conditions plasma testosterone, delta <em>4</em>, and dihydrotestosterone did not differ significantly between the two groups of subjects. Urinary Adiol G excretion was significantly higher (P less than 0.01) in IH patients [123 +/- 36 (SE) micrograms/2<em>4</em> h] than in the normal women group (<em>4</em>5 +/- 20 micrograms/2<em>4</em> h). After percutaneous administration of delta <em>4</em>, plasma delta <em>4</em> increased in both groups by nearly 600% and there was a 300% increase in Adiol G excretion in IH patients (336 +/- 57 micrograms/2<em>4</em> h), whereas only a 50% increase occurred in normal women (65 +/- 17 micrograms/2<em>4</em> h). We postulate that plasma delta <em>4</em> may be the main precursor accounting for the increased production of urinary Adiol G in women with IH, in whom hirsutism may be due to a high 5 alpha-reductase activity. Indeed, 5 alpha-reductase as measured in vitro in pubic skin was significantly higher in hirsute patients (22<em>4</em> +/- 66 fmol/mg skin X h) than in normal women (<em>4</em>5 +/- 15 fmol/mg skin X h).

A <em>4</em>6,XY phenotypically male patient with 17-ketosteroid reductase deficiency is described. The patient was a 6-month-old infant who presented with micropenis and bilateral cryptorchidism. Baseline plasma levels of testosterone (T), delta <em>4</em>-<em>androstenedione</em> (delta <em>4</em>A), and 5 alpha-dihydrotestosterone (5 alpha-DHT) were within the normal range [patient: 0.17 (T), 0.12 (delta <em>4</em>A), and 0.032 (5 alpha-DHT) ng/ml; normal infants: 0.03-0.55 (T), 0.1<em>4</em>-0.<em>4</em>5 (delta <em>4</em>A), and 0.01-0.23 (5 alpha-DHT) ng/ml]. hCG administration induced a significant rise in plasma delta <em>4</em>A levels (up to 8.39 ng/ml) and a slight increase in T and 5 alpha-DHT levels. The delta <em>4</em>A/T ratios before and during the hCG challenge were 0.86 and 55.61, respectively (controls: 0.83 and 0.13). Incubation of genital skin-derived fibroblasts from the patient with either [3H]T or [3H] delta <em>4</em>A revealed normal formation of delta <em>4</em>A from T and diminished conversion of delta <em>4</em>A to T. The development of a male phenotype despite both a testicular and peripheral 17-ketosteroid reductase deficiency is difficult to explain. It is possible that the fetal testes were the source of sufficient amounts of T during the early periods of embryonic life, and that late onset of the enzyme deficiency prevented the development of completely normal male genitalia. The in vitro finding of normal T to delta <em>4</em>A conversion by the mutant fibroblasts suggests that in this particular tissue 17 beta-reduction and dehydrogenation of androgens are mediated by two isoenzymes with distinct substrate and/or cofactor specificities.

In contrast to the human placenta, which does not secrete androgens, the rat placenta synthesizes significant amounts of these steroids. The purpose of this study was to determine why the rat placenta does not secrete androgens before day 12 of pregnancy, to ascertain whether the rat placenta secretes more <em>androstenedione</em> than testosterone, to compare the capacity of luteal and placental tissue to secrete androgen, and to determine whether the rat placental produces <em>androstenedione</em> via the delta <em>4</em>- or delta 5-steroidogenic pathway. To determine whether the inability of the rat placenta to produce <em>androstenedione</em> before midpregnancy was due to the absence of active 17 alpha-hydroxylase and 17,20-lyase enzymes and also to investigate the ontogeny of both placental production of <em>androstenedione</em> and enzyme activities, placentas were isolated from rats between days 8-21 of pregnancy and either incubated or used to determine the activities of 17 alpha-hydroxylase and 17,20-lyase. Before day 11, enzyme activity was not detectable. From day 11, both enzyme activities and placental secretion of <em>androstenedione</em> steadily increased to peak values by day 18 and declined just before parturition. To investigate the principal aromatizable androgen secreted both in vivo and in vitro approaches were used. Levels of <em>androstenedione</em> and testosterone found in the uterine vein as well as those produced by placental tissue were determined. Rat placentas secreted markedly more <em>androstenedione</em> than testosterone, both in vivo and in vitro. When placental and luteal secretion of <em>androstenedione</em> and testosterone were compared, it was found that luteal tissue had a higher capacity for androgen synthesis than did the placenta. Yet, because of its greater mass, each placenta secreted 15 times more <em>androstenedione</em> and <em>4</em>.5 times more testosterone than each corpus luteum. To determine the preferential usage of progesterone or pregnenolone as substrate by the placenta, [1<em>4</em>C] progesterone and [3H]pregnenolone were added in equimolar concentrations. The resulting 1<em>4</em>C to 3H ratio of the androgen produced indicates that the preferred substrate is progesterone. In summary, results of this investigation describe, for the first time, the development of 17 alpha-hydroxylase and 17,20-lyase activities in the rat placenta and demonstrate that the placenta does not produce androgen before day 11 due to the absence of active enzymes. The results further demonstrate that the rat placenta secretes significantly more <em>androstenedione</em> that testosterone both in vivo and in vitro, produces more androgen than the corpus luteum because of its greater mass, and forms its androgen primarily via the delta <em>4</em>-st

The possible direct effect of luteinizing hormone-releasing factor (LRF) and a potent LRF agonist, [D-Trp6,Pro9NEt]-LRF (LRF-Ag), on basal and follicle-stimulating hormone (FSH)-stimulated estrogen and progesterone production by cultured human granulosa cells was examined. Granulosa cells, collected from healthy follicles (<em>4</em> to 12 mm in diameter) in the early to midfollicular phase were grown for 2 days in serum-free medium with aromatase substrate (<em>androstenedione</em>; 10(-7) M) and human FSH (100 ng/ml), LRF (10(-10) to 10(-7) M) or in LRF-Ag (10(-7) M). Granulosa cells from each follicle responded to FSH stimulation by showing marked increases (2- to 20-fold) in estrogen and progesterone production. Concomitant treatment with high doses of LRF or LRF-Ag did not alter the steroidogenic effect of FSH. These results suggest that in contrast to the rat, LRF and its agonistic analog do not act directly on human granulosa cells to inhibit acute steroidogenic function.

To examine inhibin-F activity (FSH-suppressing activity) in human follicular fluid of polycystic ovary (PCO) patients, 13 follicles from 5 documented PCO patients and an additional 31 follicles from normal women in the follicular phase of the menstrual cycle were sampled, and inhibin-F activity was measured in rat anterior pituitary cell cultures. Inhibin-F activity was measured in follicular fluid after stripping steroids from the fluids using treatment with dextran and activated charcoal. Estrogen, progesterone, and delta <em>4</em>-<em>androstenedione</em> in the follicular fluid were also determined by RIA. Estrogen and progesterone levels in follicular fluid from PCO follicles 3.9 +/- 0.3<em>4</em> mm in diameter were comparable with those in follicular fluid obtained from viable follicles (which had a delta <em>4</em>-<em>androstenedione</em> to estrogen ratio of 10 or less) from normal women. delta <em>4</em>-<em>Androstenedione</em> levels in PCO follicles were higher (P less than 0.01) than those in viable and atretic follicles of normal women. Inhibin-F levels in PCO follicles were comparable to those in viable follicles, but significantly greater (P less than 0.01) than levels in atretic follicles of normal women. If inhibin-F levels in both atretic and viable follicles of normal women were pooled, the levels were less (P less than 0.05) compared to the level in PCO follicular fluid. As an additional control, follicular fluid was collected from 90 follicles of normal women throughout the menstrual cycle, and follicular size was determined as well as inhibin-F and steroid content. Small follicles less than 8 mm; (comparable in size to the PCO follicles examined) obtained at each stage represented 79%, 2<em>4</em>%, 0%, and 9<em>4</em>% of the total follicles obtained in the early to midfollicular, late follicular, preovulatory, and luteal phases of the cycle, respectively. Inhibin-F activity in the fluids of these follicles was less than that in PCO follicular fluid. The average inhibin content of all of the small normal follicles was 197 +/- 3<em>4</em> U/10 microliter, which was significantly less (P less than 0.05) than the level in PCO follicles (332 +/- 13 U/10 microliters). These data represent the first observation of inhibin-F activity in PCO follicular fluid and suggest the possibility of involvement of inhibin-F in bringing about low or normal basal levels of FSH in the presence of elevated basal LH levels often observed in PCO patients.

The increase in circulating estrogen concentrations that follows injection of Escherichia coli endotoxin (Endo) may be due to increased aromatase activity. We have therefore analysed the effect of the aromatase inhibitor, <em>4</em> hydroxy<em>androstenedione</em> (<em>4</em>OHA) on the steroid hormone response of male rats, particularly the dramatic increase in estrogens and decrease in androgens, induced by Endo. The concentrations of corticosterone (B), progesterone (P<em>4</em>), 17 alpha hydroxyprogesterone (17 alpha OHP<em>4</em>), <em>androstenedione</em> (delta <em>4</em>), testosterone (T), estrone (E1) and estradiol (E2) were determined 2 hours after injection of increasing doses of <em>4</em>OHA with and without Endo. The increase in serum estrogen concentrations and drop in serum androgen levels in response to Endo were blocked by a single dose of <em>4</em>OHA. The effect of <em>4</em>OHA appeared to be dose dependent. Low doses (30 mg/kg and 50 mg/kg) induced significant changes in the estrogen and androgen responses, but the high dose (100 mg/kg) blocked all changes in sex steroids induced by Endo. <em>4</em>OHA did not alter the Endo-induced changes in other steroids.

Testicular function was evaluated in forty-one prepubertal patients with male pseudohermaphroditism by determining serum concentrations of progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, <em>androstenedione</em>, testosterone and dihydrotestosterone before and after stimulation with hCG and, in some instances, ACTH. Testosterone response to hCG was normal in all subjects. In one patient, a <em>4</em>-year-old boy, a deficiency of 17,20-desmolase activity was diagnosed based on the coexistence of elevated levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone and low levels of dehydroepiandrosterone and <em>androstenedione</em>. In three other patients enzymatic blocks were suspected but not confirmed. Congenital deficiency of enzymes necessary for testosterone biosynthesis is an uncommon aetiology of male pseudohermaphroditism.

The concentrations of progesterone, <em>androstenedione</em>, testosterone, 5 alpha-dihydrotestosterone and androsterone were determined in tissue samples from the human hypothalamus, anterior pituitary, pineal, amygdala and parietal cortex, taken at autopsy from male (n = <em>4</em>) and female cadavers (n = <em>4</em>) of various ages. The measurements were performed using radioimmunoassays for the individual steroids after the chromatographic purification of solvent extracts of tissue samples on Lipidex-5000TM. Preliminary qualitative analyses of the chromatographic profiles of various steroids by radioimmunoassay demonstrated the presence of these steroids in various regions of the brain, but an immunoreactive peak corresponding to 17-hydroxyprogesterone was not found. The concentrations (ng/g tissue wet wt.) of all steroids measured were either very low or below the limit of detection in brain tissues taken from male and female infants. In the adult brain, there was no difference in the distribution of steroids between the various regions studied. There was no sex difference in the brain tissue steroid concentrations, with the exception of testosterone which was clearly much higher in brain tissues from men as compared to women. Although testosterone was undetectable in most samples taken from adult women. 5 alpha-dihydrotestosterone could be measured in almost all samples, which suggests that this is the most important androgen in the human brain. When brain tissue steroid levels are compared with serum concentrations, it can be postulated that a state of equilibrium exists between the fraction of serum steroids which are not bound to high-affinity binding proteins and the amount of steroids in brain tissues.

Previous studies have shown that concentrations of insulin-like growth factor (IGF)-binding proteins (BPs) in bovine follicles before the preovulatory surge of LH are inversely associated with estradiol concentrations, such that decreases in IGFBPs occur with advanced development of antral follicles and atresia is associated with increases in IGFBPs. In the present study, we evaluated serum and follicular fluid at different times after the preovulatory surge of LH to determine whether changes in IGF-I and IGFBPs are associated with the marked changes in the steroidogenic capacity that occur in follicle during this period. Serum and fluid from small follicles >> or = 5-mm diameter) and the preovulatory follicle were collected from cows at the onset of standing estrus or 8.5 and 20.5 h after administration of 100 micrograms GnRH at the onset of estrus (n = <em>4</em>/time). Concentrations of IGF-I (determined by RIA) did not differ (p>> 0.10) among times but were lower (p < 0.05) in fluid from small and preovulatory follicles than those in serum. Profiles of IGFBPs (evaluated by ligand blot analysis) differed among serum and fluid from small and preovulatory follicles. Preovulatory follicles contained IGFBP-3 with little or no other IGFBPs detected. Amount of IGFBP-3 and progesterone did not differ with time of sample collection, but a 13- to 15-fold reduction in estradiol-17 beta and <em>androstenedione</em> occurred between 8.5 and 20.5 h after the LH surge. Small follicles contained IGFBP-2, -3, -<em>4</em>, and -5 (determined by immunoprecipitation). The amount of 28-29-kDa IGFBPs (small form of IGFBP-5 and [or] glycosylated form of IGFBP-<em>4</em>) was inversely associated with concentrations of <em>androstenedione</em>. The 31-kDa form of IGFBP-5 was positively correlated with concentration of progesterone. Serum contained IGFBP-2, -3, and -<em>4</em>, but not IGFBP-5. Circulating IGFBP-2, but not other IGFBPs, decreased by 50% from estrus to 20.5 h after GnRH. In summary, amounts of IGF-I or IGFBPs in preovulatory follicular fluid did not change in response to an LH surge, even though estradiol and <em>androstenedione</em> decreased markedly. The absence of IGFBPs other than IGFBP-3 in bovine preovulatory follicles may allow for increased availability of IGF-I, which is proposed to be important for oocyte maturation and ovulation.

Traditional literature and textbooks generally describe that estradiol (E2) and dihydrotestosterone (DHT) are synthesized from the aromatization and 5α-reduction of testosterone (T), respectively, following a pathway in which T is an essential intermediate (Tpath). This pathway implies that the steps of aromatization and 5α-reduction follow the reaction of the androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) that catalyzes the conversion of <em>4</em>-<em>androstenedione</em> (<em>4</em>-dione) into T, and that estrogenic 17β-HSDs are not required. Contrary to this belief, the cloning of many estrogen-specific 17β-HSDs and the observation of higher affinity of aromatase and 5α-reductase for <em>4</em>-dione than T are strongly in favor of biosynthetic pathways in which the steps catalyzed by aromatase and 5α-reductase precede that catalyzed by 17β-HSDs. Such pathways do not require T as an intermediate, as demonstrated by experiments using [1<em>4</em>C]-labeled DHEA and <em>4</em>-dione as substrates and incubation with SZ95 sebaceous gland, DU-1<em>4</em>5 prostate cancer and JEG-3 choriocarcinoma cell lines cultured in the presence of inhibitors of 5α-reductase and aromatase. A review of early literature about patients with testicular 17β-HSD deficiency and of steroid metabolism appears to confirm the physiological functionality of the E2 and DHT biosynthetic pathway not requiring T as intermediate (noTpath).

Besides the measurement of circulating conjugated metabolites of dihydrotestosterone (DHT), which reflects androgenic activity, only one assay to measure androgenic bioactivity in human serum has been proposed thus far. This recombinant bioassay is based on the androgen-dependent interaction between the LBD and NT domains of AR fused to the Gal <em>4</em> DNA-binding domain, but its construction is highly complex. We have developed a mammalian cell (CHO 515) bioassay that measures total androgen bioactivity in human serum. The AR-deficient Chinese hamster ovary (CHO) cells were stably transfected with pSG5-puro-hAR and pMMTV-neo-Luc. After selection with puromycin and neomycin, five highly inducible clones were isolated and one was selected. The expression of human androgen receptor (hAR) was confirmed by Western blot and steroid-binding assays on the whole cells. The transcriptional activity of the clone was measured after 2<em>4</em> h of incubation with increasing concentrations of various androgenic and non-androgenic steroid compounds in a 96-well plate. The EC50s for each tested androgenic steroid compound were <em>4</em> x 10(-11) M, 1.5 x 10(-11) M, 1 x 10(-9) M, 2 x 10(-10) M, 3 x 10(-10) M for testosterone, DHT, dehydroepiandrosterone (DHEA), delta5-androstenediol, and delta<em>4</em>-<em>androstenedione</em>, respectively. In the physiological concentrations of the non-androgenic steroids, estradiol, cortisol, aldosterone, and progesterone, no interference was noted with the AR transactivation level. Evaluation of androgenic bioactivity in human serum was performed by incubation of CHO 515 cells with 100 microl of patient serum, diluted at 1/100 = 1% in DMEM-F12 without phenol red. The sensitivity of the assay was < 0.3 ng/ml. The mean androgenic bioactivity expressed in testosterone equivalents was 0.6 +/- 0.2 ng/ml in normal prepubertal boys, and 12.<em>4</em> +/- 2 and 1.7 +/- 0.1 ng/ml in normal pubertal boys and girls, respectively. In conclusion, this new recombinant cell bioassay is today the only assay that takes into account testosterone, DHT, DHEA, delta5-androstenediol, and delta<em>4</em>-<em>androstenedione</em>. It should be of particular use in male children with cryptorchidism, delayed puberty or hypogonadotrophic hypogonadism, i.e., in pediatric patients with low androgen levels.

Possible influences of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) upon gonadal steroidogenesis were investigated in vitro. Granulosa cells from hypophysectomized, estrogen-treated rats were cultured for 2 days in medium containing 0.1 microM <em>androstenedione</em>. Follicle-stimulating hormone (FSH) treatment increased estrogen, progesterone, and 20 alpha-hydroxypregn-<em>4</em>-en-3-one production. Concomitant TPA treatment inhibited FSH-stimulated estrogen production by up to 95% [concentration that induced 50% inhibition of steroid production (ED50), 1.1 ng/ml]. TPA also inhibited FSH-stimulated progesterone (ED50, approximately 0.6 ng/ml) and 20 alpha-hydroxypregn-<em>4</em>-en-3-one (ED50, approximately 1.1 ng/ml) production. N6O2'-dibutyryl cyclic adenosine 3':5'-monophosphate increased steroidogenesis; however, cotreatment with TPA blocked progestin but not estrogen production. The TPA inhibition of progestin biosynthesis was accompanied by decreases in FSH-stimulated pregnenolone biosynthesis and 3 beta-hydroxysteroid dehydrogenase activity without decreasing the activity of 20 alpha-hydroxysteroid dehydrogenase. In primary cultures of rat testicular cells, human chorionic gonadotropin treatment increased testosterone production <em>4</em><em>4</em>-fold, whereas concomitant treatment with TPA inhibited testosterone production by up to 86% (ED50, 10 ng/ml). Cholera toxin and N6O2'-dibutyryl cyclic adenosine 3':5'-monophosphate also increased testosterone production, while the actions of these agents were decreased by TPA. The TPA suppression of testosterone production was associated with a decrease in accumulation of 17 alpha-hydroxyprogesterone and <em>androstenedione</em> and an increase in progesterone production, suggesting a specific inhibition of 17 alpha-hydroxylase and 17,20-lyase activities. These results demonstrate the inhibitory effects of a tumor promoter upon gonadotropin-stimulated steroidogenesis by cultured rat granulosa and Leydig cells through specific regulation of steroidogenic enzymes. Additional studies may assist in further elucidation of cellular mechanisms associated with carcinogenesis and steroidogenesis.

The purpose of this study was to compare the steroidogenic potential of the granulosa, theca, and medullary tissues from polycystic and normal ovaries. These ovarian endocrine compartments were isolated from appropriate ovaries and were cultured in vitro for three days in the absence (control) and presence of follicle-stimulating hormone (FSH)/luteinizing hormone (LH) (1 lU/ml), N6,O2-dibutyryladenosine-3':5''-cyclic monophosphoric acid (Bu2cAMP) (10(-2)M), and adrenocorticotropic hormone (ACTH) (1.3 U/ml). After the incubation, steroids in the media were measured by radioimmunoassay. Granulosa cells (10(5) cells per dish) from <em>4</em> to 7 mm follicles of normal and polycystic ovaries secreted progesterone spontaneously during the culture period and the production of progesterone was markedly stimulated (between tenfold and thirtyfold) by gonadotropins and Bu2cAMP but not by ACTH. Little, if any, androgen (<em>androstenedione</em>, dehydroepiandrosterone, and testosterone) or estrogen (estrone and estradiol) accumulated in the media of any granulosa cell culture. The control cultures of theca tissue from normal and polycystic ovaries secreted large amounts of <em>androstenedione</em> and progesterone and the production of these steroids by normal and polycystic ovary theca was stimulated in most cases by LH/FSH and Bu2cAMP but not by ACTH. Both normal and polycystic ovary theca secreted some testosterone and dehydroepiandrosterone but little, if any, estrone or estradiol accumulated in any theca culture. The medullary tissue of normal and polycystic ovaries produced only trace amounts of steroids in vitro except for the results from one polycystic ovary with hyperthecosis in which case significant quantities of C19 and C18 steroids were secreted. These experiments have demonstrated that isolated granulosa and theca cells from midantral follicles of normal and polycystic ovaries have a similar capacity to secrete C21 and C19 steroids in the absence and presence of trophic agents. Therefore, it seems probable that chronic anovulation in patients with polycystic ovaries is not caused by an obvious deficiency in the de novo steroidogenic potential of the multiple midantral follicles of the polycystic ovaries or by the absence of gonadotropin receptors on the polycystic ovary follicular cells.

<em>4</em>-tert-octyphenol (OP) is a surfactant additive widely used in the manufacture of a variety of detergents and plastic products. OP has been reported to mimic the actions of estrogen in many cellular systems. The present studies evaluated the direct effects of OP on human chorionic gonadotropin (hCG)-stimulated testosterone biosynthesis by cultured precursor and immature Leydig cells from 23-day old (prepubertal) rats. Exposure to increasing OP concentrations (1 to 2000 nM) progressively decreased hCG-stimulated testosterone formation in both precursor and immature Leydig cells at higher OP concentrations (100 or 500 to 2000 nM). Testosterone levels were reduced approximately 30 to 70% below control at the highest concentration in both cell types. Similar reductions in testosterone associated with OP exposure were observed in cells stimulated with 1 mM 8-Br-cAMP, suggesting that the main actions of OP occur after the generation of cAMP. Increasing concentrations of 17beta-estradiol (1 to 1000 nM) had no effect on hCG-stimulated testosterone formation in both precursor and immature Leydig cells and the inclusion of 100 nM ICI 182,780, a pure estrogen antagonist, in precursor and immature Leydig cells exposed to OP and hCG, did not alter the inhibition by higher OP concentrations of testosterone formation in both cell types. These results suggest that OP is a hormonally active agent, but that some of its actions are distinct from those of 17beta-estradiol and are not mediated through the estrogen receptor alpha or beta pathway. To further localize the potential site(s) of action of OP, cultured precursor and immature Leydig cells were exposed to increasing concentrations of OP and hCG for 2<em>4</em> h. Next, fresh media containing 1 microM 22(R)-hydroxycholesterol, 1 microM pregnenolone, 1 microM progesterone, or 1 microM <em>androstenedione</em> was added, and the conversion of each substrate to testosterone was determined after incubation for <em>4</em> h. The conversion of <em>androstenedione</em> to testosterone was unaffected by exposure to OP, suggesting that the 17beta-hydroxysteroid dehydrogenase step is not inhibited. However, the conversion of 22(R)-hydroxycholesterol, pregnenolone and progesterone all were inhibited by prior exposure to OP and hCG. This finding suggests that the 17alpha-hydroxylase/c17-20-lyase step, which converts progesterone to <em>androstenedione</em>, is inhibited by OP, and that the cholesterol side-chain cleavage and 3beta-hydroxysteroid dehydrogenase -isomerase steps, which convert cholesterol to pregnenolone and pregnenolone to progesterone, respectively, are other potential sites of OP action. Because concomitant exposure to the antioxidants alpha-tocopherol or ascorbate did not alter the inhibition of testosterone formation by higher OP concentrations, it does not appear that OP is acting as a pseudosubstrate for the generation of free radicals, which can damage P<em>4</em>50 enzymes.

We compare testosterone (T) metabolism in primary cultures of epithelial cells and fibroblasts separated from benign prostate hypertrophy (BPH) and prostate cancer tissues. In all cultures, <em>androstenedione</em> (delta <em>4</em>) formed by oxidation of T by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) represented 80% of the metabolites recovered. The amounts of 5 alpha-dihydrotestosterone (DHT), formed by reduction of T by 5 alpha-reductase (5 alpha-R), were small: 5 and 2% (BPH) and 8 and 15% (adenocarcinoma) for epithelial cells and fibroblasts, respectively. Northern blot analysis of total RNA from epithelial cells (BPH or adenocarcinoma) attributed the reductive activity to the 5 alpha-reductase type 1 isozyme and oxidative activity to the 17 beta-HSD type 2. In cancer fibroblasts, only little 17 beta-HSD type 2 mRNA was detected. The 5 alpha-reductase inhibitors, <em>4</em>-MA (17 beta-(N,N-diethyl)carbamoyl-<em>4</em>-methyl-<em>4</em>-aza-5 alpha-androstan-3-one) and finasteride, inhibited DHT formation with a preferential action of <em>4</em>-MA on epithelial cells (BPH or adenocarcinoma) and of finasteride on fibroblasts from adenocarcinoma. Neither inhibitor acted on delta <em>4</em> formation. On the other hand, the lipido-sterol extract of Serenoa repens (LSESr, Permixon) inhibited the formation of all the T metabolites studied [IC50 S = <em>4</em>0 and 200 micrograms/ml (BPH) and 90 and 70 micrograms/ml (adenocarcinoma) in epithelial cells and fibroblasts, respectively]. These results have important therapeutic implications when selecting appropriate treatment options for BPH.

To date the molecular basis and hormonal criteria for inherited mild late-onset 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency congenital adrenal hyperplasia (CAH) have not been defined. We have thus investigated the presence or absence of mutation in the type II 3 beta-HSD gene encoding adrenal/gonadal 3 beta-HSD in each of five premature pubarche children and hirsute female patients manifesting moderately decreased adrenal 3 beta-HSD activity. ACTH-stimulated hormonal levels in all patients compared with mean levels in pubertal stage-matched normal subjects were between 2.5 and 6.5 SD for 17-hydroxypregnenolone levels, and between 2.5 and 7 SD for dehydroepiandrosterone levels in all except one patient. 17-Hydroxypregnenolone to cortisol ratios were between 2.5 and <em>4</em>.3 SD, and dehydroepiandrosterone to <em>androstenedione</em> ratios were between 3 and 8.6 SD. The type II 3 beta-HSD gene regions of a putative promoter, exons I, II, III, and IV, and exon-intron boundaries in all subjects were amplified by polymerase chain reaction and then sequenced. All patients had normal sequences of the type II 3 beta-HSD gene in both alleles. Three female patients heterozygotic for severe 3 beta-HSD deficiency CAH with one allele mutation of the gene demonstrated normal ACTH-stimulated hormone profiles. These data indicate that moderately decreased adrenal 3 beta-HSD activity resulting in modestly increased delta 5 precursor steroid levels and delta 5 to delta <em>4</em> steroid ratios in premature pubarche and hirsute patients is not caused by a mutation in the type II 3 beta-HSD gene.(ABSTRACT TRUNCATED AT 250 WORDS)

The abundance of fat tissue surrounding normal and malignant epithelial mammary cells raises the questions whether such "adipose milieu" is important in the local proinflammatory/genotoxic shift, which apparently promotes tumor development and worsens prognosis, and what conditions stimulate this shift, or "adipogenotoxicosis." We studied 95 mammary fat samples from 70 postmenopausal and 25 premenopausal breast cancer (BC) patients at a distance of 1.5-2.0 cm from tumors. The levels of leptin, adiponectin, TNFalpha and IL-6 release after <em>4</em>-hr incubation of the samples were evaluated with ELISA, nitric oxide (NO) production by Griess reaction and lipid peroxidation by determination of thiobarbiturate-reactive products (TBRP). Infiltration of fat with macrophages (CD68-positive cells) and expression of cytochrome P<em>4</em>50 1B1/estrogen <em>4</em>-hydroxylase (CYP1B1) were detected by immunohistochemistry. Aromatase (CYP19) activity in mammary fat was measured by (3)H(2)O release from (3)H-1beta-<em>androstenedione</em>. In the postmenopausal BC patients, NO and TNFalpha production by adipose tissue explants increased independent of BMI and in parallel with decreasing leptin and, especially, adiponectin release. In the premenopausal patients, higher CYP1B1 expression and TBRP level were found in mammary fat, while higher aromatase activity was combined with higher CYP1B1 expression as well as NO and IL-6 production. In the postmenopausal group, impaired glucose tolerance was associated with higher IL-6 release production by fat and with higher IL-6/adiponectin ratio. Thus, signs of adipogenotoxicosis in mammary fat can be found in both pre- and postmenopausal BC patients. This condition is likely being maintained through estrogen- and glucose-related factors and mechanisms presumably associated with less favorable types of hormonal carcinogenesis.

To assess the effect of chronic hypersecretion of corticotropin (ACTH) and other peptides derived from proopiomelanocortin , and of cortisol, on plasma adrenal androgen concentration, plasma dehydroepiandrosterone sulphate (DHEA-S), dehydroepiandrosterone (DHEA), <em>androstenedione</em> (delta <em>4</em> A), and cortisol were measured in 1<em>4</em> children and adolescents with Cushing's disease, a 9-year-old boy with an ectopic ACTH-producing tumour, and a group of normal, age-related individuals. The plasma DHEA-S concentration was normal for chronological age in 9 of 12 patients and for bone age in 7 of 10 patients. The plasma DHEA level was normal for chronological age in 12 of 1<em>4</em> patients and for bone age in 8 of 10 patients. In contrast, the concentration of plasma delta <em>4</em> A was raised for chronological age in 6 of 13 patients and for bone age in 7 of 10 patients. All patients had raised plasma cortisol levels in the afternoon and other laboratory and clinical signs of hypercortisolism. In the boy with an ectopic ACTH-producing tumour, plasma DHEA-S was moderately raised, plasma DHEA was normal, and plasma delta <em>4</em> A was very high. This patient's plasma ACTH levels ranged from 13<em>4</em>0 to 1520 pg/ml and the cortisol levels from 51 to 95 micrograms/dl. The findings suggest that a factor other than ACTH is also required for adrenal androgen secretion. Since the other proopiomelanocortin -related peptides--ie, the N-terminal peptide (1-76), beta-endorphin (beta-EP), beta-lipotropin (beta-LPH), and gamma-lipotropin (gamma-LPH)--are raised in the plasma of patients with Cushing's disease, one of these is unlikely to be that putative factor.

Osteoporosis and associated fractures are the most common and debilitating complication of glucocorticoid use. The use of alternative anti-inflammatory agents without the deleterious skeletal effects of glucocorticoids is needed. Dehydroepiandrosterone (DHEA) may have immunomodulatory as well as positive effects on bone. For our further understanding of the mechanisms of action of DHEA, as a steroid-sparing agent, we investigated and compared the effects of dexamethasone (DEX) and DHEA on the regulation of the downstream effector pathway of osteoclastogenesis; RANKL/OPG and a range of inflammatory/pro-resorbing cytokines and receptors using a human clonal osteoblastic cell line. The cells were treated with DEX, DHEA, and <em>androstenedione</em> (ANDI). The mRNA expression of RANKL and OPG was determined by real-time PCR after overnight incubation. The regulation of a broad spectrum of cytokines by DEX and DHEA was also investigated using a human cytokine/growth factor and receptor gene array consisting of 268 cytokine-related cDNAs. To confirm some of the gene expression changes, protein production was measured by ELISA. RANKL expression and RANKL/OPG ratio were increased by DEX. This effect was reversed by co-treatment with both DHEA or ANDI. Several pro-inflammatory/resorptive cytokines including IL-6, IL-<em>4</em>, IFN-gamma, macrophage inhibitory factor (MIF) were down-regulated not only by DEX but also by DHEA. In contrast to DEX, DHEA did not lead to suppression of growth factors including vascular endothelial growth factor (VEGF), fibroblast growth factor-5 (FGF-5), insulin-like growth factor-binding protein3 (IGF-BP3). Several new target genes previously documented to influence bone formation were up-regulated by DHEA such as Notch 2, insulin receptor, thrombin receptor (PAR1). The data suggest that DHEA has immunomodulatory properties without the catabolic effects on bone remodeling, observed with glucocorticoid use. DHEA may thus prove useful as a steroid-sparing agent in the management of inflammatory disorders such as SLE or rheumatoid arthritis. Further in vivo studies are indicated.

Steroid release before and during maturation was studied in Xenopus ovarian follicles under various conditions of stimulation of LH in a perifusion system. Acute stimulation by 15 micrograms of LH induces a 10-fold increase in the androgen (testosterone and <em>androstenedione</em>) level which reaches a maximum <em>4</em> hr later and then slowly decreases until the 25th hour. Repeated stimulations every 2 or <em>4</em> hr are followed by the same androgen increase during the first 8 or 10 hr and then by a slow decrease in the secretion despite new LH injections. A significant increase in progesterone secretion is seen only after at least two stimulations (8 hr). Estradiol secretion slowly increases to a moderate level during the first 5 hr and then remains stable whatever the stimulation. During continuous stimulation (LH 0.5 microgram/ml) androgen levels reach an initial maximum after <em>4</em> hr and then fluctuate with a 2-hr period. Addition of theophyllin to the medium enhances these fluctuations. After 12 hr when the progesterone has increased, androgen secretion diminishes to reach a basal level without fluctuations. Germinal vesicle breakdown occurs only in follicles that have been appropriately stimulated to secrete androgens and progesterone during the required time.

Serum levels of cortisol (F), pregnenolone (delta5-P), 17-hydroxypregnenolone (17-delta5-P), progesterone (P), 17-hydroxyprogesterone (17-P), <em>androstenedione</em> (A), testosterone (T), 5alphadihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), its sulfate (DHEA-S), estrone (E1), and estradiol-17beta (E2) were measure in 2 virilized patients with ovarian hyperthecosis. Daily morning blood samples were obtained for 6 consecutive days. Dexamethasone (Dex) 2 mg/day was administered orally starting after venipuncture on the second day and continued for 5 days. Human chorionic gonadotropin (hCG) was administered intramuscularly on the afternoon of the fourth and fifth days. Following the suppression-stimulation test, both patients underwent abdominal hysterectomy and bilateral salpingo-oophorectomy. At the time of surgery, samples of peripheral and ovarian vein blood were obtained for steroid measurements. Blood samples were also obtained postsurgery to evaluate the effect of ovariectomy on the steroid levels. Of significance were the following observations: 1)Although both patients were eumenorrheic, no corpus luteum or corpus albicans was seen on histologic examination of the ovaries. 2)Of the androgens measured, only peripheral T and DHT were elevated and did not suppress on Dex treatment, but decreased to low levels following ovariectomy, pointing toward the ovary as the source of excess T and DHT. Both patients had elevated T and DHT in the ovarian vein samples. 3)In 1 patient the ovarian vein samples shoed elevated F levels with a significant ovarian-peripheral venous gradient for this steroid, and indication of ovarian secretion of F in this patient. <em>4</em>)The levels of 17-P were elevated in both patients, did not suppress on Dex, and increased markedly following hCG, suggesting the ovary as the source of excess 17-P. Since A levels were normal and did not increase concomitantly with 17-P levels following hCG, it is likely that the patients had a decreased activity of the ovarian C17-20 desmolase, the enzyme responsible for the conversion of 17-P to A.