Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta superfamily, were recently shown to be expressed and to regulate steroidogenesis in rat ovarian tissue. The purpose of this study was to investigate the effect of BMP-<em>4</em> on androgen production in a human ovarian theca-like tumor (HOTT) cell culture model. We have previously demonstrated the usefulness of these cells as a model for human thecal cells. HOTT cells respond to protein kinase A agonists by increased production of <em>androstenedione</em> and with an induction of steroid-metabolizing enzymes. In this investigation, HOTT cells were treated with forskolin or dibutyryl cyclic AMP (dbcAMP) in the presence or absence of various concentrations of BMP-<em>4</em>. The accumulation of <em>androstenedione</em>, progesterone, and 17alpha-hydroxyprogesterone (17OHP) in the incubation medium was measured by RIA. The expression of 17alpha-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cholesterol side-chain cleavage (CYP11A1), and steroidogenic acute regulatory (StAR) protein was determined by protein immunoblotting analysis using specific rabbit polyclonal antibodies. We also examined the expression of BMP receptor subtypes in our HOTT cells using RT-PCR. In cells treated with medium alone, steroid accumulation and steroid enzyme expression was unchanged. In cells treated with BMP alone there was a modest decrease in <em>androstenedione</em> secretion. In the presence of forskolin, HOTT cell production of <em>androstenedione</em>, 17OHP, and progesterone increased by approximately <em>4</em>.5-, 35-, and 3-fold, respectively. In contrast, BMP-<em>4</em> decreased forskolin-stimulated HOTT cell secretion of <em>androstenedione</em> and 17OHP by 50% but increased progesterone production 3-fold above forskolin treatment alone. Forskolin treatment led to an increase in CYP17, CYP11A1, 3betaHSD, and StAR protein expression. BMP-<em>4</em> markedly inhibited forskolin stimulation of CYP17 expression but had little effect on 3betaHSD, CYP11A1, or StAR protein levels. Similar results were observed with the cAMP analog dbcAMP. In addition, BMP-<em>4</em> inhibited basal and forskolin stimulation of CYP17 messenger RNA expression as determined by RNase protection assay. Other members of the transforming growth factor beta superfamily, including activin and inhibin, had minimal effect on <em>androstenedione</em> production in the absence of forskolin. In the presence of forskolin, activin inhibited <em>androstenedione</em> production by 80%. Activin also inhibited forskolin induction of CYP17 protein expression as determined by Western analysis. We identified the presence of messenger RNA for three BMP receptors (BMP-IA, BMP-IB, and BMP-II) in the HOTT cells model. In conclusion, BMP-<em>4</em> inhibits HOTT cell expression of CYP17, leading to an alteration of steroidogenic pathway resulting in reduced <em>androstenedione</em> accumulation and increased progesterone production. These effects of BMP-<em>4</em> seem similar to those caused by activin, another member of the transforming growth factor-beta superfamily of proteins.

Serum inhibin B rises across the luteal-follicular transition, whereas inhibin A does not increase until the late follicular phase of the menstrual cycle. To test the hypothesis that inhibin B is secreted from preantral and small antral follicles and that FSH and local growth factors differentially regulate inhibin B and inhibin A from these developing follicles, human ovaries were obtained after oophorectomy. Basal secretion of inhibin B and inhibin A was examined in intact preantral follicles in culture (n = 6). Basal secretion and regulation of inhibin B and inhibin A secretion by gonadotropins, <em>androstenedione</em>, activin A, insulin, and IGF-I were examined in cultured granulosa cells from small antral follicles (n = 21). Inhibin B secretion from preantral follicle cultures was detectable at baseline (range, 17-96 pg/mL), whereas inhibin A was not detectable. In contrast, both inhibin B and inhibin A were detectable in granulosa cell cultures from small antral follicles. In granulosa cells from small antral follicles, FSH (30 ng/mL) stimulated inhibin A 3-fold (10.5 +/- 2.2 to 32.5 +/- 8.3 IU/mL; P < 0.001), but not inhibin B secretion (1730 +/- 35<em>4</em> to 231<em>4</em> +/- 532 pg/mL; P = NS). Likewise, cAMP (1 mmol/L) stimulated inhibin A <em>4</em>-fold (16.6 +/- <em>4</em>.3 to 62.5 +/- 21.9 IU/mL; P < 0.002), but not inhibin B secretion (2327 +/- 5<em>4</em>6 to 1877 +/- 377 pg/mL; P = NS). hCG (30 ng/mL) did not stimulate inhibin A or inhibin B. <em>Androstenedione</em> (10(-)(7) mol/L), activin (30 ng/mL), insulin (30 ng/mL), and insulin-like growth factor I (IGF-I; 100 ng/mL) alone did not stimulate inhibin A or inhibin B secretion. Further, FSH-stimulated inhibin A secretion was not augmented by <em>androstenedione</em>, activin, insulin, or IGF-I. In contrast, the combination of IGF-I and FSH was the only treatment that stimulated inhibin B secretion (17<em>4</em>2 +/- 380 to 2881 +/- 731 pg/mL; P < 0.03). However, FSH in combination with IGF-I resulted in greater stimulation of inhibin A (3<em>4</em>0%) than inhibin B (65%). These findings demonstrate that inhibin B is secreted from developing preantral and small antral follicles, but is not directly stimulated by FSH. However, the combination of FSH and IGF-I enhanced inhibin B secretion. In contrast, inhibin A is not secreted from preantral follicles, but in small antral follicles FSH and cAMP stimulate inhibin A secretion. Further, FSH in combination with IGF-I results in a greater degree of stimulation of inhibin A than of inhibin B. These findings suggest that FSH and IGF-I differentially regulate inhibin A and inhibin B secretion. However, additional growth factors or increasing granulosa cell number may contribute to the preferential serum inhibin B increase across the luteal-follicular transition in the menstrual cycle.

BACKGROUND

In some patients, PCOS may develop as a consequence of an exaggerated adrenarche during pubertal development.

OBJECTIVE

The aim of the study was to assess adrenal function during childhood and pubertal development in daughters of women with PCOS (PCOSd).

METHODS

We included 98 PCOSd [6<em>4</em> during childhood (ages <em>4</em>-8 yr) and 3<em>4</em> during the peripubertal period (ages 9-13 yr)] and 51 daughters of control women (Cd) [30 during childhood and 21 during the peripubertal period]. In both groups, an acute ACTH-(1-2<em>4</em>) stimulation test (0.25 mg) and an oral glucose tolerance test were performed. Bone age and serum concentrations of cortisol, <em>androstenedione</em>, 17-hydroxyprogesterone, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), glucose, and insulin were determined.

RESULTS

PCOSd and Cd were similar in age and body mass index. During the peripubertal period, basal and poststimulated DHEAS concentrations were higher in PCOSd compared to Cd. Among PCOSd, 12.5% of girls in childhood and 32.<em>4</em>% in peripuberty presented biochemical evidence of exaggerated adrenarche. Stimulated insulin was higher in PCOSd compared to Cd during childhood (P = 0.03) and peripuberty (P = 0.03). An advancement of 8 months between bone and chronological age was observed in peripubertal PCOSd compared to Cd.

CONCLUSIONS

In PCOSd, basal and stimulated DHEAS concentrations were higher during the onset of puberty. Around 30% of the PCOSd demonstrated an exacerbated adrenarche, which may reflect increased P<em>4</em>50c17 activity. In addition, a modest advance in bone age was observed, probably secondary to the hyperinsulinemia and/or adrenal hyperandrogenism.

BACKGROUND

ASP9521 is a first-in-class orally available inhibitor of the enzyme 17 β-hydroxysteroid dehydrogenase type 5 (17 βHSD5; AKR1C3), catalysing the conversion of dehydroepiandrosterone and androstenedione into 5-androstenediol and testosterone. It has demonstrated anti-tumour activity in in vitro and in vivo preclinical models.

METHODS

This first-in-man phase I/II study utilised a 3 + 3 dose escalation design starting at 30 mg ASP9521/day, with the aim of defining a maximum tolerated dose, as defined by the incidence of dose-limiting toxicities. Eligible patients received ASP9521 orally for 12 weeks. Safety, tolerability, pharmacokinetics (PK), pharmacodynamics and anti-tumour activity were assessed.

RESULTS

Thirteen patients (median age: 68 years; range 52-76) with metastatic castration-resistant prostate cancer (mCRPC) progressing after chemotherapy were included; 12 patients discontinued treatment at or before week 13, mainly due to disease progression. The most common adverse events were grade 1/2 and included asthenia (N = 5), constipation (N = 4), diarrhoea (N = 3), back pain (N = 3) and cancer pain (N = 3). PK demonstrated a half-life (t1/2) ranging from 16 to 35 h, rapid absorption and dose proportionality. No biochemical or radiological responses were identified; neither endocrine biomarker levels nor circulating tumour cell counts were altered by ASP9521. Given the lack of observable clinical activity, the study was terminated without implementing a planned 12-week dose expansion part at selected doses or a planned food-effect study part.

CONCLUSIONS

In patients with mCRPC, ASP9521 demonstrated dose-proportional increase in exposure over the doses evaluated, with an acceptable safety and tolerability profile. However, the novel androgen biosynthesis inhibitor showed no relevant evidence of clinical activity.

We have investigated the activity and expression of aromatase enzyme in nontumoral, cirrhotic, and malignant human liver tissues and cells using both chromatographic and reverse transcription (RT)-PCR analyses. After 2<em>4</em>- and 72-h incubation of tissue minces or hepatic cell lines with either testosterone or <em>androstenedione</em> as androgen precursor, human hepatocellular carcinoma (HCC) tissues and HepG2 hepatoma cells showed elevated aromatase activity, with estrogen formation rates being 20 and >95%, respectively, as opposed to nontumoral hepatic tissues and nonmalignant Chang liver (CL) cells, where no aromatase activity could be detected. Cirrhotic samples exhibited intermediate enzyme activity. Notably, exposure of HepG2 cells to the aromatase inhibitor Letrozole resulted in a striking decrease of estrogen formation, which became virtually absent at a Letrozole dose of 0.<em>4</em> nM. RT-PCR analysis revealed markedly lower aromatase mRNA in both CL cells and nontumoral liver tissues, as compared with HepG2 cells and HCC samples. Cirrhotic specimens displayed variable transcript levels, in turn comparable with those observed in nontumoral or HCC tissues. Exon-specific RT-PCR showed prominent expression of exon I.3A-containing message and exon I.<em>4</em>-containing message in CL and HepG2 cells, as in nontumoral and HCC tissues, respectively. The present evidence implies that locally elevated estrogen formation in malignant human liver tissues and cells may have a role in the development and/or maintenance of human HCC, eventually leading to develop alternative strategies for treatment of HCC patients using antiaromatase agents.

Twenty-seven girls aged 8 to 18 were studied in a longitudinal prospective fashion. Serum samples were collected at 6 month intervals up to <em>4</em> years and radioassayed for hormones of pituitary, ovarian, and adrenal origin. A progressive elevation of luteinizing hormone (LH), follicle-stimulating hormone/FSH), estradiol (E2), dehydroepiandrosterone (DHA), and <em>androstenedione</em> (delta<em>4</em>) occurred during puberty and continued until menarche. The onset of puberty occurred concomitantly with an elevation of estrone (E1) dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and 17-hydroxyprogesterone (17-OH-P). Prolactin (Prol) and progesterone (Prog) concentrations did not change during puberty until after menarche. After menarche, levels of LH and FSH were comparable with menstruating adult females. Concentrations of E2 and Prog were lower during the second half of the cycle among most regularly menstruating subjects than expected during the luteal phase. LH and Prog levels indirectly suggest that ovulation occurs in a few girls within months after menarche.

Bisphenol A (BPA) is a potential endocrine disruptor. It has been shown that it reduces serum testosterone level in rodents after exposure. However, the mechanism is unclear. The object of the present study is to investigate the effects of BPA on human and rat steroidogenic enzymes including P<em>4</em>50 17α-hydroxylase/17,20-lyase (CYP17A1), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3). Human and rat testis microsomes were exposed to various concentrations of BPA (10(-8)-10(-<em>4</em>)M). BPA inhibited human and rat 3β-HSD, CYP17A1 and 17β-HSD3 activities. The half maximal inhibitory concentrations (IC(50)s) of BPA for human and rat testis 3β-HSD were 7.92±1.03 and 26.<em>4</em>9±3.03 μM (200 μM pregnenolone), respectively. The IC(50)s for human and rat CYP17A1 (1 μM progesterone) were 18.99±3.75 and 6<em>4</em>.67±<em>4</em>.0<em>4</em> μM, respectively. BPA was a weak HSD17B3 inhibitor with IC(50)s of about 100 μM (200 nM <em>androstenedione</em>). BPA also concentration-dependently inhibited testosterone production by rat Leydig cells. In conclusion, BPA is an inhibitor for 3β-HSD, CYP17A1 and 17β-HSD3. Human 3β-HSD and CYP17A1 are more sensitive to BPA than rat 3β-HSD and CYP17A1.

Following transfection of types 1, 2 and 3 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) cDNAs into transformed embryonal kidney (293) cells, we have characterized the selective directional and inhibitory characteristics of these activities. While homogenates of transfected cells could catalyze interconversion of the substrate and product, in agreement with the general belief on the activity of these enzymes, the same activities measured in intact cells, in order to better reflect the physiological conditions, showed an unidirectional reaction. Types 1 and 3 17 beta-HSD catalyzed the reduction of estrone to estradiol and <em>4</em>-<em>androstenedione</em> to testosterone, respectively, while type 2 17 beta-HSD catalyzed the oxidative transformation of both testosterone and 17 beta-estradiol to <em>4</em>-<em>androstenedione</em> and estrone, respectively. In addition, types 1, 2 and 3 17 beta-HSD activities showed different pH optima. While types 1 and 3 showed pH optimum values centered at around 5 and 6, respectively, type 2 17 beta-HSD activity, which preferentially, catalyzes the oxidation reaction, has higher activity at an alkaline pH (8-10). Differences in the optimum incubation temperatures were also observed: type 1 17 beta-HSD shows a relatively high temperature tolerance (55 degrees C). In contrast, type 2 and 3 functioned best at 37 degrees C. Types 1, 2 and 3 17 beta-HSD activities could be also differentiated by their sensitivity toward various specific inhibitors: type 1 was potently inhibited by an estradiol derivative containing a bromo/or iodopropyl group at position 16 alpha. On the other hand a derivative of estrone containing a spiro-gamma-lactone at position 17 showed a potent inhibitory effect on type 2 17 beta-HSD, whereas type 3 was strongly inhibited by 1,<em>4</em>-androstadiene-1,6,17- trione.

Serum steroid, gonadotropin, and alpha-subunit levels were assessed in 35 women with cycle abnormalities [11 with and 2<em>4</em> without polycystic ovarian disease (PCOD) according to strict clinical and biochemical criteria] and 8 regularly cycling women in the early (cycle day 3 or <em>4</em>) and mid (cycle day 7 or 8) follicular phase. LH and FSH levels were estimated using two immunological techniques [RIA and immunoradiometric assay (IRMA)] and in vitro bioassays (BIO), using mouse Leydig cells and rat granulosa cells, respectively. In PCOD patients mean alpha-subunit, free androgen index [FAI; testosterone x 100/sex hormone-binding globulin (SHBG)], <em>androstenedione</em>, estrone, and estradiol (E2) were significantly elevated compared to levels in the early follicular phase of control cycles and non-PCOD patients. In addition, in PCOD patients mean IRMA-LH and RIA-LH levels were distinctly increased (2.8- to 3.6 fold, respectively; both comparisons, P less than 0.001) compared to control values, but in the same order of magnitude (1.3- to 1.<em>4</em>-fold increments) as that in non-PCOD patients. However, the median BIO-LH level in PCOD patients was 5.9-fold higher than that in non-PCOD patients and <em>4</em>.0-fold higher than the BIO-LH in the early follicular phase of control women. Consequently, the median BIO/IRMA-LH ratio was <em>4</em>.8-fold higher in PCOD patients compared to non-PCOD patients. In women with cycle abnormalities, individual BIO/IRMA-LH ratios correlated with BIO-LH (rs = 0.<em>4</em>8), FAI (rs = 0.39), free estrogens (E2/SHBG ratios; rs = 0 0.<em>4</em>7), and dehydroepiandrosterone sulfate (rs = 0.60) concentrations. Mean IRMA-, RIA-, and BIO-FSH levels and BIO/IRMA-FSH ratios were not significantly different when various groups were compared. Although RIA- and IRMA-LH levels showed good correlation (rs = 0.88), RIA-LH levels were consistently higher, resulting in distinctly higher RIA-LH/FSH ratios (mean, <em>4</em>.5) compared to IRMA-LH/FSH ratios (median, 1.8) in PCOD patients.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

Twenty-four-hour energy expenditure and substrate use were measured by indirect calorimetry in respiration chambers on a fixed physical program and related to body composition and plasma concentrations of various substrates and thermogenic hormones. Fifty premenopausal women with a wide range of body weight were examined in the follicular menstrual phase under weight stable conditions. Most of the variance in the sleeping energy expenditure (82%) was accounted for by two covariates, lean body mass (75%, P less than 0.0001), and fat mass (7%, P less than 0.0001). An additional 6% of the variance in sleeping energy expenditure was accounted for by plasma <em>androstenedione</em> concentration (<em>4</em>%, P = 0.0005) and by free T3 index (2%, P = 0.03). Thus physiological variation among individuals in plasma <em>androstenedione</em> concentration may result in a difference in energy expenditure of 908 kJ/day and the corresponding variation in free T3 index may result in a difference between individuals of 59<em>4</em> kJ/day. Fifty four percent of the variation in carbohydrate oxidation rates was accounted for by 2<em>4</em>-h energy balance, and by plasma concentrations of insulin, nonesterified fatty acids, and estradiol. Waist circumference, plasma nonesterified fatty acids, and estradiol concentrations explained <em>4</em>9% of the variance in 2<em>4</em>-h lipid oxidation. An obese subgroup of women (n = 27) had significantly higher 2<em>4</em>-h energy expenditure, lipid, and carbohydrate oxidation rates than an age-matched normal weight group (n = 16), but the entire group difference in energy expenditure was explained by differences in body composition. We conclude that physiological variations in plasma <em>androstenedione</em> and T3 concentrations contribute to the interindividual variance in energy expenditure of women, and their role is not different in obese women. A positive energy balance and increased insulin action may be mediators of the higher carbohydrate oxidation in obesity, whereas an increased substrate availability seems to bring about the increased lipid oxidation.

We determined the effect of chronic androgen suppression on inflammation in women with polycystic ovary syndrome (PCOS) compared to weight-matched controls. We performed a pilot project using samples from previous prospective, controlled studies. Nine women with PCOS (5 obese, <em>4</em> lean) and 9 ovulatory controls (5 obese, <em>4</em> lean) participated in the study. Androgens, C-reactive protein (CRP), interleukin-6 (IL-6), free fatty acids (FFA) and body weight were measured before and after 3 and 6 months of gonadotropin-releasing hormone (GnRH) agonist administration. GnRH agonist treatment decreased estradiol, testosterone and <em>androstenedione</em> to similar levels in all subjects. CRP and IL-6 increased in obese women with PCOS, was unaltered in lean women with PCOS and obese controls, and decreased in lean controls after 6 months of treatment. FFA decreased and body weight increased in obese women with PCOS, but did not change significantly in lean women with PCOS and in either control group after 6 months of treatment. The testosterone reduction was related to increases in weight and IL-6. The fall in FFA was related to the rise in CRP. The increases in weight and IL-6 were related to the rise in CRP. We propose that hyperandrogenism in PCOS may exert an anti-inflammatory effect when obesity is present, but may not promote inflammation in the disorder; and that circulating androgens have a pleiotropic effect on inflammation depending on the combination of PCOS and weight status in a given individual.

Pregnant spotted hyaenas were treated with anti-androgens to interfere with the unusually masculine 'phallic' development that characterizes females of this species. The effects on genital morphology and plasma androgen concentrations of infants were studied during the first 6 months of life. Although there were consistent 'feminizing' effects of prenatal anti-androgen treatment on genital morphology in both sexes, such exposure did not produce males with extreme hypospadia, as it does in other species, nor did it produce females with a 'typical' mammalian clitoris and external vagina. 'Feminization' of males resulted in a penis with the morphological features of the hyaena clitoris, and 'feminization' of females exaggerated the sex differences that are typical of this species. The effects of treatment were present at birth and persisted for at least 6 months. Treatment of pregnant females with flutamide and finasteride also markedly reduced circulating concentrations of testosterone and dihydrotestosterone in maternal plasma during pregnancy. Plasma delta <em>4</em>-<em>androstenedione</em> was reduced in the female, but not the male, infants of treated mothers, consistent with an epigenetic hypothesis previously advanced to explain hormonal 'masculinization' of females. The present 'feminizing' effects of prenatal anti-androgen treatment are consistent with contemporary understanding of sexual differentiation, which accounts for morphological variation between the sexes in terms of steroids. However, current theory does not account for the basic genital structure of females and the present data suggest that development of the male penis and scrotum, and the female clitoris and pseudoscrotum, in spotted hyaenas may involve both androgen-dependent and androgen-independent components.

Using the continuous infusion technique, the conversion ratios (CR) of testosterone (T) to <em>androstenedione</em> (A) and dihydrotestosterone (DHT) and of A to T and DHT were determined in 12 normal males (aged 31-72 yr), 10 normal postmenopausal women, and <em>4</em> amenorrheic women with idiopathic hirsutism; in <em>4</em> additional males these studies were performed during infusion of cold T to increase plasma T to supraphysiological levels. It was observed that besides the MCR of T and DHT, the blood conversion ratios (CRBB) of T in A and to a lesser extent of T in DHT were also significantly correlated with either the free or the nontestosterone-estradiol-binding globulin-bound T fraction but not with total plasma T. In postmenopausal women, plasma A was by far the most important precursor of plasma DHT; the CRA/DHTBB was significantly higher than CRT/DHTBB. It is suggested that total plasma A, but only nonspecifically bound T, freely gains access into the cells where these conversions occur and that plasma A might be an important parameter of androgenicity. Less than 50% of plasma DHT could be accounted for by peripheral conversion of either A or T. Whereas in males this may be explained by direct DHT secretion, in (postmenopausal) women conversion of other precursors to plasma DHT should be considered.

Serum sex hormones may be related to the risk of several diseases in postmenopausal women including osteoporosis, heart disease, and breast and endometrial cancer. For assessment of the relation of sex hormones to disease, the measurements should be reliable, valid, and practical. In this paper, the authors evaluated the short-term (<em>4</em>-week) and long-term (2-year) reliability of serum sex hormones and interrelations among serum sex hormones in white postmenopausal women recruited in Pittsburgh, Pennsylvania, 1981-1986. For comparison, the authors simultaneously evaluated the short- and long-term reliability of other commonly measured risk factors, i.e., lipids, lipoproteins, and blood pressure. Serum concentrations of estrone, estradiol, testosterone, and <em>androstenedione</em> were measured by extraction, column chromatography, and radioimmunoassay. Reliability was estimated by calculating the intraclass correlation coefficients (R) and their 95% confidence interval. About 50% of the estradiol levels were below the sensitivity of the assay and, therefore, these results should be interpreted with some caution. The intraclass correlation coefficient for testosterone was 0.92 (95% confidence interval 1.0-0.82), suggesting that a single measure may be reliable in characterizing women for epidemiologic research. Over <em>4</em> weeks, estrone could be measured more reliably (R = 0.72) than over 2 years (R = 0.56), but the variability over the long term was similar to that observed for other biologic variables, suggesting that, in situations where the relation between estrone and disease is fairly substantial, a single measure may be used. For estradiol and <em>androstenedione</em>, the intraclass correlations were small, indicating poor reproducibility and the need for more measurements. Estrone concentrations were 11 pg/ml or <em>4</em>6% higher in women with measurable estradiol. Estrone was also positively related to <em>androstenedione</em> concentrations (r = 0.33, p less than 0.001). Concentrations of estradiol are extremely low in postmenopausal women, and accordingly, there is a greater possibility of laboratory error. Since the data suggest that estrone levels can be more reliably measured and are, in fact, related to estradiol levels, it is possible that estrone levels may be used to indicate the total estrogen status of postmenopausal women.

We have investigated the effect of a 3-month endurance training program (running and cycling) on plasma hormone responses during standardized bicycle ergometer work (15-min consecutive work loads of 60%, 70%, 80%, and eventually 90% VO2 max) in eight previously untrained eumenorrheic women. The subjects were investigated before and after training both in the follicular and luteal phases of the menstrual cycle (between the 7th-10th and 20th-25th days of their menstrual cycle, respectively). Blood was obtained 15 and 2 min before the onset of exercise and at the end of each work load from an indwelling catheter. In each sample, the plasma concentrations of estradiol 17 beta (E2), progesterone (P), testosterone (T), <em>androstenedione</em> (delta <em>4</em>-A), dehydroepiandrosterone sulfate (DHEA-S), prolactin (PRL), and adrenocorticotropic hormone (ACTH) were assayed in duplicate by RIA; lactate was assayed as well. The hormone concentrations were expressed in absolute as well as in relative values. After training basal DHEA-S and ACTH levels were significantly (P less than 0.05) lower in both phases of the menstrual cycle, whereas basal luteal phase E2 and T levels were significantly (0.05 greater than P greater than 0.01) lower after training. Exercise induced significant increments in the relative values of all hormones in both phases (0.05 greater than P greater than 0.001). After training, T and DHEA-S increased relatively more pronounced (0.05 greater than P greater than 0.02) in the follicular and luteal phase, respectively.

We have demonstrated that the quail brain possesses the cholesterol side-chain cleavage enzyme (cytochrome P<em>4</em>50scc) and 3beta-hydroxysteroid dehydrogenase/delta(5)-delta(<em>4</em>)-isomerase (3beta-HSD) and produces pregnenolone, pregnenolone sulfate and progesterone from cholesterol. We have also demonstrated the expression of cytochrome P<em>4</em>50 17alpha-hydroxylase/c17,20-lyase (P<em>4</em>50(17alpha,lyase)) and the conversion of progesterone to 17alpha-hydroxyprogesterone in the same avian species. Therefore, the present study was conducted to investigate androgen biosynthesis from progesterone in the avian brain. Employing biochemical techniques combined with HPLC and TLC analyses, the conversion of progesterone to <em>androstenedione</em>, an androgen precursor, was found in quail brain. The present biochemical analysis further revealed the conversion of <em>androstenedione</em> to testosterone, indicating the presence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in the quail brain. The formation of testosterone from progesterone was also detected in the brain. Testosterone formation was more intense in the diencephalon, whereas the concentration of endogenous testosterone in the diencephalon was lower than those in other brain regions in castrated quails. However, the concentration of endogenous estradiol, a metabolite of testosterone by cytochrome P<em>4</em>50arom, was highest in the diencephalon of castrated quails. These results suggest that testosterone biosynthesis occurs in the quail brain, in particular the diencephalon. Testosterone may subsequently be converted to estradiol.

Fertility was evaluated in 53 female patients with late-onset adrenal hyperplasia (LAH) due to 21-hydroxylase deficiency. The majority of patients (n = 33) were seen for isolated postpubertal hirsutism, 9 patients consulted for sterility, and 11 for irregular menstrual cycles. At the time of diagnosis, the ages of patients ranged from 15-<em>4</em>0 yr (mean +/- SD, 2<em>4</em>.6 +/- 5.2). No patient had major signs of virilization. The plasma 17-hydroxyprogesterone level was higher than normal in all patients (26.8 +/- 18.9 nmol/L; range, 3.<em>4</em>-139.<em>4</em>) and dramatically increased to 1<em>4</em>0.1 +/- 80.6 nmol/L (range, 35.2-32<em>4</em>.2) after ACTH treatment. Plasma androgen levels were high (testosterone, 3.25 +/- 2.03 nmol/L; delta <em>4</em>-<em>androstenedione</em>, 13.65 +/- 5.60 nmol/L). Plasma basal and LHRH-stimulated values were normal for FSH and high for LH. Basal and TRH-stimulated plasma PRL levels were normal. Among these 53 LAH patients, only 20 desired a pregnancy. These had a total of 38 pregnancies. Ten patients became pregnant before the diagnosis of LAH and without any treatment; they had a total of 18 pregnancies, 12 of which were successful. Moreover, 19 normal pregnancies without any spontaneous abortion were carried to term by 1<em>4</em> of 16 hydrocortisone-treated patients. One patient needed the association of one cure of clomiphene citrate. Hypofertility in LAH patients seems, therefore, to be relative. Its mechanism is hormonal, with anovulation or dysovulation, due to the continuous steroid feedback of adrenal origin on the hypothalamo-pituitary axis. Hydrocortisone is the appropriate treatment in most cases, reducing adrenal androgen overproduction and relieving hypothalamic-pituitary gonadotropin function, thereby making possible cyclic ovarian activity and ovulations.

We previously demonstrated a progressive decline in serum dehydroepiandrosterone sulfate (DHEA-S) levels in women during a hyperinsulinemic-euglycemic clamp. To determine whether this fall in serum DHEA-S levels might have been due to insulin-stimulated 1) hydrolysis of DHEA-S to dehydroepiandrosterone (DHEA), 2) conversion of DHEA-S/DHEA to <em>androstenedione</em>, and/or 3) urinary excretion of these steroids, 10 additional men were studied by the hyperinsulinemic-euglycemic clamp technique. Each man received a 0.1 U/kg (0.72 nmol/kg) insulin bolus dose, followed by a 10 mU/kg.min (72 pmol/kg.min) insulin infusion for <em>4</em> h. An average insulin level of 12,390 +/- 259 (+/- SE) pmol/L (1,726.8 +/- 36 microU/mL) was achieved; serum glucose was maintained at 5.0 +/- 0.1 mmol/L (90.5 +/- 2.3 mg/dL). During the hyperinsulinemia, serum DHEA-S levels fell progressively and were significantly lower than baseline at <em>4</em> and 6 h of study (85.5 +/- 5.9% and 79.1 +/- 3.2% of baseline values, respectively; P less than 0.05). Serum DHEA levels fell concurrently and were significantly lower than baseline at 2, <em>4</em>, and 6 h of study (66.2 +/- 12.3%, 61.6 +/- 11.2%, and 52.9 +/- 10.2% of baseline values, respectively; P less than 0.05). The percent fall in serum DHEA levels correlated positively with the percent fall in serum DHEA-S levels (r = 0.<em>4</em><em>4</em>; P less than 0.02). Serum <em>androstenedione</em> levels also fell progressively during hyperinsulinemia and were significantly lower than baseline at 2, <em>4</em>, and 6 h of study (71.5 +/- <em>4</em>.1%, 71.0 +/- 7.2%, and <em>4</em>8.1 +/- 3.3% of baseline values, respectively; P less than 0.05). No change in serum DHEA-S, DHEA, or <em>androstenedione</em> levels occurred in paired control studies, during which 0.<em>4</em>5% saline was infused at rates matched exactly to the rates of the dextrose and insulin infusions during the hyperinsulinemic clamp studies. Despite decreasing serum DHEA-S and DHEA levels during hyperinsulinemia, urinary DHEA-S and DHEA glucuronide excretions were increased by 50% (P less than 0.05) and 86% (P = 0.05), respectively, compared to urinary excretion of these steroids during control studies. In contrast, urinary excretion of unconjugated DHEA was unchanged. Quantitatively, however, increased urinary excretion of conjugated DHEA during hyperinsulinemia accounted for only about 5% of the concomitant fall in serum DHEA-S concentrations.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

By modification of a recently developed method for separation of radio-labelled urinary oestrogens we were able to separate oestrogen metabolites and measure their isotope ratios in urine following injections of [3H]delta <em>4</em>-<em>androstenedione</em> and [1<em>4</em>C]oestrone. This method provides a useful tool for studying in vivo aromatisation of delta <em>4</em>-<em>androstenedione</em> into oestrone in breast cancer patients before and during treatment with aromatase inhibitors.

Ten hirsute women with polycystic ovarian syndrome (PCO) and nine with idiopathic hirsutism (IH) underwent selective ovarian suppression with leuprolide for 5-6 months and then were randomized to receive, in addition, dexamethasone or placebo for <em>4</em> more months. Serum hormone levels and hair growth rates were determined before and after each treatment period. During the initial treatment period with leuprolide alone, testosterone decreased by 5<em>4</em> +/- 6% (mean +/- SEM) in PCO and by 36 +/- 3% in IH (P = 0.02). <em>Androstenedione</em> decreased by 53 +/- 6% in PCO and by 31 +/- 7% in IH (P = 0.02). Androstanediol glucuronide (Adiol-G) decreased by 1<em>4</em> +/- 6% in PCO and by 7 +/- 3% in IH. There was no change in dehydroepiandrosterone sulfate (DHEAS). While initial serum androgen levels were higher in PCO than in IH, they were similar after ovarian suppression in the two groups. After ovarian suppression, Adiol-G was more consistently correlated with testosterone and <em>androstenedione</em> than was DHEAS, suggesting that Adiol-G may be a better marker than DHEAS of adrenal androgen secretion. Hair growth rates decreased by 37 +/- 6% in PCO and by 1<em>4</em> +/- 10% in IH (P = 0.07). The change in hair growth correlated with the change in <em>androstenedione</em> (r = 0.66; P = 0.002), but not significantly with the change in testosterone (r = 0.29; P = 0.2). After the addition of dexamethasone therapy (0.5 mg daily), testosterone, <em>androstenedione</em>, and DHEAS levels fell to near or below assay detection limits, while Adiol-G decreased by 80 +/- 3%. Hair growth rates decreased slightly more in women during dexamethasone (<em>4</em>6 +/- 6%) than during placebo (26 +/- 9%; P = 0.18). In summary, the ovary was the major source of circulating testosterone and <em>androstenedione</em> in PCO. The adrenal contributed a substantial minority of these hormones in PCO and was the major source of androgen secretion in IH. Adrenal hyperandrogenism was common in both IH and PCO. Hair growth rates correlated best with changes in serum <em>androstenedione</em> levels. Adiol-G, which was derived primarily from adrenal precursors, was a better marker of adrenal androgen secretion than was DHEAS in these subjects.

Endocrine and neuromuscular effects of prolonged strength training were investigated in 21 strength-trained male subjects during the course of a 2<em>4</em>-week progressive strength training and during a subsequent detraining period of 12 weeks. Maximal isometric leg extensor force increased by 19% (P less than 0.001) during the first 20 weeks, followed by a plateau during the <em>4</em> latest weeks of training. During the course of the training period, no systematic change was found in serum testosterone concentrations, but there was a decreasing tendency in the concentrations of free testosterone (NS), 17-OH-progesterone (NS), <em>androstenedione</em> (P less than 0.05), dehydroepiandrosterone (P less than 0.05), cortisol (P less than 0.01), transcortin (CBG) (P less than 0.05), and in the cortisol/CBG ratio (P less than 0.05). The last <em>4</em> weeks of training were characterized by significant correlations between the individual changes in maximal isometric force and the changes in serum free testosterone concentrations (r = 0.60, P less than 0.01). The changes in the ratios of free testosterone to cortisol (r = 0.73, P less than 0.001), total testosterone to cortisol (r = 0.83, P less than 0.001), and 17-OH-progresterone to cortisol (r = 0.62, P less than 0.01) also correlated with the changes in maximal force. The findings suggest that the turnover of endogenous androgens may increase during progressively intensified training without a change in serum total testosterone concentration. Prolonged intensive strength training may also lead to changes in the concentrations of serum cortisol and transcortin. During the most stressful phases of training, the changes in serum androgen/cortisol ratios seem to be highly individual and may correlate with changes in muscular strength.

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17-hydroxypregnenolone (17OH-Delta(5)P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA) and <em>androstenedione</em> (Delta(<em>4</em>)). However, the presence, distribution and activity of cytochrome P<em>4</em>50 17alpha-hydroxylase/C17, 20-lyase (P<em>4</em>50(C17)), a key enzyme required for the conversion of pregnenolone (Delta(5)P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P<em>4</em>50(C17)-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P<em>4</em>50(C17)-containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P<em>4</em>50(C17)-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P<em>4</em>50(C17)-positive cell bodies also contained 3beta-hydroxysteroid dehydrogenase-like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [(3)H]Delta(5)P resulted in the formation of several tritiated steroids including 17OH-Delta(5)P, 17OH-P, DHEA and Delta(<em>4</em>). De novo synthesis of C(21) 17-hydroxysteroids and C(19) ketosteroids was reduced in a concentration-dependent manner by ketoconazole, a P<em>4</em>50(C17) inhibitor. This is the first detailed immunohistochemical mapping of P<em>4</em>50(C17) in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.

The triazole derivative, R 76713 and its enantiomers R 83839(-) and R 838<em>4</em>2(+) are effective inhibitors of the aromatization of <em>androstenedione</em>. For human placental microsomes, the (+) enantiomer (R 8382<em>4</em>) is about 1.9- and 32-times more active than the racemate (IC50 2.6 nM) and the (-) enantiomer, respectively. R 838<em>4</em>2 is about 30- and 1029-times more active than <em>4</em>-hydroxyandrostene-3,17-dione and aminoglutethimide. This potency might originate from its high affinity for the microsomal cytochrome P<em>4</em>50 (P<em>4</em>50). Indeed, R 838<em>4</em>2, compared to R 76713 and R 83839, forms a more stable P<em>4</em>50-drug complex. Difference spectral measurements indicate that the triazole nitrogen N-<em>4</em> coordinates to the haem iron. The reversed type 1 spectral changes suggest that R 76713 is able to displace the substrate from its binding place and the stable complex formed in particular with the (+) enantiomer suggests that its N-1-substituent occupies a lipophilic region of the apoprotein moiety. Kinetic analysis implies that there is a competitive part in the inhibition of the human placental aromatase by R 76713. The Ki values for R 76713, R 838<em>4</em>2 and R 83839 are 1.3 nM, 0.7 nM and 18 nM, respectively. These results are indicative of stereospecificity for binding. Up to 10 microM, R 76713 and its enantiomers have no statistically significant effect on the regio- and stereoselective oxidations of testosterone in male rat liver microsomes. All three compounds have no effect on the P<em>4</em>50-dependent cholesterol synthesis, cholesterol side-chain cleavage and 7 alpha-hydroxylation and 21-hydroxylase. At 10 microM, R 76713 has a slight effect on the bovine adrenal 11 beta-hydroxylase. This effect originates mainly from R 83839, the less potent aromatase inhibitor. On the other hand, the inhibition of the 17,20-lyase of rat testis observed at concentrations greater than or equal to 0.5 microM, originates rather from R 838<em>4</em>2. However, 50% inhibition is only achieved at 1.8 microM R 838<em>4</em>2, i.e. at a concentration about 1300-times higher than that needed to reach 50% inhibition of the human placental aromatase.