Effect of acute ACTH administration on plasma steroid levels in the dog

Production of adrenal steroids in intact and castrated dogs is stimulated acutely by ACTH. While the increase in plasma cortisol, 17-hydroxypregnenolone and 17-hydroxyprogesterone is not affected by castration, the increment of dehydroepiandrosterone is totally abolished. On the other hand, administration of 17-hydroxypregnenolone in adrenalectomized dogs caused an increase in plasma C-19 steroids such as dehydroepiandrosterone, androstenedione and testosterone indicating that this C-21 progestin in plasma is rapidly converted. The site of this conversion is likely the testis. Furthermore, acute hCG administration in adrenalectomized dogs resulted in a marked increase in the levels of plasma 17-hydroxypregnenolone and dehydroepiandrosterone. However, our data show an ACTH-induced rise in 5-androstene-3 beta. 17 beta-diol in intact and castrated dogs, thus suggesting that this steroid is a good parameter to assess in the stimulation of adrenal steroidogenesis by ACTH.     

An adrenocortical tumor secreting weak mineralocorticoids

An adrenocortical carcinoma (15.5 g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i.e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.     

Testicular steroidogenesis in the baboon Papio anubis.

We recently reported that the baboon testis converts pregnenolone to testosterone through the delta-4 pathway. The present studies were to determine the metabolism of intermediates of the delta-4 and delta-5 pathway by the baboon testis. Fragments (50 mg) were incubated for 3 hr with 10 muCi of the following tritium-labelled substrates: pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, or testosterone. Pregnenolone was converted to testosterone primarily through the delta-4 pathway, with accumulation of progesterone, 17-hydroxyprogesterone and 20alpha-dihydroprogesterone as predominant intermediates. Similar results were obtained in progesterone incubations. 17-hydroxyprogesterone was not efficiently metabolized by the fragments, while 17-hydroxypregnenolone and dehydroepiandrosterone were efficiently converted into testosterone and androstenedione. Androstenedione was metabolized primarily to testosterone, while testosterone was not a suitable substrate. Some 5alpha-androstanediol was identified in each incubate. These results suggest that although testosterone is formed from pregnenolone through the delta-4 pathway, the delta-5 intermediates are more suitable substrates for testosterone synthesis in the baboon testis.     

Age-related changes in serum 17-hydroxypregnenolone and 17-hydroxypregnenolone sulfate concentrations in human infancy and childhood

In order to clarify some of the developmental processes of the human adrenal cortex or steroidogenesis in infancy and childhood, serum concentrations of 17-hydroxypregnenolone, 17-hydroxypregnenolone sulfate and 17-hydroxyprogesterone were measured by means of a combined radioimmunoassay method, and the age-related changes in these steroids were also examined. The actual ranges of serum concentrations of 17-hydroxypregnenolone, 17-hydroxypregnenolone sulfate and 17-hydroxyprogesterone in umbilical cord blood were 27.1-80.5, 1,560-5,030 and 53.3-304 nmol/l, respectively. These values subsequently decreased to nadirs of 0.95-2.09 nmol/l of 17-hydroxypregnenolone in subjects 1 to 2 years old, 0.93-7.03 nmool/l of 17-hydroxypregnenolone sulfate in subjects 3 to 6 years old and 0.18-0.78 nmol/l of 17-hydroxyprogesterone in subjects 1 to 2 years old, respectively, and they were followed by gradual increases to the adult levels. This study thus revealed the age-related changes in 17-hydroxypregnenolone and its sulfate concentrations in infancy and childhood and indicated that, in the process in which the adrenal cortex was differentiated to the definitive form, the decrease in the activity of steroid sulfotransferase in infancy and childhood occurred more slowly than the increase in that of 3 beta-hydroxysteroid dehydrogenase.     

Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450C17) in the frog brain and pituitary

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 androstenedione (Delta(4)). However, the presence, distribution and activity of cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450(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 P450(C17)-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450(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, P450(C17)-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450(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(4). De novo synthesis of C(21) 17-hydroxysteroids and C(19) ketosteroids was reduced in a concentration-dependent manner by ketoconazole, a P450(C17) inhibitor. This is the first detailed immunohistochemical mapping of P450(C17) in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.     

Steroid hormone formation in bovine ovarian follicles.

In an attempt to assess histophysiological implication of the follicular compartment of the bovine ovary in steroid hormone formation and the effect of human chorionic gonadotropin (hCG) in vitro on follicular steroidogenesis, minces of follicular tissues from non-gravid bovine ovaries were incubated with radioactive testosterone or acetate in the presence and absence of hCG. Significant amounts of estrone and estradiol-17beta were formed on incubation with testosterone-4-14C; hCG decreased the conversion approximately by 30%. The major radioactive products formed from acetate-l-14C were androstenedione and testosterone with lesser amounts of dehydroepiandrosterone and 17-hydroxyprogesterone. In addition, small amounts of progesterone, 17-hydroxypregnenolone, estrone and estradiol-17beta were formed. Histology of the dissected follicle specimens was characterized by dominant theca cells undergoing luteinization with small amounts of granulosa cells, which showed neither proliferation nor luteinization. The pattern of distribution of radioactivity among the steroids formed from acetate-14C was considered to represent steroidogenic profile of bovine atretic follicles. The addition of hCG in vitro increased the overall incorporation of radioactive acetate into the steroids approximately by 50%, although the range of increase was not uniform in the individual steroids under the exprimental conditions.     

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.     

Steroidogenesis in ovarian follicles of chub mackerel, Scomber japonicus

We incubated different radiolabeled steroid precursors with intact chub mackerel ovarian follicles to clarify the synthetic pathways of steroid hormones during vitellogenesis and following final oocyte maturation (FOM). During vitellogenesis, estradiol-17beta (E2) was synthesized from pregnenolone via 17-hydroxypregnenolone, 17-hydroxyprogesterone, androstenedione, and testosterone. The physiological significance of the intermediate metabolites of E2 in the ovarian follicles was examined by comparing follicular steroidogenesis between gonochoric and hermaphroditic fish species. After vitellogenesis, the steroidogenic pathway shifted from E2 to maturation-inducing hormone (MIH) production owing to the inactivation of 17,20-lyase and the activation of 20 beta-hydroxysteroid dehydrogenase. Of the new steroids produced during FOM, 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) was most effective at inducing germinal vesicle breakdown in vitro. Circulating levels of 17,20beta-P increased specifically around the time of germinal vesicle migration, while another FOM-specific 20beta-hydroxylated progestin, 17,20beta,21-trihydroxy-4-pregnen-3-one, was present at consistently low levels during FOM. These results indicate that 17,20beta-P is the MIH of chub mackerel.     

Intratesticular steroid levels and their hormonal control

Litter-mate adult male rats were treated with daily intramuscular injections of ACTH (10.5 micrograms), dexamethasone (2.0 mg), ethynyl estradiol (1.7 micrograms) and hCG (5 IU) for three consecutive days. The animals were sacrificed on the fourth day and the intratesticular and peripheral plasma steroid levels were analyzed. The steroids measured by radioimmunoassay included pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone and dihydrotestosterone. In addition, the sulphoconjugated forms of pregnenolone, dehydroepiandrosterone, testosterone and dihydrotestosterone were estimated in the peripheral blood. The administration of ACTH diminished the intratesticular levels of all steroids studied. Also dexamethasone and ethynyl estradiol treatment suppressed all intratesticular steroid levels, except that of pregnenolone (the former) and of 17-hydroxyprogesterone (the latter). The suppressive effect of ethynyl estradiol was strongest on the levels of the delta 5-steroids and that of dexamethasone on the delta 4-steroids; the latter was significantly stronger than the effect of ACTH. The stimulatory effect of hCG was limited to the metabolism of progesterone and was restricted to the sequence: 17-hydroxyprogesterone----androstenedione----testosterone---- dihydrotestosterone. Dexamethasone-suppression, and hCG-stimulation of the intratesticular levels of delta 4-steroids, was mirrored by corresponding changes in the peripheral plasma levels, with the exception of the plasma levels of androstenedione which were not influenced by any of the treatments studied. Also the suppression of intratesticular testosterone and dihydrotestosterone levels by ACTH, dexamethasone, or ethynyl estradiol was closely reflected by their plasma levels both in the unconjugated and sulphoconjugated forms. On the hand, the administration of ACTH diminished the intratesticular levels of pregnenolone and progesterone but significantly increased those in the plasma. Moreover, both ACTH and ethynyl estradiol reduced the levels of all delta 5-steroids in testicular tissue, but not in the peripheral plasma, although they decreased the circulating levels of pregnenolone sulphate and dehydroepiandrosterone sulphate. The data are interpreted as suggesting that the hormonal agents studied interfere with testicular steroidogenesis through different mechanisms.     

Serum 17-hydroxypregnenolone and 17-hydroxypregnenolone sulfate concentrations in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Serum concentrations of 17-hydroxypregnenolone, 17-hydroxypregnenolone sulfate and 17-hydroxyprogesterone were measured simultaneously in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, using a combined radioimmunoassay method. All these precursor steroids were found to be markedly elevated in the sera of untreated patients with a salt-losing form of the disease, whereas, in untreated patients with a simple virilizing form, only the concentration of unconjugated steroids was increased and the 17-hydroxypregnenolone sulfate concentration remained within the normal range. Among the patients with a salt-losing form under maintenance therapy, these steroids were all still significantly increased in those on insufficient control, whereas only 17-hydroxyprogesterone was significantly but slightly increased in those on adequate control. Although the mechanism whereby the serum 17-hydroxypregnenolone sulfate concentration is not increased in the untreated simple virilizers is unknown, both a milder degree of 21-hydroxylase deficiency and a role of 17-hydroxypregnenolone sulfate in adrenal steroid production as a kind of supplier are suggested as possible explanations, especially in the neonatal period and early infancy. Thus, this study showed the serum concentrations of 17-hydroxypregnenolone and its sulfate together with 17-hydroxyprogesterone in patients with 21-hydroxylase deficiency in various conditions.     

Testicular steroidogenesis in the rhesus monkey (Macaca mulatta.

Studies were undertaken to investigate testicular steroidogenesis in the Rhesus monkey Macaca mulatta. Testicular fragments (50 mg) were incubated for 3 hr with pregnenolone-7-3H or with progesterone-7-3H. The major metabolite of pregnenolone was progesterone (70.1%), with a lesser conversion to 17-hydroxyprogesterone (1.6%), androstenedione (3.3%), and testosterone (7.2%). The delta-5 intermediates 17-hydroxypregnenolone (4.6%) and dehydroepiandrosterone (8.6%) were also identified in the pregnenolone incubates. A majority of the progesterone substrate was not metabolized by the testicular fragments (80.1%), while some conversion to 17-hydroxyprogesterone (3.4%), androstenedione (4.8%), and testosterone (11.7%) occurred in the incubates. These results suggest that testicular fragments from the Rhesus monkey may convert pregnenolone to testosterone through both the delta-4 and the delta-5 pathways.     

Effects of chronic ethanol ingestion on steroid profiles in the rat testis

Testosterone, seven of its potential precursors, three of its metabolites and estradiol were analyzed in testes from rats given ethanol for 23 days in a nutritionally adequate liquid diet. The results were compared to those obtained with pair-fed control rats. The concentrations of pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were markedly lowered in four of the five rats given ethanol. The concentrations of the other 3 beta-hydroxy-delta 5 steroids and estradiol were unchanged, resulting in significantly increased ratios between 17-hydroxypregnenolone and 17-hydroxyprogesterone (P less than 0.025) and between androstenediol and testosterone (P less than 0.025) in the ethanol-treated rats. The results indicate that chronic ethanol administration reduces formation of testosterone by affecting a step prior to pregnenolone. There may also be an effect on the conversion of some 3 beta-hydroxy-delta 5 to the corresponding 3-oxo-delta 4 steroids. The levels of testosterone and three other steroids in testes of rats given the liquid diet were significantly lower than those in testes of animals fed a standard rat chow. This indicates a dietary influence on testicular steroid concentrations.     

Steroid hormone formation in human ovarian follicles in vitro.

Ovarian follicles of 5 to 15 mm in diameter were isolated from 45 ovaries of 34 patients in the follicular and luteal phases of the cycle. Three experiments were done. In the first, follicles were minced and incubated in Krebs-Ringer bicarbonate buffer containing 1 to 2muCi of testosterone-4-14C in the presence or absence of 100 IU human chorionic gonadotropan (hCG). In the second, minced follicles were incubated with 100 muCi of sodium acetate-I-14C under identical conditions. In the third, ten follicles from a single patient in the late proliferative stage of endometrial dating were cut in halves and incubated with 100 muCi of acetate-I-14C under identical conditions. The minced follicle preparation was capable of aromatizing testosterone-4-14C into radioactive estrone and estradiol in significant amounts. Incorporation of radioactive acetate into pregenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, estradiol and estrone was assessed by reverse dilution analysis with recrystallization to constant specific activity. The major radioactive products formed were androstenedione and 17-hydroxyprogesterone in the latter two experiments. Dehydroepiandrosterone was one of the major steroids in the second experiment. The minor products were testosterone, progesterone and pregnenolone. Smaller, but definite incorporations of radioactive acetate into estradiol and estrone occurred in the second experiment. On histological examination, the follicles were characterized by atretic changes. This distribution pattern of radioactive acetate among the steroids was considered to represent the steroidogenic profile of unstimulated or atretic follicles.     

Effects of 17-hydroxyprogesterone caproate (17-OHPC) administration to pregnant squirrel monkeys (Saimiri boliviensis boliviensis)

Poor reproductive performance of the squirrel monkey (Saimiri boliviensis boliviensis) in captivity and a relative progesterone (P) deficiency in pregnancy have been reported. Since premature births may contribute to pregnancy wastage, we attempted to measure the effectiveness of 17-hydroxyprogesterone caproate (17-OHPC) treatment of pregnant squirrel monkeys to prevent early deliveries. Based on clearance studies of nonpregnant animals, 25 mg of 17-OHPC was administered at 6-day intervals to a test group of 31 pregnant monkeys while the control group of 29 received saline. Venous blood was obtained at 6- to 12-day intervals for measurement of 17-hydroxyprogesterone (17-OHP), P, 17-B estradiol (E), and androstenedione (A), and dihydroepiandrosterone (DHEA) levels by radioimmunoassays. The treated group had a significant increase in serum 17-OHP (P < 0.001), P (P < 0.01), and DHEA (P < 0.05) levels compared to controls. The numbers of live births, stillbirths, or neonatal deaths did not differ significantly between groups. Although 17-OHPC administration appeared to increase P and 17-OHP levels, this did not alter the duration of pregnancy nor delay the onset of labor. A significant fall in 17-OHP, P, and E levels was observed 6–12 days before delivery.     

Increased adrenal steroid secretion in response to CRF in women with hypothalamic amenorrhea

OBJECTIVE: To evaluate adrenal steroid hormone secretion in response to corticotropin-releasing factor (CRF) or to adrenocorticotropin hormone in women with hypothalamic amenorrhea. DESIGN: Controlled clinical study. SETTING: Department of Reproductive Medicine and Child Development, Section of Gynecology and Obstetrics, University of Pisa, Italy. PATIENT(S): Fifteen women with hypothalamic amenorrhea were enrolled in the study. Eight normal cycling women were used as control group. INTERVENTION(S): Blood samples were collected before and after an injection of ovine CRF (0.1 microg/kg iv bolus) or after synthetic ACTH (0.25 mg iv). MAIN OUTCOME MEASURE(S): Plasma levels of ACTH, 17-hydroxypregnenolone (17OHPe), progesterone (P), dehydroepiandrosterone (DHEA), 17-hydroxyprogesterone (17OHP), cortisol (F), 11-deoxycortisol (S) and androstenedione (A). RESULT(S): Basal plasma concentrations of ACTH, cortisol, 11-deoxycortisol, DHEA and 17OHPe were significantly higher in patients than in controls, whereas plasma levels of progesterone and 17-OHP were significantly lower in patients than in controls. In amenorrheic women the ratio of 17-OHPe/DHEA, of 17-OHPe/17-OHP and of 11-deoxycortisol/cortisol were significantly higher than in controls, while a significant reduction in the ratio of 17-OHP/androstenedione, of 17-OHP/11-deoxycortisol was obtained. In response to corticotropin-releasing factor test, plasma levels of ACTH, cortisol, 17-OHP, 11-deoxycortisol, DHEA and androstenedione were significantly lower in patients than in controls. In response to adrenocorticotropin hormone, plasma levels of 17-OHP, androstenedione and androstenedione/cortisol were significantly higher in patients than in controls. CONCLUSIONS: Patients suffering for hypothalamic amenorrhea showed an increased activation of hypothalamus-pituitary-adrenal (HPA) axis, as shown by the higher basal levels and by augmented adrenal hormone response to corticotropin-releasing factor administration. These data suggest a possible derangement of adrenal androgen enzymatic pathway.     

Chronic cigarette smoking alters circulating sex hormones and neuroactive steroids in premenopausal women

Chronic smoking alters the circulating levels of sex hormones and possibly also the neuroactive steroids. However, the data available is limited. Therefore, a broad spectrum of free and conjugated steroids and related substances was quantified by GC-MS and RIA in premenopausal smokers and in age-matched (38.9+/-7.3 years of age) non-smokers in the follicular (FP) and luteal phases (LP) of menstrual cycle (10 non-smokers and 10 smokers, in the FP, and 10 non-smokers and 8 smokers in the LP). Smokers in both phases of the menstrual cycle showed higher levels of conjugated 17-hydroxypregnenolone, 5alpha-dihydroprogesterone, conjugated isopregnanolone, conjugated 5alpha-pregnane-3beta,20alpha-diol, conjugated androstenediol, androstenedione, testosterone, free testosterone, conjugated 5alpha-androstane-3alpha/beta,17beta-diols, and higher free testosterone index. In the FP, the smokers exhibited higher levels of conjugated pregnenolone, progesterone, conjugated pregnanolone, lutropin, and a higher lutropin/follitropin ratio, but lower levels of cortisol, allopregnanolone, and pregnanolone. In the LP, the smokers exhibited higher levels of free and conjugated 20alpha-dihydropregnenolone, free and conjugated dehydroepiandrosterone, free androstenediol, 5alpha-dihydrotestosterone, free and conjugated androsterone, free and conjugated epiandrosterone, free and conjugated etiocholanolone, 7alpha/beta-hydroxy-dehydroepiandrosterone isomers, and follitropin but lower levels of estradiol and sex hormone binding globulin (SHBG) and lower values of the lutropin/follitropin ratio. In conclusion, chronic cigarette smoking augments serum androgens and their 5alpha/beta-reduced metabolites (including GABAergic substances) but suppresses the levels of estradiol in the LP and SHBG and may induce hyperandrogenism in female smokers. The female smokers had pronouncedly increased serum progestogens but paradoxically suppressed levels of their GABA-ergic metabolites. Further investigation is needed concerning these effects.     

Steroid biosynthesis by zona glomerulosa-fasciculata cells in primary culture of guinea-pig adrenals

Steroidogenesis was studied in guinea-pig glomerulosa-fasciculata cells maintained in primary culture for up to 7 days. The basal secretion which remained stable for the first 2 days in culture rapidly rose to reach a plateau on day 4 at levels 6-7-fold higher than those observed during the first 2 days of culture while the maximal response to ACTH in terms of cortisol and androstenedione secretion was fairly stable throughout the 7-day period. Exposure of glomerulosa-fasciculata cells to ACTH caused a stimulation of pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, corticosterone, 11-deoxy-corticosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione, 11 beta-hydroxyandrostenedione and aldosterone while, after 48 h of incubation, a marked accumulation of end-products, namely cortisol and 11 beta-hydroxyandrostenedione, was observed. The half-maximal steroidogenic response to ACTH occurred at concentrations varying between 1.7 x 10(-11) and 1.1 x 10(-10) mol/l for the 12 steroids examined. Addition of 8-bromoadenosine 3', 5'-cyclic monophosphate stimulated steroid secretion in a dose-dependent manner. Maximal response to 8-bromoadenosine 3', 5'-cyclic monophosphate was obtained at 1 mmol/l, and no further rise of steroid secretion was observed after addition of ACTH. Incubation of glomerulosa-fasciculata cells with labeled corticosterone, cortisol and androstenedione indicates that only androstenedione can be converted into 11 beta-hydroxyandrostenedione, thus suggesting that this end-product is a good parameter of the C-19 steroid production by guinea-pig glomerulosa-fasciculata cells in primary culture. The present data confirm that guinea-pig glomerulosa-fasciculata cells in primary culture provide an interesting model for the study of the regulation of C-19 steroid formation by the adrenals.     

Regulation of serum testosterone in men with steroid sulfatase deficiency: response to human chorionic gonadotropin

Human chorionic gonadotropin (hCG; 5000 IU) was administered to 6 control men and 6 patients with recessive x-linked ichthyosis (RXLI) with verified 3 beta-hydroxysteroid sulfate sulfatase (3 beta-HSS) deficiency in their skin biopsy samples. Concentrations of steroids and their sulfate conjugates were determined in peripheral serum specimens collected a day before and 4 days after hCG administration. Testosterone concentrations were identical in patients and controls. Baseline serum LH concentrations were also identical in the 2 groups showing that there were no major differences in the regulation of the hypothalamic-pituitary-gonadal axis. The significantly increased (31-82%) serum concentrations of sulfated pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone and 5-androstene-3 beta,17 beta-diol in patients compared with controls indicated that their circulating concentrations were regulated by 3 beta-HSS. This is in line with the fact that the baseline concentrations of the same unconjugated steroids were significantly lower (32-90%) in patients with RXLI, suggesting that a proportion of these circulating steroids were derived from the corresponding sulfated precursors. The response patterns and actual concentrations of testosterone, 17-hydroxyprogesterone and estradiol were similar in the patients and the controls after hCG. The decreased concentrations of testosterone sulfated at carbon 17 under baseline conditions and after hCG in patients with RXLI remains enigmatic. In conclusion, testosterone production and the response to hCG seem to be identical in patients with RXLI and controls despite the fact that significant differences were observed in the circulating concentrations of several unconjugated and sulfated testosterone precursors.     

Xenopus laevis CYP17 regulates androgen biosynthesis independent of the cofactor cytochrome b5

The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17alpha-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity). Most mammalian CYP17 isoforms have high 17alpha-hydroxylase relative to 17,20-lyase activities and preferentially mediate one of the two 17,20-lyase reactions. In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary. CYP17 isoforms generally rely on the cofactor cytochrome b(5) for the 17,20-lyase reaction, suggesting that the high lyase activity of Xenopus CYP17 might be due to a lesser dependence on b(5). The kinetics of Xenopus CYP17 expressed in yeast microsomes were therefore examined in the absence and presence of Xenopus on human b(5). Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b(5), confirming that the activity did not require b(5). However, both Xenopus and human b(5) slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b(5). Surprisingly, only the human b(5) cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17. Studies using human/Xenopus chimeric b(5) proteins revealed that human b(5) residues 16-41 were important for the specific regulation of the lyase activity of HuCYP17, possibly serving as an interacting domain with the enzyme. CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.     

Gas chromatographic-mass spectrometric study of metabolites of C21 and C19 steroids in neonatal porcine testicular microsomes.

Microsomal fractions obtained from testes of 3-week-old piglets have been incubated, separately, with progesterone, 17-hydroxyprogesterone, 5-pregnene-3 beta,20 beta-diol, 16 alpha-hydroxypregnenolone, 5-androstene-3 beta,17 alpha-diol and dehydro-epiandrosterone. The metabolites, after derivatization, have been separated by capillary gas chromatography and identified by mass spectrometry. Quantification was by selected ion monitoring. Progesterone was shown to be 17-hydroxylated and also converted into 4,16-androstadien-3-one (androstadienone). The major metabolite of 17-hydroxyprogesterone was 4-androstene-3,17-dione (4-androstenedione), but little, if any, androstadienone was formed, indicating that this particular biosynthesis did not require 17-hydroxylation. The metabolites of 5-pregnene-3 beta, 20 beta-diol were found to be 17-hydroxypregnenolone, 3 beta-hydroxy-5,16-pregnadien-20-one (16-dehydropregnenolone) and 5,16-androstadien-3 beta-ol. Dehydroepiandrosterone and 5-androstene-3 beta,17 alpha-diol were interconvertible but neither steroid acted as a substrate for 16-androstene formation. However, dehydroepiandrosterone was metabolized to a small quantity of 4-androstenedione. Under the conditions used, no metabolites of 16 alpha-hydroxypregnenolone could be detected. The present results, together with those obtained earlier, indicate that the neonatal porcine testis has the capacity to synthesize weak androgens, mainly by the 4-en-3-oxo steroid pathway. Although 16-androstenes cannot be formed from C19 steroids, progesterone served as a substrate and may be converted directly to androstadienone, without being 17-hydroxylated first. The pathway to 5,16-androstadien-3 beta-ol, however, involves 17-hydroxypregnenolone and 16-dehydropregnenolone as intermediates.