Preovulatory changes of steroidogenesis in isolated rabbit follicles (author's transl)

In an attempt to investigate the effect of ovulating hormone on the steroidogenesis of mature follicles in the course of ovulation, transitory changes of steroidogenesis in isolated rabbit follicles have been studied at several intervals after injection of an ovulatory dose of human chorionic gonadotropin (hCG). Five to ten follicles of approximately 1-2 mm in diameter were isolated from ovaries of a mature rabbit (2.5-3.0 kg) under streomicroscope, before and at the 3rd, 6th, 9th and 12th hours after intravenous injection of of 100 IU/kg of hCG. Follicles were incubated with 100 muCi of acetate-1-14C in 2 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) at 37 degrees C for 3 hours under 95% oxygen plus 5% carbon dioxide. Each incubation was terminated by quick freezing and stored forzen at -20 degrees C until eighty follicles had been collected for each time period before commencement of analysis. Incorporation of radioactive acetate into pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone., 20 alpha-dihydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone and estradiol-17beta were analysed by the reverse dilution technique and identified in radiochemically pure form by recrystallization to constant specific activities. The steroidogenic activity of the follicles was evaluated by overall as well as fractionated incorporations. A peak in the overall incorporation of 14C- acetate into the ten steroids at the 3rd hour after hCG injection, followed by gradual decrease up to the 9th hour was observed. The incorporation decreased markedly to a minimum level at the 12th hour after hCG injection, which was below the level of preinjection control. Comparable quantitative fluctuations were found with the fractionated incorporation of 14C-acetate into the C21 and C18 steroids in the time sequence following hCG injection. However, the fractionated incorporation into C19 steroids reached to a maximum at the 6th hour after hCG injection. 5istribution patterns of incorporation among the individual steroids were varied at each interval of time. In the non-injected control, mature follicles synthesized predominantly estradiol-17beta, testosterone and androstenedione. Divergent steroids were formed from radioactive acetate at the 3rd hour after hCG injection. These included porgestogen, androgen and estrogen, but pregnenolone and 17hydroxyprogesterone were the two principal steroids produced. There was no essential difference in the steroidogenic patterns between the 6th and 9th hour, the major products being C21 and C19 steroids such as pregnenolone, 17hydroxyprogesterone, dehydroipiandrosterone and testosterone. The three androgens were the major steroids formed at the 12th hour after hCG injection. Thus the chages in the steroidogenic profile of the follicle was obvious in the course of ovulation. The basis of qualitative changes in follicular steroidogenesis during the process of ovulation have been discussed in connection with an accompanying effect of an ovulatory dose of hCG.     

Capillary gas chromatography with chemical ionization negative ion mass spectrometry in the identification of odorous steroids formed in metabolic studies of the sulphates of androsterone, DHA and 5alpha-androst-16-en-3beta-ol with human axillary bacterial isolates

The products of metabolism of the sulphates (0.5 micromol/l) of androsterone, dehydroepiandrosterone (DHA) and 5alpha-androst-16-en-3beta-ol have been investigated after incubation with 72 h cultures of human axillary bacterial isolates for 3 days at 37 degrees C. The medium used, tryptone soya broth (TSB), contained yeast extract and Tween 80. The isolates used were Coryneform F1 (known previously to metabolize testosterone and to be involved in under-arm odour (UAO) production, i.e. UAO +ve), Coryneform F46 (inactive in both the testosterone metabolism and UAO tests, i.e. UAO -ve) and Staphylococcus hominis/epidermidis (IIR3). Control incubations of TSB alone, TSB plus each of the steroid sulphates and TSB plus each of the bacterial isolates were also set up. After termination of reactions and addition of internal standards, 5alpha-androstan-3beta-ol and 5alpha-androstan-3-one (50 ng each), extracted and purified metabolites were subjected to combined gas chromatography-mass spectrometry with specific ion monitoring. Steroidal ketones were derivatized as their O-pentafluorobenzyl oximes; steroidal alcohols (only androst-16-enols in this study) were derivatized as their tert-butyldimethylsilyl ethers. Analysis was achieved by negative ion chemical ionization mass spectrometry for the pentafluorobenzyl oximes at [M-20]- and electron impact positive ion mass spectrometry for the tert-butyldimethylsilyl ethers at [M-57]+. The incubation broth contained two compounds which had gas chromatographic and mass spectrometric properties identical to those of DHA and 4-androstenedione. It was not possible, therefore, to show unequivocally that DHA sulphate (DHAS) was converted microbially into DHA, although this is implied by the finding of small quantities of testosterone and 5alpha-dihydrotestosterone in incubations with F1. With androsterone S, no free androsterone was recorded and only very small (5 pg or less) amounts of testosterone. Two odorous steroids, androsta-4,16-dien-3-one and 5alpha-androst-2-en-17-one (Steroid I) were formed (mean quantities 40 and 45 pg, respectively). The sulphate of 5alpha-androst-16-en-3beta-ol was metabolized with F1 into large quantities of the odorous steroids, 5alpha-androst-16-en-3-one and Steroid I. In addition, much smaller quantities of androsta-4,16-dien-3-one were formed. In contrast, incubations of DHAS with F46 resulted in no metabolites except, possibly, DHA, but the sulphate moiety of androsterone S was also cleaved to yield the free steroid together with large amounts of Steroid I. In incubations of DHAS and androsterone S with F1, no 16-unsaturated steroids were formed, although 5alpha-androst-16-en-3beta-yl S was de-sulphated and the free steroid further metabolized. No evidence was obtained for androst-16-ene metabolism in incubations with F46. In incubations with S. hominis/epidermidis (IIR3), androsterone S was converted into androsterone and, in high yield, to Steroid I plus some 5alpha-androst-16-en-3-one. Both DHAS and androsterone S were converted into androst-16-enols. Sulphatase activity was also manifested when 5alpha-androst-16-en-3beta-yl S was utilized as substrate with IIR3, large quantities of Steroid I and 5alpha-androst-16-en-3-one being formed, together with further metabolism of androst-16-enes. In view of the fact that both DHAS and androsterone S occur in apocrine sweat, the metabolism of these endogenous substrates by human axillary bacteria to several odorous steroids may have important implications in the context of human odour formation.     

The 16,17-double bond is needed for irreversible inhibition of human cytochrome p45017alpha by abiraterone (17-(3-pyridyl)androsta-5, 16-dien-3beta-ol) and related steroidal inhibitors

Abiraterone (17-(3-pyridyl)androsta-5,16-dien-3beta-ol, 1) is a potent inhibitor (IC50 4 nM for hydroxylase) of human cytochrome P45017alpha. To assist in studies of the role of the 16,17-double bond in its mechanism of action, the novel 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) and 17beta-(3-pyridyl)-16,17alpha-epoxy-5alpha-androst-3beta-ol (6) were synthesized. 3beta-Acetoxyetienic acid was converted in three steps into 5 via photolysis of the thiohydroxamic ester 8. Oxidation of an appropriate 16,17-unsaturated precursor (21) with CrO3-pyridine afforded the acetate (23) of 6. Inhibition of the enzyme by 1, the similarly potent 5,6-reduced analogue 19 (IC50 5 nM), and the 4, 16-dien-3-one 26 (IC50 3 nM) and by the less potent (IC50 13 nM) 3,5, 16-triene 25 is slow to occur but is enhanced by preincubation of the inhibitor with the enzyme. Inhibition following preincubation with these compounds is not lessened by dialysis for 24 h, implying irreversible binding to the enzyme. In contrast under these conditions the still potent (IC50 27 nM) 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) showed partial reversal after 5 h of dialysis and complete reversal of inhibition after 24 h. This behavior was also shown by the less potent 16,17-reduced 3-pyridyl compounds 3 and 24. Further, in contrast to the compounds (1, 19, 25, 26) with the 16,17-double bond, the inhibition of the enzymic reaction was not enhanced by preincubation either with 5 or with the 17beta-pyridyl analogues 3, 4, and 24 which also lack this structural feature. The results show that the 16,17-double bond is necessary for irreversible binding of these pyridyl steroids to cytochrome P45017alpha. However oxidation to an epoxide is probably not involved since epoxide 6 was only a moderately potent inhibitor (IC50 260 nM).     

Synthesis of androst-5-en-7-ones and androsta-3,5-dien-7-ones and their related 7-deoxy analogs as conformational and catalytic probes for the active site of aromatase

A series of androst-5-en-7-ones and androsta-3,5-dien-7-ones and their 7-deoxy derivatives, respectively, were synthesized and tested for their abilities to inhibit aromatase in human placental microsomes. All the steroids inhibited the enzyme in a competitive manner with Ki's ranging from 0.058 to 45 microM. The inhibitory activities of 17-oxo compounds were much more potent than those of the corresponding 17 beta-alcohols in each series. Steroids having an oxygen function (hydroxy or carbonyl) at C-19 were less potent inhibitors than the corresponding parent compounds having a 19-methyl group. 3,5-Dien-7-one 24 and its 19-hydroxy and 19-oxo derivatives (12 and 13) as well as 19-oxo-5-en-7-one 3 caused a time-dependent inactivation of aromatase only in the presence of NADPH in which the kinact values of 19-als 3 and 13 (0.143 and 0.189 min-1, respectively) were larger than those of the corresponding 19-methyl (23 and 24) and 19-hydroxy (1 and 12) steroids, respectively. 19-Nor-5-en-7-one 4 but not its 3,5-diene derivative 14 also inactivated the enzyme in a time-dependent manner. In contrast, 7-deoxy steroids 21 and 27, having a 19-methyl group, did not cause it. The inactivations were prevented by the substrate androstenedione, and no significant effects of L-cysteine on the inactivations were observed in each case. The results suggest that oxygenation at C-19 would be at least in part involved in the inactivations caused by the inhibitors 23 and 24. The conjugated enone structures should play a critical role in the inactivation sequences.     

Effects of ascorbic acid on in vitro steroidogenesis in guinea pigs

Guinea pig ovarian whole tissue homogenates were incubated with [14C]-labelled cholesterol, pregnenolone, and progesterone. Testicular homogenates were incubated with [14C]-progesterone. All incubations were carried out in the presence of 0, 0.5, 1.0, or 2.0 mM ascorbic acid. The conversion of cholesterol to pregnenolone was significantly decreased in testosterone and progesterone production. The addition of 0.5 mM ascorbic acid increased the conversion of pregnenolone to delta 4 steroids and decreased its conversion to delta 5 steroids, relative to the other ascorbic acid treatments. The conversion of progesterone to 17 A-hydroxyprogesterone was significantly decreased in the presence of 1.5 mM ascorbic acid over the O mM treatment. The data supports a general inhibitory effect of high ascorbic acid on the steroid hydroxylations, and a possible regulatory role of ascorbic acid on the conversion of pregnenolone to delta 4 and delta 5 steroids.     

3 beta-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5 alpha-androstane-3 beta,17 beta-diol and dehydroepiandrosterone as substrates

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3 beta-HSD/delta 5-->4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3 beta-HSD activity. For this purpose, we compared the efficiencies of a 3 beta-hydroxy-5-ene steroid (DHEA) and a 3 beta-hydroxy-5 alpha-reduced steroid (5 alpha-androstane-3 beta,17 beta-diol, 5 alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (Km) for 5 alpha-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent Km of placental 3 beta-HSD for 5 alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5 alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5 alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to Kms and rates of product formation was obtained by use of purified placental 3 beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone) as substrates: the Km of 3 beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein.min and, with epiandrosterone, 127 nmol/mg protein.min. With placental homogenate as the source of 3 beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [3H]5 alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [3H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5 alpha-A-diol (40 mumol/l).(ABSTRACT TRUNCATED AT 400 WORDS)     

6-Alkylandrosta-4,6-diene-3,17-diones and their 1,4,6-triene analogs as aromatase inhibitors. Structure-activity relationships

Two series of 6-alkylandrosta-4,6-diene-3,17-diones (5) and their 1,4,6-triene analogs 6 were synthesized as aromatase inhibitors to gain insight into the structure-activity relationship between varying the 6-n-alkyl substituents (C1-C7) and inhibitory activity. All of the steroids synthesized were extremely powerful competitive inhibitors of aromatase in human placental microsomes, with apparent Ki values for the 6-alkyl-4,6-diene steroids 5 ranging from 17 to 36 nM and with those for the 1,4,6-triene steroids 6 ranging from 2.5 to 58 nM. The 6-ethyl-1,4,6-triene compound 6b (Ki = 2.5 nM) was the most potent inhibitor among them. The 6-alkyl-1,4,6-triene steroids 6, except for the 6-methyl analog 6a, and higher affinity for aromatase than the natural substrate androstenedione (K(m) = 24 nM), and their inhibitory activities were more potent than the corresponding 4,6-diene steroids 5. In a series of the 4,6-diene steroids 5, compounds 5c-f with the n-alkyl chain substituents (C3 to C6) also had slightly higher affinity than androstenedione for dromatase. All of the 1,4,6-triene steroids 6 inactivated aromatase in a time-dependent manner, with k(inact) values ranging from 0.021 to 0.074 min-1; in contrast, the 4,6-diene analogs 5 did not. The inactivation was prevented by androstenedione, and no significant effect of L-cysteine on the inactivation was observed in each case. These results indicate that the length of the n-alkyl substituent at C-6 of androsta-1,4,6-triene-3,17-dione (6h), rather than its 4,6-diene analog 5h, plays a critical role in tight binding to the active site of aromatase. No significant correlation was observed between affinity for the enzyme and the inactivation ability of the 6-alkyl-1,4,6-trienes.     

Immunocytochemical localization and biological activity of hydroxysteroid sulfotransferase in the frog brain

Biosynthesis of the neuroactive steroids pregnenolone sulfate (delta5PS) and dehydroepiandrosterone sulfate (DHEAS) is catalyzed by the enzyme hydroxysteroid sulfotransferase (HST), which transfers the sulfonate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) on the 3-hydroxy site of steroids. Although high concentrations of delta5PS and DHEAS have been detected in the rat brain, the anatomical localization of HST in the CNS has never been determined. Using an antiserum against rat liver HST, we have investigated the distribution of HST-like immunoreactivity in the CNS of the frog Rana ridibunda. Two populations of HST-immunoreactive neurons were observed in the hypothalamus, and several bundles of positive nerve fibers were visualized in the telencephalon and diencephalon. Incubation of frog brain homogenates with [35S]PAPS and [3H]pregnenolone yielded the formation of several 3H,35S-labeled compounds, including delta5PS and testosterone sulfate. When [3H]dehydroepiandrosterone and [35S]PAPS were used as precursors, one of the 3H,35S-labeled metabolites coeluted with DHEAS. Neosynthesis of [3H]delta5PS and [3H]DHEAS was reduced significantly by 2,4-dichloro-6-nitrophenol, a specific inhibitor of sulfotransferases. The present study provides the first immunocytochemical mapping of HST in the brain. Our data also demonstrate for the first time that biosynthesis of the highly potent neuroactive steroids delta5PS and DHEAS occurs in the CNS of nonmammalian vertebrates.     

Ovarian enzymatic divergence in patients with polycystic ovary syndrome excreting urinary pregnanetriolone

When ovarian mitochondria from patients with polycystic ovary syndrome (POS) were incubated with [7-3H]17alpha-hydroxypregnenolone and [4-14C]-17alpha-hydroxyprogesterone, 11beta-hydroxylated metabolites were obtained. The mitochondria, prepared from pooled, frozen, polycystic ovarian tissue of 5 patients, converted [7-3H]17alpha-hydroxypregnenolone to 3beta, 11beta, 17alpha--trihydroxy-5-pregnen-20-one (yield 0.065%) and to 3beta, 17alpha-dihydroxy-5-pregnene-11,20-dione (0.22%), while [4-14C]17alpha-hydroxyprogesterone was converted to 21-deoxycortisol (0.1%). Incubation of mitochondria, prepared from 4 pooled samples of frozen, normal ovarian tissue, yielded no evidence of 11beta-hydroxylation of either of the substrates. Mitochondria obtained from fresh, polycystic ovarian tissue of a single patient with POS converted [7-3H]17alpha-hydroxypregnenolone to 3beta,17alpha-dihydroxy-5-pregnene-11,20-dione (2.1%) and [4-14C]17alpha-hydroxyprogesterone to 21-deoxycortisol (0.1%). When the same mitochondrial preparation was incubated simultaneously with [7-3H]17alpha-hydroxypregnenolone and [4-14C]11-deoxycortisol, it converted 17alpha-hydroxypregnenolone to 3beta,17alpha-dihydroxy-5-pregnene-11,20-dione (1.9%), but no 11beta-hydroxylated derivatives of 11-deoxycortisol were found. These results demonstrate that ovaries of patients with POS contain an 11beta-hydroxylase active towards C-21-deoxysteroids but inert to C-21-hydroxysteroids such as 11-deoxycortisol.     

Hepatic and extrahepatic dehydrogenation/isomerization of 5-cholestene-3 beta,7 alpha-diol: localization of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in pig tissues and subcellular fractions

Conversion of 5-cholestene-3 beta,7 alpha-diol (7 alpha-hydroxycholesterol) into 7 alpha-hydroxy-4-cholesten-3-one was studied with microsomes from different pig tissues and with liver subcellular fractions. Dehydrogenase/isomerase activity was efficient in microsomes from liver, ovary and lung, but less efficient in microsomes from adrenal gland and kidney. Microsomes from these tissues, with the exception of lung, were also active in dehydrogenation/isomerization of dehydroepiandrosterone and pregnenolone. Inhibition studies were carried out with trilostane, a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenases active in steroid hormone biosynthesis (C19/C21-dehydrogenases), and a monoclonal antibody raised against a purified hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase. The results showed that the C27-dehydrogenase activity in the tissues was not dependent on the C19/C21 dehydrogenases, but was dependent on the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase. Liver mitochondria, cytosol and peroxisomes lacked dehydrogenase/isomerase activity towards 7 alpha-hydroxycholesterol when microsomal contamination was taken into account. Immunoblotting experiments with monoclonal antibodies raised against the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase showed immunoreactivity only with protein in liver microsomes. Immunohistochemical studies showed localization of the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in the bile duct epithelium. It is concluded that 7 alpha-hydroxycholesterol is converted into 7 alpha-hydroxy-4-cholesten-3-one by the microsomal 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in liver and extrahepatic tissues.     

The orphan receptors COUP-TF and HNF-4 serve as accessory factors required for induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids

In the present study we examined the coupling of NADPH oxidation to substrate hydroxylation and the effects of steroids on this process in reconstituted P450scc and P450c11 systems. To determine the relative rates of substrate hydroxylation vs electron leakage we assayed both the steroid product and H2O2 in the same sample. For both P450 systems the rates of steroid product and superoxide formation increased as NADPH concentration was increased. However, P450c11 was found to be more leaky. The leakage from the P450scc system was not affected by pregnenolone, the product of cholesterol side chain cleavage. In contrast, corticosterone, the product of P450c11, increased the rate of futile NADPH oxidation by the P450c11 system. We also tested a series of steroids to analyze the stereospecificity of their effects. Relative to the control without steroid, both C-19 and C-21 steroids with 11 alpha-hydroxy groups (11 alpha-OH-testosterone and 11 alpha-OH-cortisol) decreased leakage, and those with 11 beta-OH groups (11 beta-OH-testosterone and cortisol) stimulated both NADPH oxidation and electron leakage as measured by H2O2 formation. The results revealed a correlation between the effects previously observed in living cells and in our reconstituted systems. These findings provide further evidence that mitochondrial P450 systems indeed function as a significant source of oxygen radicals in steroidogenic cells.     

The effects of epidermal growth factor, interleukin-1, and phenytoin, alone and in combination, on C19 steroid conversions in fibroblasts

The formation of the biologically active metabolite 5 alpha-dihydrotestosterone (DHT) from testosterone in response to phenytoin (Ph), interleukin-1 (IL-1), and epidermal growth factor (EGF) was investigated. The androgen DHT stimulates matrix synthesis in connective tissue and bone. Duplicate incubations were performed with confluent human gingival fibroblasts, 14C-testosterone, and optimal stimulatory concentrations of IL-1 (5 IU/ml), EGF (10 ng/ml), Ph (5 micrograms/ml), Ph + EGF, and Ph + IL-1 respectively for 24 hours in Eagle's MEM at 37 degrees C. The medium was then analyzed for radioactive metabolites. Similar incubations were performed with human gingival tissue using 14C-4-androstenedione as substrate in the presence or absence of EGF, Ph, and EGF + Ph. In the cell lines studied, EGF stimulated DHT and 4-androstenedione synthesis by 20% (n = 5; P < 0.01; Wilcoxon signed rank statistic for paired observations). IL-1 stimulated DHT and 4-androstenedione synthesis by 2-fold (n = 6; P < 0.01). Ph stimulated DHT and 4-androstenedione synthesis by 2-fold increases (n = 3; P < 0.01). Combinations of phenytoin and EGF stimulated DHT and 4-androstenedione synthesis by 33% and 37% greater than the effect of phenytoin alone (n = 3; P < 0.01). Combinations of Ph and IL-1 caused a 45% increase in the amount of DHT formed and a 66% increase in 4-androstenedione when compared to the effect of phenytoin alone (n = 3; P < 0.01). 14C-4-androstenedione was converted to DHT and testosterone by human gingival tissue. There were 2-fold, 4-fold, and 2.5-fold increases in DHT synthesis and 5-fold, 2-fold, and 6-fold increases in the formation of testosterone in response to EGF, Ph, and EGF + Ph respectively (n = 3; P < 0.01). EGF and IL-1 present in inflammatory exudate may have implications on phenytoin-induced overgrowth via the steroid metabolic pathway.     

A simple method for the assay of eight steroids in small volumes of plasma

A simple method is described for the simultaneous radioligand assay of four delta5-3beta-hydroxysteroids adjacent to one another on the biosynthetic pathway (pregnenolone [1], 17alpha-hydroxypregnenolone, dehydroepiandrosterone and 5-androsterone-3beta, 17beta-diol), and their four delta4-3keto products (progesterone, 17alpha-hydroxyprogesterone, 4-androstene-3, 17-dione and testosterone). Two plasma aliquots are extracted and fractionated each for four steroids and individual corrections are made for losses. For fractionation, maximum use is made of the high resolution and reproducibility of celite minicolumns, using propylene glycol as stationary phase, and a discontinuous gradient of ethyl acetate in iso-octane as mobile phase. The fractions are then assayed in the appropriate radioligand end-assay system. Each assay was finally validated by demonstrating coincidence of peaks of immuno- and radioactive steroid in extracts of female plasma. Results in pre-pubertal girls and women in the follicular phase of the menstrual cycle suggest that the major change in adrenal steroid production at puberty may be an increase in 17, 20-desmolase activity. There appears to be little reversal of this change in adrenal function after ovariectomy.     

Steroid 5alpha-reductase type 1 immunolocalized in the rat peripheral nervous system and paraganglia

Steroid 5alpha-reductase is an enzyme that converts a number of steroids with a C-4, 5 double bond and C-3 ketone to 5alpha-reduced metabolites. This enzyme has been suggested to play a role in brain development and myelination in the rat nervous system. In the present study, we examined the cellular and subcellular localization of the enzyme immunocytochemically in the rat peripheral nervous system and paraganglia using a polyclonal antibody against rat 5alpha-reductase type 1. Light and electron microscopical studies localized 5alpha-reductase in the Schwann cells of myelinated and unmyelinated nerve fibres, the satellite cells of the ganglia, the enteric glial cells and the supporting/sustentacular cells of the paraganglia. In the myelinated nerve fibres, immunoreactivity was observed in the outer loops, the nodes of Ranvier and the Schmidt-Lanterman incisures. Subcellularly, the immunoreactivity was localized in the cytoplasm of various glial cells. No immunoreactivity was observed in the myelin membrane, the axon or the neuronal perikaryon. These findings suggest that 5alpha-reductase is widely distributed in glial cells, and that, in addition to myelination, 5alpha-reduced steroids play a role in some glial functions in the peripheral nervous system.     

C19 steroids estrogenic activity in human breast cancer cell lines: importance of dehydroepiandrosterone sulfate at physiological plasma concentration

The estrogenic action of C19 steroids on breast cancer cells was measured by bioluminescence in stably transfected human breast cancer MCF-7 and T47D cell lines with a reporter gene that allows expression of the firefly luciferase enzyme under control of an estrogen regulatory element. The "estrogenic activity" of C19 steroids, such as dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), androst-5-en-3 beta,17 beta-diol, androst-4-en-3,17-dione, dihydrotestosterone, testosterone, and 5 alpha-androstan-3 beta,17 beta-diol was studied. This showed that DHEAS, at concentration observed in physiological conditions (10(-6) M), had a high "estrogen-like effect" in MCF-7 and T47D cell lines. Other C19 steroids, at physiological plasma concentration, alone or together did not have any significant effect on the luciferase activity. Moreover aminoglutethimide, an inhibitor of the aromatase enzyme, in the presence of C19 steroids, partially decreased the luciferase activity. These results suggest that MCF-7 and T47D cell lines could convert DHEAS to estrogen-like compounds by different enzymatic systems.     

The influence of prostaglandin F-2alpha on pregnenolone metabolism by the autotransplanted ovary of the ewe

Eight ewes each with an autotransplanted ovary received infusions of tritium-labelled pregnenolone (41 muCi/hr) for 8 hr into the artery supplying the ovary, together with prostaglandin (PG) F-2alpha (30 mug/hr) for 3 hr beginning 2 hr after the start of the pregnenolone infusion. All animals exhibited oestrus 2-3 days after the start of the experiment. During the PGF-2alpha infusion blood flow through the ovaries was increased by 13%, but subsequently returned to pre-infusion levels. Secretion rates of endogenous progesterone and 20alpha-hydroxypregn-4-en-3-one dropped rapidly 5 hr after the PGF-2alpha infusion had started from values of 250 mug/hr and 25 mug/hr to values below 60 mug/hr and 8 mug/hr, respectively. At this time the conversion of radioactive pregnenolone to progesterone was reduced by 50% of its initial value, but the secretion of endogenous pregnenolone and the formation of radioactive metabolites other than progesterone were not diminished. In 4 control animals, which received pregnenolone only, no changes in ovarian blood flow, steroid secretion rates, or in the conversion of labelled pregnenolone were observed. These results suggest a possible involvement of PGF-2alpha in the regulation of progesterone biosynthesis by an action on the 3beta-hydroxysteroid oxidoreductase-delta5(-4) isomerase enzyme system.     

Studies of the neuronal transdifferentiation process in cultured human pheochromocytoma cells: effects of steroids with differing functional groups on catecholamine content and cell morphology

The neuronal differentiation of adrenal pheochromocytoma cells from human subjects was studied in vitro for periods of up to 65 days. Changes with time in culture were observed in both intracellular catecholamine content (progressive decreases in epinephrine, norepinephrine, and dopamine, except for a possible transient early increase in the latter) and in morphology (increases in neurite outgrowth) of cells cultured in control medium; supplementation of cultures with nerve growth factor resulted in a substantial increase in neurite formation. The effects on these changes of the presence in the culture medium of various steroids were examined. The addition of 11-oxygenated steroids (aldosterone, corticosterone, cortisol, or dexamethasone) at 10(-5) M concentrations caused at least 2.5-fold increases in mean intracellular dopamine and norepinephrine levels; with dexamethasone, 9-10-fold increases were observed. Intracellular epinephrine content was also enhanced by 11,17-oxygenated steroids (dexamethasone and cortisol), but not by the other 11-oxygenated compounds studied. These two 11,17-oxygenated glucocorticoids also inhibited the morphologic changes seen with extended periods in culture, decreasing the outgrowth of neurite projections and causing cells to attain a vacuolated and granular appearance; the presence of dexamethasone strongly inhibited the morphologic changes induced by nerve growth factor. 11-Deoxy steroid intermediates (pregnenolone, 11-deoxycorticosterone, and 11-deoxycortisol) had little or no effect on catecholamine content or on morphology. Preliminary observations suggest that C-18 and C-19 sex steroid hormones (17 beta-estradiol and testosterone) may have morphologic effects opposite to those of the 11-oxygenated compounds, showing a slight stimulatory influence on the formation of neurite projections, but no significant effect on catecholamine content.     

Biosynthesis of pteridines. NMR studies on the reaction mechanisms of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, and sepiapterin reductase

GTP cyclohydrolase I catalyzes a ring expansion affording dihydroneopterin triphosphate from GTP. [1',2',3',4',5'-13C5, 2'-2H1]GTP was prepared enzymatically from [U-13C6]glucose for use as enzyme substrate. Multinuclear NMR experiments showed that the reaction catalyzed by GTP cyclohydrolase I involves the release of a proton from C-2' of GTP that is exchanged with the bulk solvent. Subsequently, a proton is reintroduced stereospecifically from the bulk solvent. This is in line with an Amadori rearrangement mechanism. The proton introduced from solvent occupies the pro-7R position in the enzyme product. The data also confirm that the reaction catalyzed by pyruvoyltetrahydropterin synthase results in the incorporation of solvent protons into positions C-6 and C-3' of the enzyme product. On the other hand, the reaction catalyzed by sepiapterin reductase does not involve any detectable incorporation of solvent protons into tetrahydrobiopterin.     

Kinetic analysis of successive reactions catalyzed by bovine cytochrome p450(17alpha,lyase)

Bovine P450(17alpha,lyase) containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin. The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum. In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone (Delta5-steroid) to 17alpha-hydroxypregnenolone and further to dehydroepiandrosterone. The enzyme catalyzed 17alpha-hydroxylation of progesterone (Delta4-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17alpha-hydroxyprogesterone to androstenedione. The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17alpha-hydroxypregnenolone that did not dissociate from the enzyme. The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17alpha-hydroxyprogesterone was 10 times faster than that of 17alpha-hydroxypregnenolone. The release of the intermediate metabolite of Delta4-steroid is sufficiently faster than the lyase reaction to prevent further reaction by the P450. It is concluded that the dissociation rates of the first hydroxylation metabolites regulate the successive reactions of P450(17alpha,lyase).