Showing papers on "Pregnenolone published in 1972"

The Effect of Ether Anaesthesia Stress on Cholesterol‐Side‐Chain Cleavage and Cytochrome P450 in Rat‐Adrenal Mitochondria

Abstract: Cholesterol side-chain cleavage has been studied in intact adrenal mitochondria from rats subjected to stress by ether anaesthesia as a means of raising the plasma adrenocorticotrophin levels. Control rats were either injected with cycloheximide or kept in a quiescent state. After initiation of cholesterol side-chain cleavage by addition of isocitrate, pregnenolone formation from endogenous cholesterol in intact rat-adrenal mitochondria follows a biphasic time-course of formation with an initial rapid phase lasting 3–5 min followed by a much slower rate of formation. Pregnenolone formation from [4-14C]cholesterol is linear if the tracer substrate is added to the mitochondria together with isocitrate. Preincubation of the mitochondria with [4-14C]cholesterol prior to addition of isocitrate results in a biphasic [4-14C]pregnenolone formation; the rate of the initial rapid phase depending on the duration of preincubation. The effect of stress is to increase 2–3 times the rate of pregnenolone formation in the initial phase compared to the rates observed in mitochondria from quiescent or cycloheximide-treated rats. Depletion of cholesterol from adrenal mitochondria follows a similar pattern to pregnenolone formation, but the initial cholesterol content of the mitochondria is not affected by the pretreatment. On the basis of these results it is suggested that only a fraction of the total mitochondrial cholesterol is readily available for cholesterol side-chain cleavage and that this pool is increased by stress. The cytochrome P450 type II spectral changes induced by addition of pregnenolone and isocitrate to the intact rat adrenal mitochondria have also been studied. The effect of stress was to increase pregnenolone-induced difference spectrum in adrenal mitochondrial two- to three-fold compared with cycloheximide-treated animals. Similar results were found for the isocitrate-induced type II difference spectrum. The spectral changes are interpreted to mean that stress causes an increase in the cholesterol complex of side-chain cleavage cytochrome P450. Metabolism of the remainder of the cholesterol in the mitochondria is limited by the rate of transport, or binding, to this reactive centre. It is proposed that the acute effect of stress, mediated by adrenocorticotrophin, is to increase the proportion of mitochondrial cholesterol in the readily available form perhaps by an increase in the rate of transport or binding of cholesterol to sites from which it can be readily metabolised.

Steroid 17, 20‐desmolase deficiency: a new cause of male pseudohermaphroditism

Abstract: SUMMARY In a child with male pseudohermaphroditism (ambiguous external genitalia, XY sex chromosomal constitution and normal adrenocortical function), incubations of testicular tissue with pregnenolone/progesterone, 17α-hydroxy-pregnenolone/17α-hydroxyprogesterone and androstenedione/dehydroepiandrosterone showed that testosterone could be formed from androstenedione and dehydroepiandrosterone only, but not from other substrates. In urine, testosterone did not increase after HCG, but small amounts of pregnanetriolone were found, which increased after HCG and ACTH. There was no DHA increment after ACTH. It is concluded that this patient, as well as a first cousin and a gonadectomized maternal 'aunt' with the same clinical and urinary steroid findings have testicular and adrenal steroid 17,20-desmolase deficiency, causing a defect of androgen biosynthesis, which has not previously been described. The heredity of this condition seems to be autosomal or X-chromosomal.

Mechanism of ACTH action

Abstract: ACTH regulates both the differentiated function (steroidogenesis) and the growth and replicative potential of the adrenal cortex. Cyclic AMP (cAMP) serves as the mediator of ACTH action within the cell. The binding of cAMP to its specific receptor activates a protein phosphokinase enzyme with resultant phosphorylation and altered function of several important substrates. The regulation of steroidogenesis requires protein synthesis and affects the conversion of cholesterol to pregnenolone, the rate-limiting step under hormonal control. In normal adrenal cortical tissue, ACTH stimulates DNA synthesis and replication, and in a functional adrenal tumor in tissue culture, ACTH inhibits DNA synthesis and replication. ACTH and cAMP thus appear to direct the adrenal cell toward the differentiated, maximally functional state.

Studies of the human testis. I. Biosynthetic pathways for androgen formation in human testicular tissue in vitro.

Abstract: Thirty-four incubations of minced testis tissue from five patients with prostatic carcinoma were performed for different periods of time with labeled precursors pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone (DHA), androst-4-ene-3,17-dione (Δ4-dione), and androst-5-ene-3β, 17β-diol (Δ5-diol) singly or in varying combinations. Testosterone (T) and the Δ5 and Δ4 intermediates which have been suggested as possible precursors of T were identified. Metabolism of pregnenolone or 17-hydroxypregnenolone yielded steadily increasing T with a bell shaped curve depicting the Δ5 intermediates, whereas the recovery of Δ4 intermediates remained low throughout the incubation period. From a comparison of the 3H/14C ratio of intermediates with the 3H/14C ratio of T following incubations with two labeled precursors, it appeared that T is formed more readily from pregnenolone than progesterone; from 17-hydroxypregnenolone than pregnenolone, progesterone or 17-hydroxyprogest...

A Pregnenolone Radioimmunoassay Utilizing a New Fractionation Technique for Sheep Antiserum

Abstract: Antibodies against pregnenolone were generated in a ewe immunized with a pregnenolone 3-bovine serum albumin conjugate. Upon fractionation of the resultant antiserum using QAE-Sephadex chromatography, a 7-8S highly negatively-charged antibody fraction was isolated possessing substantially increased sensitivity towards pregnenolone relative to the crude antiserum. On the basis of this novel fractionation procedure it was possible to devise a radioimmunoassay sensitive to 20 pg of pregnenolone. Normal serum concentrations in ng/ml obtained with this assay were: men (1.05 ± 0.19 se); women, follicular phase, (0.96 ± 0.16 se); women, luteal phase, (1.23 ± .25 se). These mean values were not significantly different from each other. It is speculated that our purified antibody preparation may represent a previously unidentified subclass of IgG in the sheep, and that the chromatographic method described may be generally useful for isolating highly sensitive antibodies against steroids or other hapten-BSA...

Ultrastructural studies and metabolic regulation of isolated adrenocortical carcinoma cells of rat.

Abstract: With a trypsin digestion method, an isolated adrenocortical carcinoma cell preparation has been achieved. The electron microscopic examination of tumor cells shows the sparsity of the lipid granules and mitochondria. The cristae of the mitochondria are tubular, in contrast to the vesicular cristae in the normal cells. Other ultrastructural features of these cells are presented. The adrenocortical carcinoma cells did not respond for corticosteroidogenesis to adrenocorticotropic hormone, cyclic adenosine 3′,5′-monophosphate, cyclic inosine 3′,5′-monophosphate, cyclic thymidine 3′,5′-monophosphate, cyclic uridine 3′,5′-monophosphate, cyclic guanosine 3′,5′-monophosphate, cyclic cytidine 3′,5′-monophosphate, and cyclic N 6-2′- O -dibutyryladenosine 3′,5′-monophosphate. Similarly, no response was observed to the varying concentrations of calcium ion. A 7-fold stimulation of corticosteroidogenesis was obtained when the adrenal carcinoma cells were incubated with pregnenolone and progesterone. Adrenocorticotropic hormone was found to inhibit the formation of corticosterone from pregnenolone. Epinephrine, glucagon, insulin, and proinsulin did not stimulate the formation of corticosterone.

The influence of an anti-steroidogenic drug (aminoglutethimide phosphate) on pregnancy maintenance.

Abstract: Aminoglutethimide phosphate (AGP) blocks the conversion of cholesterol to pregnenolone in steroidogenic tissue. Administration of this drug to pregnant animals should interfere with pregnenolone synthesis in the corpus luteum and reduce plasma progesterone (P) to levels insufficient to permit pregnancy to be established or maintained. A single ip dose of AGP (ISO mg/kg) caused abortion in only 25% of treated animals although plasma P was depressed 80% in 30 min. Recovery was slow but steady; return to normal plasma P values occurred by 48 hr. Lower doses (SO, 100 mg/kg) were ineffective and there were no abnormalities in surviving fetuses. Continued suppression of plasma P was achieved by increasing the dose. AGP, 100 and 150 mg/kg × 3, caused abortion in 100% of treated animals in 72 hr. Concomitant injection of 5 mg P/day to AGP-treated animals (intact or castrate) prevented pregnancy wastage but 5β-dihydro P, LH, HCG, Prolactin and F did not protect against AGP. Thus, AGP administered at proper dose, t...

Early adrenal response to ACTH; Plasma concentrations of pregnenolone, 17-hydroxypregnenolone, progesterone, and 17-hydroxyprogesterone.

Abstract: The peripheral venous plasma concentrations of pregnenolone (Δ5-P), 17-hydroxypregnenolone (Δ5-17-OHP), progesterone (P), 17-hydroxyprogesterone (17-OHP), and cortisol were measured in 5 normal men during a 3-hour infusion of ACTH The concentrations of Δ5-P, Δ5-17-OHP reached levels 4 to 6 times those of control samples, whereas progesterone, 17-OHP, and cortisol concentrations had only doubled We propose that ACTH causes preferential stimulation of the Δ5 pathway for cortisol biosynthesis

On the rate-limiting step in microsomal hydroxylation of steroids.

Abstract: The rate-limiting step in the microsomal, NADPH-dependent 24- and 26-hydroxylations of 5β-cholestane-3α,7α,12α-triol, 6β-hydroxylation of testosterone and 16α-hydroxylation of pregnenolone has been studied. Possible isotope effects were determined when the hydrogen in the substrate which is removed in the hydroxylation was substituted with deuterium; when the 4A- or 4B-hydrogen in NADPH was substituted with deuterium; and when the incubation was carried out in deuterated water. 1 6β-Hydroxylation of [6β-2H, 1,2-3H2]-plus [4-14C]labeled testosterone and 26-hydroxylation of 5β-[26-2H3]-cholestane-3α,7α,12α-triol did not involve significant isotope effect, whereas 16α-hydroxylation of [16α-2H, 3α-3H]-plus [4-14C]-labeled pregnenolone and 24-hydroxylation of 5β-[24-2H2]cholestane-3α,7α,12α-triol involved isotope effects with a KH/KD of about 3—4. The isotope effects were essentially unchanged when the reactions were partially inhibited with carbon monoxide. 2 None of the hydroxylations was significantly inhibited when NADPH was substituted with [4A-2H]- or [4B-2H]-labeled NADPH. 3 When the reactions were carried out in deuterated water, there was no significant inhibition of 24-hydroxylation of 5β-cholestane-3α,7α,12α-triol, whereas 16α-hydroxylation of pregnenolone was inhibited by 15%, 6β-hydroxylation of testosterone by 30% and 26-hydroxylation of 5β-cholestane-3α,7α,12α-triol by 60%. As splitting of a C-2H bond in the rate-limiting step is expected to decrease the rate of the overall reaction to less than one-half, it was concluded that the rate-limiting step in the 16α-hydroxylation of pregnenolone and 24-hydroxylation of 5β-cholestane-3α,7α,12α-triol is the splitting of the C-H bond in the substrate. Some other step is rate-limiting in the 6β-hydroxylation of testosterone and the 26-hydroxylation of 5β-cholestane-3α,7α,12α-triol. In the case of 26-hydroxylation of 5β-cholestane-3α,7α,12α-triol, water might participate in the rate-limiting step by hydration or protonolysis of the enzyme. It is suggested that a common feature of hydroxylation in which splitting of the C-H bond in the substrate is rate-limiting is a relatively low sensitivity towards carbon monoxide.

Studies on the biosynthesis in vivo and excretion of 16-unsaturated C19 steroids in the boar

Abstract: 1. In one experiment [7α-3H]pregnenolone was infused continuously for 12min into the left spermatic artery of a sexually mature boar and blood was collected during this period by continuous drainage from the spermatic vein. After infusion, the testis was removed and immediately cooled to −196°C. 2. From both the testicular tissue and the spermatic venous plasma, 3H-labelled 16-unsaturated C19 steroids were isolated and characterized and their radiochemical purity was established. 5α-Androst-16-en-3α- and 3β-ol occurred mainly as sulphate conjugates and to a lesser extent as free steroids. Only traces of these alcohols occurred as glucosiduronate conjugates. 5α-Androst-16-en-3-one was found in the free (ether-extractable) fraction. 3. The isotope concentration of each of the 3H-labelled 16-unsaturated C19 steroids in testicular tissue was different from that in spermatic venous plasma. 4. The ratios of tritiated 5α-androst-16-en-3α- and 3β-ol (free steroids) to their respective sulphate conjugates in the testicular tissue were less than the ratios of the same compounds in the spermatic venous plasma. The possibility that the sulphates are partially hydrolysed by testicular sulphatases before secretion is discussed. 5. In a second experiment, a continuous close-arterial infusion of [7α-3H]pregnenolone into the left testis was performed over a 200min period and all the urine that accumulated during the infusion was collected for analysis. 6. No 3H-labelled 16-unsaturated C19 steroids were detected in the urine as free steroids. Only a trace of 5α-androst-16-en-3α-ol was detected conjugated as glucosiduronate, whereas the corresponding 3β-alcohol occurred mainly as glucosiduronate and to a lesser extent as sulphate. 7. The absence of 5α-androst-16-en-3β-ol glucosiduronate in the spermatic venous blood and its presence in considerable amount in the urine may be attributed to hepatic glucuronyl transferase activity.

Histochemical Studies on Hydroxysteroid Dehydrogenase Activity of Fetal Pig Testes

Abstract: Histochemical studies were undertaken for the detection and localization of �‘-3fl-hydroxysteroid dehydrogenases (HSD) and 17t9-HSD in gonads of male pig fetuses of 1.529.0 cm crown-rump (C-R) length. The steroid substrates used for �5’-3$-HSD were pregnenolone, dehydroepiandrosterone, and androstenediol, and for 17fl-HSD the substrates were testosterone, estradiol-17$, and androstenediol. Considerable differences were found in both intensity and location of reactions within the gonads, depending on the age of fetus and the substrate used. A’-3�5-HSD activity, which might be related to steroid hormone formation at a very early stage in fetal development, was seen clearly in gonads from fetuses of 1.5-cm C-R length. An increase was observed with pregnenolone, dehydroepiandrosterone, and androstenediol at 2.5-3.0-cm C-R stages. The histochemical demonstration of &-3�-HSD activity in the 2.5-3.0-cm fetuses was remarkable in two ways. First, it appeared to be stronger before the early differentiation of the Leydig cells. Second, the HSD activity seemed to be confined to the forming sex cords, with no reaction for the added substrates in the mature and immature Leydig cells and in mesenchymal cells of the interstitium. Substrate utilization was low, or absent, in fetuses from midpregnancy for all steroids. HSD reactions were present again at the 19.0-cm C-R stage and activity increased toward the end of gestation for all substrates except testosterone. Estradiol was the best substrate in fetal testes in late pregnancy where its utilization was confined to the interstitial cells. These findings are discussed in relation to steroidogenesis in the fetal testes.

Steroid hormone metabolism in developing rats.

Abstract: 1 The metabolism of [4-14C]pregnenolone in male and female rats 9, 14, 21, 28, 35, 49, 63 and 85 days of age has been studied. Rats 9, 14 and 21 days of age excreted large amounts of monoand disulphurylated [4-14C]pregnenolone metabolites and no sexual differences were seen in the distribution of radioactive metabolites between free steroid, monosulphate and disulphate fractions. At the age of 28 days female rats excreted more steroid sulphates than male rats and in older rats this sexual difference became successively more apparent. 2 The excretion of endogenous steroid metabolites in faeces and urine from male and female rats 28–34, 35–41, 42–48 and 49–55 days of age has been investigated. Sexual differences were found already at an age of 28–34 days; at this time female rats excreted several metabolites identical to those previously described in faeces and urine from adult female rats. Female rats 42–48 and 49–55 days of age had a pattern of steroids identical to that in adult rats. Only small amounts of endogenous steroids were found in male rats; these compounds were not identified. 3 The capacity of adrenals and gonads from rats of various ages to oxidize pregnenolone into progesterone has been studied. The specific 3β-hydroxy-Δ5-steroid-oxidoreductase activity in adrenals from female and male rats as well as in ovaries increased about 5–10 fold from 7 to 56 days of age; the most pronounced increase occurred between 28 and 42 days. There was only a small increase in specific testicular 3β-hydroxy-Δ5-steroid-oxidoreductase activity with age. No significant sexual differences were found in the adrenal 3β-hydroxy-Δ5-steroid-oxidoreductase activity.

The intermediary role of 5-pregnene-3β,20β-diol in the biosynthesis of 16-unsaturated C19 steroids in boar testis

Abstract: 1. The possible involvement of 5-pregnene-3beta,20beta-diol in 16-unsaturated C(19) steroid biosynthesis has been investigated. 2. 5,16-Androstadien-3beta-ol (andien-beta) formation from [4-(14)C]pregnenolone (3beta-hydroxy-5-pregnen-20-one), 5-pregnene-3beta,20alpha-diol and 5-pregnene-3beta,20beta-diol was studied in homogenates of boar testis and the mean yields obtained were 25.6, 2.7 and 16.0% respectively. 3. Short-term kinetic studies with pregnenolone and 5-pregnene-3beta,20beta-diol separately and together suggested that the latter compound might be an intermediate in the biosynthesis of andien-beta. 4. In agreement with this interpretation, radioactive 5-pregnene-3beta,20beta-diol has been isolated during andien-beta biosynthesis from [4-(14)C]pregnenolone in the presence of NADPH, more radioactivity being trapped under limiting conditions of andien-beta formation with NADH present as cofactor. 5. Further, 5-pregnene-3beta,20beta-diol and andien-beta have been shown to inhibit the formation of the 16-unsaturated C(19) steroid from [4-(14)C]pregnenolone, the yield of radioactive 5-pregnene-3beta,20beta-diol increasing in the presence of added unlabelled andien-beta. 6. It is concluded that there may be two pathways leading to 16-unsaturated C(19) steroid formation from pregnenolone, one of these involving 5-pregnene-3beta,20beta-diol as an intermediate. Possible mechanisms are presented and discussed.

Conversion of Pregnenolone and Pregnenolone Sulfate to Other Steroid Sulfates by the Human Fetus Perfused at Midgestation

Abstract: Two previable human fetuses (16th and 19th weeks of gestation) were perfused in situ with a combination of 4-14C-pregnenolone and 3H-pregnenolone sulfate. Approximately 70% of the perfused radioactive material was recovered from the various fetal tissues and perfusates in both experiments. Only approximately 20–40% of the 3H- and 10–25% of the 14C-labeled material administered was recovered from the perfusate. Very little 3H-labeled material was detected in any of the extracts of the fetal tissues and perfusates as unconjugated (free) material. Approximately 30% of the 14C- and 0.07% of the 3H-labeled conjugated material recovered from the perfusate was isolated as dehydroepiandrosterone sulfate (DHAS). Double-labeled dihydropregnenolone sulfate, 16α-hydroxypregnenolone sulfate, DHAS and pregnenolone sulfate (PS) were identified in the liver, whereas from the adrenal only PS and DHAS were isolated. These results are interpreted as demonstrating that the midtrimester human fetus is capable of effe...