Showing papers on "Pregnenolone published in 1974"

Testosterone Formation and Metabolism During Male Sexual Differentiation in the Human Embryo

Abstract: The formation by the gonads of [3H] testosterone from [7α-3H]pregnenolone and [1,2-3H] progesterone and the metabolism of [1,2-3H]testosterone by various tissues have been studied in 33 human fetuses that varied in age from phenotypically undifferentiated stages (1–3 cm crown-rump length) to sexually differentiated male and female embryos greater than 21 cm in length. In the first series of studies utilizing thinlayer and celite column chromatography for quantification of the metabolic products following incubation of the gonads with the C21 steroids, it was concluded that testosterone is the principal androgen formed by the fetal testis at the time of male sexual differentiation. The capacity for testosterone formation from these precursors was shown to rise from undetectable levels at 1–3 cm of development to maximal rates of about 150 pinoles/10 mg tissue/2 hr in the testes obtained from embryos of 7.1–9 cm crown-rump length, a sequence that correlates closely with the androgenmediated events ...

Spectral properties of rat adrenal-mitochondrial cytochrome P-450.

Abstract: Difference spectra produced by the binding of steroids to rat adrenal-mitochondrial cytochrome P-450 indicate distinct interactions with, respectively, pregnenolone, 11-deoxycorti costerone and 25-hydroxycholesterol. These so-called type I (deoxycorticosterone and hydroxy-cholesterol) or type II (pregnenolone) spectral changes have been compared in bovine adrenalcortex mitochondria and in adrenal mitochondria from rats. In the latter species the animals were cortex mitochondria and in adrenal mitochondria from rats. In the latter species the animals were subjected to one of three pretreatments; rats were either given an ether stress to increase the plasma concentration of adrenocorticotrophic hormone or were injected with the protein-synthesis inhibitor cycloheximide, or kept in a quiescent state. These experiments substantiate the theory that distinct P-450 cytochromes are involved in cholesterol side-chain cleavage (P-450SCC) and steroid 11β-hydroxylation (P-45011β). A pH-sensitive cholesterol complex of cytochrome P-450SCC was quantitated by means of the pregnenolone difference spectrum. Adrenal mitochondria from “stressed” rats had about twice as much of this cholesterol complex of cytochrome P-450SCC as adrenal mitochondria from rats kept quiescent or subjected to cycloheximide treatment. The pH-dependence of the cytochrome P-450SCC-cholesterol complexs was unaffected by the pretreatment of the animals. The type-I spectral change produced by 25-hydroxycholesterol was unaffected by stress or by changes in pH but was almost removed by sonication. The binding of 25-hydroxycholesterol to adrenal mitochondrial cytochrome P-450 was competitively inhibited by pregnenolone but was unaffected by deoxycorticosterone. The 25-hydroxycholesterol difference spectrum may detect a distinct form of cytochrome P-450SCC. Sonication of adrenal mitochondria from quiescent and cycloheximide-treated rats increased both the absorbance change produced by pregnenolone and the cholesterol side-chain cleavage activity to values approaching those found in mitochondria from stressed rats. The rate of side-chain cleavage of added 25-hydroxycholesterol was greater than that observed with endogenous cholesterol and this rate was unaffected by prior stress to the rats. It is suggested that the low cholesterol side-chain cleavage activity in adrenal mitochondria from quiescent rats and rats given cycloheximide may be due to the existence within the mitochondrial membrane of specific “restraints” upon the interaction between a certain fraction or pool of cholesterol and cytochrome P-450SCC. The availability (or restraint) of this choleterol pool may be altered by the action of adrenocorticotrophic hormone, and mediated by a labile protein factor.

The Stoichiometry of the Conversion of Cholesterol and Hydroxycholesterols to Pregnenolone (3β-Hydroxypregn-5-en-20-one) Catalysed by Adrenal Cytochrome P-450

Abstract: The stoichiometry of the side-chain cleavage of cholesterol (cholest-5-en-3β-ol), 20α-hydroxycholesterol (cholest-5-ene-3β,20α-diol), and 20α,22-dihydroxycholesterol (cholest-5-ene-3β,20α,22-triol) has been examined with respect to TPNH, oxygen and H+. Side-chain cleavage of cholesterol can be described by the following equation: Cholesterol + 3TPNH + 3H+ + 3O2 → Pregnenolone (3β-hydroxypregn-5-ene-20-one) + isocapraldehyde + 3 TPN+ 4H2O Stoichiometry of 20 α-hydroxycholesterol is 2TPNH and 2O2 per mole of cleavage and with 20α, 22-dihydroxycholesterol the values are 1:1:1. In addition 1 mole of H+ is consumed per mole of TPNH oxidized in each of these reactions. These observations are in keeping with a mechanism previously proposed in the literature, namely: Cholesterol → 20α-OH-Cholesterol → 20α, 22-di-OH-Cholesterol → pregnenolone Discordant observations are reviewed and it is concluded that insufficient data are available to sustain objections to this pathway.

The in vitro and in vivo uptake and metabolism of steroids in human adipose tissue.

Abstract: The uptake and metabolism of various steroids in adipose tissue have been studied using both in vitro and in vivo techniques. The in vitro uptake of progesterone and testosterone (60%) was found to be much higher than the uptake of cortisol and estriol (13%). In vivo, the uptake of testosterone, androstenedione, estrone and estradiol by the total adipose mass was much greater in obese patients than in non obese individuals. Estrone and estradiol were found to be interconvertible following incubation with human adipose tissue slices. In vitro, progesterone was metabolized to a variety of compounds with 20α-dihydroprogesterone4 being the major metabolite. No conversion of cholesterol or cholesterol sulfate to pregnenolone or pregnenolone sulfate could be demonstrated in the brown adipose tissue of a newborn infant. In human adult subcutaneous adipose tissue, there was no in vitro conversion of progesterone to testosterone and no aromatization of androstenedione or testosterone labeled with 14C was ...

The effect of inhibitors of protein synthesis on cholesterol side-chain cleavage in the mitochondria of luteinized rat ovaries.

Abstract: 1 Conversion of cholesterol to pregnenolone, termed cholesterol side-chain cleavage, has been studied in isolated mitochondria from luteinized rat ovaries. 2 Cholesterol in these mitochondria in the presence of cyanoketone is converted to one product pregnenolone on addition of a suitable electron donor. The percentage conversion of total mitochondrial cholesterol to pregnenolone is comparable to that measured by the percentage conversion of [4-14C]cholesterol to [4-14C]pregnenolone when the label has been preincubated at 29°C for 10 min with the mitochondria. 3 In mitochondrial incubations the depletion of endogenous cholesterol follows the production of pregnenolone. The initial rate of production of pregnenolone (0.8 nmol × mg protein−1× min−1) was maintained in incubations for 5–6 min. Addition of free cholesterol to incubations allowed the production of pregnenolone to continue at the initial rate for at least 30 min. 4 Treatment of rats with cycloheximide 20 min prior to killing, caused a 58% inhibition of luteal mitochondrial cholesterol side-chain cleavage and a small but significant rise in the cholesterol content of isolated mitochondria. The cycloheximide treatment did not affect the total luteal mitochondrial cytochrome P-450 but caused a 56% decrease in the inverted type I pregnenolone-binding spectrum, suggesting cholesterol binding to cytochrome P-450 had been reduced. 5 Rats treated with chloramphenicol 3 and 10 h before killing exhibited no differences in total luteal mitochondrial cytochrome P-450 or mitochondrial cholesterol metabolism when contrasted with saline-injected animals. 6 Puromycin was shown to bind to the rat luteal mitochondrial cytochrome P-450 to produce a type I1 difference spectrum and to inhibit luteal mitochondrial cholesterol side-chain cleavage in vitro. 7 The results are discussed in terms of a protein which is rapidly turning over and which may be synthesised extra-mitochondrially and in some way affect the binding of cholesterol to luteal mitochondrial cytochrome P-450.

Measurement of the dynamics of stimulation and inhibition of steroidogenesis in isolated rat adrenal cells by using column perfusion

Abstract: Isolated adrenal cells were perfused in a small column by using Bio-Gel polyacrylamide beads as an inert supporting matrix, and the time-course of the response to various stimuli was observed by measuring fluorogenic 11-hydroxycorticosteroids in the effluent. A small but significant response was observed 1 min after stimulation with physiological concentrations of ACTH (adrenocorticotrophin), but the response did not start to build up rapidly for 3–4min and eventually reached a plateau after 9–10min. A similar pattern of events was observed for the decay of the steroid output on removal of ACTH. ACTH analogues, including one with a long duration of action in vivo, were found to produce responses with similar kinetics. However, cyclic AMP caused a more rapid increase in steroidogenesis and its effects were more short-lived after withdrawal. If, as present evidence suggests, cyclic AMP is produced rapidly after ACTH stimulation the delayed build-up of the steroidogenic response to ACTH would indicate that cyclic AMP may not be the intracellular mediator. When inhibitors were applied during ACTH stimulation, aminoglutethimide, which blocks mitochondrial conversion of cholesterol into pregnenolone (3β-hydroxypregn-5-en-20-one), caused a rapid fall in steroid output (1 min), whereas cycloheximide took longer to achieve its full effect. Nevertheless, the response had fallen by 50% in 2 min, indicating a much shorter half-life than that previously reported for the labile protein implicated in steroidogenesis. In addition the rapid response to cyclic AMP makes it unlikely that steroid production is induced as a result of initiation of protein synthesis. This suggests that the labile protein plays an obligatory but permissive role in the development of the response. Column perfusion has proved to be a simple technique which can readily yield accurate data on responses of cells to stimulants and inhibitors.

Androgen formation from pregnenolone sulfate by the human fetal ovary.

Abstract: To assess the capacity of the human fetal ovary to utilize pregnenolone sulfate as a precursor of free steroids, homogenates of fetal ovaries from 3 fetuses (88–124 days) were incubated with 3H-pregnenolone sulfate. The rate of cleavage of pregnenolone sulfate in ovaries was compared to that in adrenals and liver. The rate of cleavage was between 3 and 7 times greater in ovaries than in adrenals and liver from the same fetus. No difference in the rate of cleavage was observed in the ovaries from fetuses of different ages. The various free steroid products from the incubations of the ovaries were isolated, identified and the amounts produced in 2 hr expressed as pmoles/mg protein incubated. Pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone and androstenedione were identified as products in each incubation of fetal ovarian tissue. Although progesterone, testosterone, estradiol and estrone were sought extensively, they were not found as products of pregnenolone sulfate metabolism.

Biochemical and clinical observations in a pregnancy with placental sulphatase and other enzyme deficiencies

Abstract: SUMMARY The oestrogen excretion of a pregnant woman, subsequently shown to have placental sulphatase deficiency, was only 1-2 mg/24 hr, whereas pregnanediol and 17-oxogenic steroid excretion were within the normal ranges. Plasma concentrations of oestradiol-17β, progesterone, 17α-hydroxyprogesterone, 11β-hydroxycortico-steroids and corticosteroid-binding globulin were subnormal. Placental sulphatase activity in vitro towards the sulphates of DHA, pregnenolone and oestrone was negligible, although extracts of a normal placenta showed high activity under identical conditions. The activities of placental enzymes concerned with metabolism of non-conjugated steroids (3β-hydroxysteroid dehydrogenase-isomerase for DHA and for pregnenolone, aromatase complex and 17β-hydroxysteroid for oestradiol-17β) were less than those of a normal placenta but were not diminished as severely as sulphatase activity. Satisfactory uterine contractions were achieved during infusion of prostaglandin E2 or Syntocinon but the cervix failed to dilate. A healthy male infant (3330 g) was delivered at term by Caesarean section. From the evidence obtained during the study of this patient it appears that (i) the presence of appropriate non-conjugated substrates may be required for the induction of placental enzymes concerned with the metabolism of free steroids; (ii) measurement of maternal 17-oxogenic steroid excretion may help to distinguish patients with placental sulphatase deficiency from those whose fetus has adrenal hypoplasia; (iii) further investigations, possibly involving steroid replacement therapy, are required to identify which, if any, of the alterations in plasma steroid concentrations are associated with the failure to achieve cervical dilatation.

Effect of calcium ions on steroid-binding spectra and pregnenolone formation in rat-adrenal mitochondria.

Abstract: The effect of calcium ions on steroid-induced difference spectra of cytochrome P-450, and on pregnenolone formation, has been studied in rat adrenal mitochondria. In the presence of phosphate, addition of calcium chloride to suspensions of these mitochondria resulted in an increase in the inverted type-I difference spectrum induced by pregnenolone or methylandrostenediol, and a disappearance of the type I spectrum induced by 25-hydroxycholesterol. The difference in the inverted type I spectrum between mitochondria from ether-stressed animals and those given cycloheximide was abolished. Cholesterol side-chain cleavage was increased, providing an NADPH generator was used as source of reducing equivalents. The difference in side-chain cleavage activity between mitochondria from ether-stressed animals and those given cycloheximide, was maintained. In the absence of phosphate, addition of calcium chloride again resulted in a loss of the type I difference spectrum induced by 25-hydroxycholesterol, but this time there was almost no change in the inverted type I spectrum induced by pregnenolone in mitochondria from rats subjected to ether anaesthesia, and the change in cycloheximide-treated rats was only about 50%, of the change in the 25-hydroxycholesterol-induced difference spectrum. These observations are interpreted in terms of interconversions of the various forms of cytochrome P-450 associated with side-chain cleavage proposed to exist in intact adrenal mitochondria. Factors such as bovine serum albumin and EDTA, which increase respiratory control in these mitochondria, also stimulate cholesterol side-chain cleavage activity, as do magnesium ions, but without altering the magnitude of the steroid-binding spectra.

Effects of Metyrapone on Pregnenolone Biosynthesis and on Cholesterol-Cytochrome P-450 Interaction in the Adrenal1

Abstract: The effects of the steroid 11²- hydroxylase inhibitor metyrapone on the conversion of cholesterol-4-14C to labeled pregnenolone have been examined in time-course incubation studies using a preparation of rat adrenal mitochon-dria. Metyrapone inhibited the cholesterol →pregnenolone reaction in a dose-related manner, the effect varying from 17 to 78‰ with 0.025 to 1.0 mM inhibitor concentrations, respectively. No effect of this agent on the conversion of 20α-hydroxycholesterol to pregnenolone was apparent. Studies of the effects of metyrapone on the changes in difference spectra resulting from cholesterol-cytochrome P-450 interaction with a preparation of acetone-extracted submitochondrial particles of bovine adrenal cortex demonstrated that metyrapone competitively inhibited the binding of cholesterol to the hemoprotein. It thus appears that inhibition by metyrapone of the two major enzyme systems of adrenal mitochondria has similar characteristics. (Endocrinology 94: 1451, 1974)

Cholesterol side-chain cleavage activity and levels of high-spin cytochrome P-450 in adrenal-regeneration hypertension (ARH).

Abstract: Whatever steroids are involved in the pathogenesis of adrenal-regeneration hypertension (ARH), cholesterol side-chain cleavage (SCC) activity is a prerequisite for their synthesis. Both cholesterol SCC and 11β-hydroxylase activities were compared in our experiments. By combining the use of cyanoketone, a 3β-ol dehydrogenase inhibitor, with a sensitive radioimmunoassayfor pregnenolone, we were able to measure pregnenolone formed from endogenous cholesterol in adrenal mitochondrial incubations. The results from a quiescent sacrifice indicated greater SCC activity in the ARH group (0.75 nmoles prognenolone⁄minute⁄mg protein) than in controls (0.45 nmoles pregnenolone⁄minute⁄mg protein). This was related to the significantly greater (p < 0.001) amount of SCC cytochrome P-450 in the high spin state observed in adrenal mitochondria from the ARHgroup (determined by the pregnenolone-induced modified Type II spectral, change). After ether stress, both control and ARH groups had comparableamounts of high-spin SCC c...

Plasma 17-OH-Pregnenolone in Normal Subjects

Abstract: A radioimmunoassay for 17-OH-pregnenolone was developed and applied to the study of this steroid in human plasma. Steroids that cross-react with the antiserum could be readily separated from 17-OH-pregnenolone; therefore the assay was highly specific. In normal subjects plasma 17-OH-pregnenolone is mainly of adrenal origin. It was largely suppressible with dexamethasone, readily responsive to ACTH and followed a diurnal rhythm similar to that of plasma fluorogenic corticosteroids with which there was a significant correlation (p < 0.001). Testicular secretion of 17-OH-pregnenolone could be inferred from the observation that while dexamethasone suppressed plasma concentrations incompletely combined treatments with dexamethasone and methyltestosterone caused the steroid to disappear completely. In contrast, women receiving dexamethasone alone had similar levels of 17-OH-pregnenolone to women receiving dexamethasone plus estrogen. Ovarian secretion of 17-OH-pregnenolone was suggested by the finding ...

Testicular Endocrine Function in a Pubertal Boy with 3β-Hydroxysteroid Dehydrogenase Deficiency

Abstract: Plasma pregnenolone, progesterone, 17-hydroxyprogesterone, testosterone and 5α-dihydrotestosterone were determined in the peripheral and spermatic venous blood of a pubertal boy with 3β-hydroxysteroid dehydrogenase deficiency in order to evaluate his testicular endocrine function The peripheral testosterone concentration was 028 ng/ml, and rose only to 060 ng/ml in response to human chorionic gonadotropin (HCG) administration The spermatic venous testosterone concentration, however, was about 15 times greater than in peripheral blood, which is a clear indication of testicular testosterone secretion in this patient In addition, testicular secretion of 17-hydroxyprogesterone and 5α-dihydrotestosterone was also evident, thus, the testis has some 3β-hydroxysteroid dehydrogenase activity In addition to the weak response to HCG administration, a very high spermatic venous pregnenolone concentration as compared to normal males and a slightly elevated plasma luteinizing hormone (LH) concentration s

Effect of 5 alpha-dihydroprogesterone, pregn-5-ene-3 20-dione, pregnenolone and related progestins on ovulation in PMSG-treated immature rats.

Abstract: Work on pregnant mares serum gonadotropin PSMG treated rats and induction of ovulation with progesterone is reviewed. The progesterone pregnenolone and related metabolites were examined for the facilitative action in 22 day old rats treated with a nonovulatory dose of PMSG. Progesterone (.5 mg) induced ovulation in all rats treated with 12 iu of PMSG. 1.5 mg of pregnenolone given at 10 hours on Day 24 produced ovulation in 80% of the animals. Neither 17 alpha-hydroxy pregnenolone nor 17 alpha-hydroxyprogesterone had any facilitative effect. 1.5 and 2 mg of 5 alpha-dihydrogesterone caused ovulation in all treated animals. 3 alpha-hydroxy-5 alpha-pregnan 20-one had no effect. The action of pregn-5-ene-3 20-dione was equal to that of progesterone in doses of .25 mg or higher.

Metabolic regulation and adenyl cyclase activity of adrenocortical carcinoma cultured cells.

Abstract: Summary In order that the mechanisms involved in the altered control of steroidogenesis by adrenocorticotropic hormone (ACTH) in the adrenal tumor 494 might be elucidated, various metabolic properties of this tumor maintained in tissue culture have been studied. It has been shown in the cultured cells that: ( a ) neither ACTH nor adenosine cyclic 3′,5′-monophosphate stimulates steroidogenesis; ( b ) the normal adrenal biosynthetic pathway from pregnenolone to corticosterone is intact; ( c ) the biosynthesis of deoxycorticosterone to corticosterone from pregnenolone is at a reduced level as compared to the normal adrenal cell, indicating a reduced 11β-hydroxylase activity; and ( d ) (20 S )-20-hydroxycholesterol can be converted to corticosterone, thus indicating a defect in the tumor system normally stimulated by ACTH to convert cholesterol to (20 S )-20-hydroxycholesterol. The adenylate cyclase activity of the cultured cells has been evaluated with an improved methodology. In confirmation of previous studies by others, it is herein shown that high concentrations of ACTH (460 milliunits) and epinephrine (10 -5 m) stimulate the activity of the whole homogenate 2.9-fold and 4.1-fold, respectively, whereas 20 mm fluoride has no effect on its activity. In addition it has been demonstrated that a partially purified particulate fraction of the cultured tumor cells containing plasma membrane was stimulated only 32% by ACTH, whereas the stimulation by epinephrine was 108% suggesting more sensitivity of the membrane receptor to epinephrine than to ACTH. Similarly, epinephrine significantly stimulated the adenylate cyclase activity of isolated nuclei, but no stimulation by ACTH was obtained. It is, therefore, proposed that distinct changes in tumor receptors have taken place. Such modified tumor receptors are at least as sensitive to epinephrine as they are to ACTH.

Serum steroid levels following a large intravenous dose of a steroid sulfate precursor during the second trimester of human pregnancy. II. Pregnenolone sulfate.

Abstract: Serum levels of dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone (DHEA), 16α-hydroxydehydroepiandrosterone (16α-OH DHEA), estrone (E1), estradiol-17β (E2), and estriol (E3) were measured by radioimmunoassay after intravenous infusion of a large dose of unlabeled DHEA-S to 11 healthy women during midtrimester pregnancy. Subjects were divided into three groups. The first group consisted of 5 subjects with a live fetus and intact placenta in whom the DHEA-S infusion was carried out before the pregnancy was disturbed. The second group consisted of 3 subjects with a dead fetus and an intact placenta in whom the DHEA-S infusion was performed after hypertonic saline amnioinfusion. The third group consisted of 3 subjects in whom the DHEA-S infusion was performed following passage of the fetus and placenta. DHEA-S levels fell exponentially following infusion. The mean DHEA-S half-life for all subjects with an intact placenta regardless of fetal viability was 3.86 ± 0.52 hr. With removal of ...

The Influence of FSH and FSH + LH on Steroidogenic Enzymes in the Immature Mouse Ovary

Abstract: Most evidence indicates that FSH alone does not stimulate estrogen biosynthesis in immature ovaries in spite of increased follicular growth. We studied the influence of FSH free of biological LH activity compared with the same dose of FSH supplemented by LH on the intermediate steps of steroid biosynthesis in the immature mouse ovary. The steroidogenic profile in the immature ovary revealed very active hydroxysteroid dehydrogenases, moderately active Δ4-reductases and low activities of mixed function oxidases. The Δ4-rather than the Δ5-pathway was shown to be the predominant route of androgen formation from pregnenolone, and Δ4-androstenedione was preferred to testosterone as androgen precursor of aromatization to estrogens. FSH acted primarily as a stimulator of general growth: it largely induced increases in enzyme activities in proportion to the general increase in protein and the protein per mg tissue did not increase. FSH † LH caused marked differential increases in cholesterol side-chain splitting, ...

Pregnenolone in man: plasma levels in states of normal and abnormal steroidogenesis.

Abstract: Using a recently described radioimmunoassay, plasma pregnenolone was measured in subjects with normal and abnormal steroidogenesis. Plasma levels were found to be higher in premenopausal women than men and higher in men than in postmenopausal women. Adrenal suppression decreased plasma pregnenolone in normal subjects and gonadal suppression decreased plasma pregnenolone in premenopausal women and men but not in postmenopausal women. Adrenal plus gonadal suppression reduced plasma pregnenolone levels below the sensitivity of the assay i.e., <0.1 ng/ml. Plasma pregnenolone was similar during the follicular and luteal phases of 4 menstrual cycles. Plasma pregnenolone rose in response to the administration of large doses of ACTH to 5 normal subjects. A diurnal rhythm of plasma pregnenolone was observed only in postmenopausal women and in premenopausal women taking estrogen. This suggests that pregnenolone of ovarian origin may have obscured a diurnal rhythm of adrenal pregnenolone in premenopausal wo...