Showing papers on "Pregnenolone published in 1968"
TL;DR: These cells provide a viable and highly reproducible system for the in vitro study of ACTH action and a fluorometric assay has been developed that permits precise quantitation of steroid output within several minutes' incubation.
135 citations
TL;DR: It is concluded that cycloheximide blocks the action of ACTH by preventing the conversion of cholesterol to pregnenolone (3β-hydroxypregn-5-ene-20-one).
127 citations
TL;DR: In this article, gas chromatography-mass spectrometry and radio-gas chromatography were used to identify metabolites of [14C]corticosterone and [ 14C]pregnenolone in faeces and urine from germfree and conventional male rats.
Abstract: Gas chromatography-mass spectrometry and radio-gas chromatography were used to identify metabolites of [14C]corticosterone and [14C]pregnenolone in faeces and urine from germfree and conventional male rats. The metabolites were generally more polar than those in female rats and the main part of those which could be identified were found in the free steroid fraction. In the conventional male rats a number of pregnane-3,11,16,20,21-pentol isomers constituted the major corticosterone metabolites identified both in urine and in faeces. In addition 3α(and 3β),20β-dihydroxy-5α-pregnan-11-one and 5α-pregnane-3α(and 3β),11β,20β,21-tetrol were excreted in faeces. In the germfree male rats, minor amounts of some C19O3 and C21O3 steroids were excreted as monosulphates in faeces. However, 5α-pregnane-3α(and 3β),11β,21-tetrols were the predominant steroids in this fraction. These compounds also constituted the major part of steroids identified in the disulphate fraction, which also contained 3α(and 3β),11β,21-trihydroxy-5α-pregnan-20-one. All radioactive steroids excreted in the urine of germfree male rats were unconjugated. Although none of these compounds could be fully identified evidence was obtained for the presence of several C19O5, C21O4, C21O5, C21O6 and C21O7 steroids.
The findings are discussed in relation to previous information on the steroid metabolizing activities of the intestinal flora and sex differences in the metabolism of steroids in rats.
95 citations
TL;DR: Aminoglutethimide was found to be a competitive inhibitor of the enzymatic conversion of cholesterol to pregnenolone by the adrenals due wholly or in part to an inhibition of the 20-hydroxylation of cholesterol.
Abstract: SummaryAminoglutethimide was found to be a competitive inhibitor of the enzymatic conversion of cholesterol to pregnenolone by the adrenals. This effect is due wholly or in part to an inhibition of the 20-hydroxylation of cholesterol.
68 citations
TL;DR: A pathway is proposed for the biosynthesis of 3alpha- and 3beta-Hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.
Abstract: 1. The formation of androst-16-enes from [4-(14)C]progesterone has been investigated with long-term incubations and short-term kinetic studies. After 4hr., 1.7 and 10.3% respectively of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes were formed in boar testis minces, but much smaller yields were obtained in boar adrenal. Both tissues formed small quantities of androsta-4,16-dien-3-one. 2. The amounts of androst-4-ene-3,17-dione and testosterone isolated were small, suggesting that androst-16-ene formation may occur preferentially in the boar testis. 3. In the absence of tissue no radioactive androst-16-enes were formed. 4. Incubation of both [4-(14)C]pregnenolone and [7alpha-(3)H]progesterone resulted in 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes containing (3)H/(14)C ratios of near unity and confirmed that both C(21) steroids were precursors. A similar incubation with 17alpha-hydroxy[4-(14)C]-progesterone and [7alpha-(3)H]progesterone gave the same Delta(16)-alcohols, but they contained only (3)H, indicating that side-chain cleavage of pregnenolone and progesterone occurred before 17alpha-hydroxylation. 5. Dehydroepiandrosterone, testosterone, testosterone acetate and 16-dehydroprogesterone were not found to be precursors of Delta(16)-steroids. 6. A pathway is proposed for the biosynthesis of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.
54 citations
TL;DR: The action of adenosine 3′,5′-cyclic-monophosphoric acid (3′5′AMP) and other nucleotides (AMP, ADP, ATP) was tested and it was found that AMP markedly stimulates the conversion of cholesterol to pregnenolone and decreases the synthesis of progesterone.
Abstract: Cholesterol side chain cleavage reaction has been studied in ovaries of immature female rats pretreated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin. The cleavage enzymes were found to be in the mitochondria and to have the characteristics of a mixed function oxidase requiring NADPH and oxygen. Using [4-14C]cholesterol as substrate the main product obtained was progesterone while pregnenolone and 20α-hydroxypregn-4-en-3-one, were formed in smaller amounts. This shows that ovarian mitochondria contains a Δ5–3β-hydroxysteroid-dehydrogenase, a Δ 5–6/4–5 isomerase and a 20α-hydroxy steroid dehydrogenase. The proposed intermediates in cholesterol side chain cleavage (20α-hydroxy cholesterol and 20α, 22ɛ-dihydroxy cholesterol) were not detected.
Mitochondrial production of progesterone in the presence of NADPH was further stimulated by succinate while fumarate was without effect. In the presence of NADPH, NADH was found to be inhibitory while NAD+ gave rise to an increase in pregnenolone, the overall reaction being unaffected. Excess of NADPH causes an accumulation of pregnenolone and slightly decreases the overall reaction. The action of reduced glutathione (GSH) and ascorbic acid-like reducing agents of physiological occurrence has been studied. It was found that GSH in small amounts stimulates and in larger amounts inhibits the side chain cleavage reaction. Ascorbic acid in physiological amounts produced an inhibitory effect.
The action of adenosine 3′,5′-cyclic-monophosphoric acid (3′5′AMP) and other nucleotides (AMP, ADP, ATP) was tested. 3′5′AMP markedly stimulates the conversion of cholesterol to pregnenolone and decreases the synthesis of progesterone. The action of AMP shows some similarities with that of 3′5′AMP but the action of ADP and ATP was found to be different.
47 citations
TL;DR: In this article, the synthesis from smilagenin, progesterone, and pregnenolone of Δ14-3β-hydroxy-5β-etienic acid and some of its ester derivatives, as well as of β-anhydrodigitoxigenin acetate is described.
Abstract: The synthesis, from smilagenin, progesterone, and pregnenolone, of Δ14-3β-hydroxy-5β-etienic acid and some of its ester derivatives, as well as of β-anhydrodigitoxigenin acetate, is described. Sinc...
42 citations
TL;DR: Cholesterol side chain cleavage has been shown to be dependent on the electron carrier chain involving a cytochrome P-450 reductase system, flavoprotein and nonheme iron-protein of the ferredoxin type, and the oxygen-activating cyto Chrome oxidase system.
42 citations
TL;DR: Kinetic and spectral analyses of 20β-hydroxysteroid dehydrogenase indicate that this enzyme mediates an Ordered BiBi reaction with cofactor binding first and has a higher affinity for NADH than for NAD + .
38 citations
TL;DR: The results suggest that progesterone may be na intermediate in the biosynthesis of cardenolides from pregnenolone.
36 citations
TL;DR: In vitro cycloheximide treatment was less selective than inhibition of protein synthesis in vivo in that the synthesis of all steroid intermediates was suppressed below control levels and the hexose monophosphate shunt is less active at 36°C than at 32°C based on the in vitro yields of 14CO2 from glucose-1-14C and glucose-6- 14C.
Abstract: The normal temperature of the rat testis in a scrotal position is approximately 32°C. In vitro incubation of normal testicular tissue at various temperatures has demonstrated a 32°C optimum for the incorporation of lysine and glucose into protein. A thermal optimum of 32°C was also found for the conversion of cholesterol to components of both the Δ4- and Δ5- steroidogenic pathways except for pregnenolone synthesis which has a positive temperature co-efficient between 28°C and 36°C. The Δ5-pathway may be more sensitive than the Δ4-pathway. In vivo inhibition of protein synthesis with cycloheximide simulated a temperature effect. Compared to saline injected control, tissue from inhibitor treated animals converted less cholesterol to steroids when incubated at 32°C, except for pregnenolone. In vitro cycloheximide treatment was less selective than inhibition of protein synthesis in vivo in that the synthesis of all steroid intermediates was suppressed below control levels. In addition, the hexose monophosphate shunt is less active at 36°C than at 32°C based on the in vitro yields of 14CO2 from glucose-1-14C and glucose-6-14C.
On the basic of these experiments, a hypothesis was proposed which can serve as a foundation for further study concerning the mode of action of temperature in regulating testicular function. The hypothesis is that a thermal environment above or below 32°C in the rat interferes with some aspect of protein synthesis, thus reducing the level of one or more enzyme related to the steroidogenic pathways beyond pregnenolone.
TL;DR: Homogenates of human ovaries obtained from fetuses at 20 and 22 weeks of gestational age were incubated with sodium acetate-l-14C and small, but definite quantities of radioactive pregnenolone and progesterone were identified.
Abstract: Homogenates of human ovaries obtained from fetuses at 20 and 22 weeks of gestational age were incubated with sodium acetate-l-14C. The extracts were analyzed for sterols and steroids by the reverse isotope dilution technique. Purification and identification of the radioactive metabolites were achieved by paper and thin-layer chromatography, derivative formation and crystallization to constant specific activity. Evidence has been obtained for the formation of lanosterol and cholesterol. Small, but definite quantities of radioactive pregnenolone and progesterone were identified. No evidence was found for the formation of dehydroepiandrosterone, androstenedione, testosterone and estrogens.
TL;DR: Rat testis interstitial tissue, freed from tubule structures, and whole testis tissue were incubated with [7α- 3 H]pregnenolone and [4- 14 C]progesterone, and it was deduced that under the conditions chosen, the preferred pathway for the formation of testosterone from pregnanolone was pregnenol one…progestersterone…17α-hydroxyprogesTERone…androstenedione.
TL;DR: In this paper, the ovarian extract of Lacerta sicula was analyzed using thin-layer and gas-liquid chromatography, and it was found that pregnenolone, 17α-hydroxypregnenolones and 17α -hydroxyprogesterone were present in the ovarian tissue.
TL;DR: In mitochondria from beef adrenal cortex, which transform cholesterol-4-14C to pregnenolone, progesterone and more polar compounds, the presence of 3′,5′-cyclic AMP results in an accumulation of label in the pregnanolone pool with concomitant decrease in labeling of pools subsequent to pregnancies.
Abstract: 3′,5′-Cyclic AMP has been found to inhibit the transformation of pregnenolone to progesterone in rat adrenal homogenates. The inhibition of this transformation also takes place in microsome preparations and is reversed by NAD+. In mitochondria from beef adrenal cortex, which transform cholesterol-4-14C to pregnenolone, progesterone and more polar compounds, the presence of 3′,5′-cyclic AMP results in an accumulation of label in the pregnenolone pool with concomitant decrease in labeling of pools subsequent to pregnenolone. These effects of 3′,5′-cyclic AMP take place at concentrations higher than those reported to be present in adrenal tissue. (Endocrinology 82: 620, 1968)
TL;DR: The results are interpreted as demonstrating a direct pathway of neutral steroid sulfate metabolism in the Fetal adrenal gland under conditions in which hydrolysis of the sulfate ester is inhibited.
Abstract: Three human fetal adrenal homogenates, from fetuses of 12, 15 and 19 weeks of gestational age, were separately incubated with pregnenolone-7α-3H-sulfate under conditions in which hydrolysis of the sulfate ester is inhibited. Three metabolites (minimal estimates of conversion in parentheses) were identified: 17α-hydroxypregnenolone sulfate (8.5%), dehydroepiandrosterone sulfate (1.4%) and 16α-hydroxydehydroepiandrosterone sulfate (1.1%). The pattern of metabolism was identical in the 3 experiments. The results are interpreted as demonstrating a direct pathway of neutral steroid sulfate metabolism in the fetal adrenal gland.
TL;DR: In the foetus, the lack of 17-hydroxypregnenolone dehydrogenase seems to account for the relatively minute yields of3 H -corticosteroids from 3 H -pregnenolate and the pattern of 14 C -progesterone metabolism in thefoetus was the same as in the adult.
TL;DR: The results suggest, but do not prove conclusively, that LH and cyclic AMP may stimulate progestin synthesis in the rabbit ovary by mechanisms other than LH, and Adenosine-3′,5′-monophosphate failed to stimulate progesterone synthesis when added directly to mitochondria, under the experimental conditions employed.
Abstract: Mitochondria isolated from rabbit ovarian interstitial tissue metabolize both endogenous and exogenous cholesterol to progesterone in the presence of NADPH. Normally, little pregnenolone accumulated during a standard 60 min incubation, indicating an active mitochondrial 3β-hydroxysteroid dehydrogenase. Luteinizing hormone (LH) failed to stimulate this conversion under the mitochondrial incubation conditions employed, whether incubated directly with mitochondria, or preincubated with ovarian slices prior to isolation of mitochondria. In addition, addition, progesterone synthesis in mitochondria prepared from ovaries of rabbits administered LH in vivo did not differ from that of control values. Adenosine-3′,5′-monophosphate (cyclic AMP) failed to stimulate progestin synthesis when added directly to mitochondria, under the experimental conditions employed. These results suggest, but do not prove conclusively, that LH and cyclic AMP may stimulate progestin synthesis in the rabbit ovary by mechanisms other tha...
TL;DR: Estrogen formation appeared to be associated with the outer or cortical portion of the ovary in the single specimen examined this way, among the 5 ovarian specimens incubated.
Abstract: . The pathways of biosynthesis of androgens have been explored in vitro in the stromal tissue (interstitium) of normal human ovaries by simultaneous incubation of substrate amounts of δ5-pregnenolone-3H and progesterone- 14C. From the distribution of 3H and 14C in the 8–9 radioactive steroids examined, both the δ4-3-ketosteroid and the δ5-3β-hydroxysteroid pathways appear to be involved in the synthesis of δ4-androstene-3,17-dione, the most prominent steroid formed. Among the 5 ovarian specimens incubated, tissue from a pregnant woman and to a lesser extent, tissue from a patient in the 18th day of the menstrual cycle were the most active; tissues from a menopausal patient and from the follicular phase of the cycle were considerably less active. Estrogen formation appeared to be associated with the outer or cortical portion of the ovary in the single specimen examined this way.
TL;DR: The Koritz-Hall hypothesis for the regulation of steroidogenesis by ACTH was analyzed by testing whether it is compatible with experimental data on the dynamic relations between the concentration of ACTH in adrenal arterial blood and cortisol secretion rate.
Abstract: The Koritz-Hall hypothesis for the regulation of steroidogenesis by ACTH was analyzed by testing whether it is compatible with experimental data on the dynamic relations between the concentration of ACTH in adrenal arterial blood and cortisol secretion rate. The hypothesis is based on the inhibition of cholesterol hydroxylation by pregnenolone in a mitochondria extract, and proposes that ACTH acts to increase mitochondrial permeability to pregnenolone, thereby lowering intramitochondrial pregnenolone concentration and relieving the inhibition on steroidogenesis. These ideas were incorporated into a dynamic model of steroidogenesis. A search was made for a set of physically realistic kinetic parameters for the model which would permit it to simulate the dynamic responses of the perfused canine adrenal to ACTH. The in vitro data do not show pregnenolone to have a wide enough range of inhibitory action on cholesterol hydroxylation to be compatible with the range over which cortisol secretion rate varies in t...
Journal Article•
TL;DR: It is postulated that DHA-S is the principal precursor of the urinary metabolites isolated, and that its metabolism in this case led to a relatively small contribution of androsterone and etiocholanolone, in contrast to other steroids measured, particularly 16-hydroxy-DHA.
Abstract: In vivo and in vitro studies on a 6-year-old boy with a virilizing adrenal adenoma are reported Of particular interest was the modest elevation of urinary 17-ketosteroids The awareness of this observation is emphasized The main biochemical findings were: elevated dehydroepiandrosterone sulfate (DHA-S), normal testosterone and androstenedione levels in plasma, and increased urinary excretion of dehydroepiandrosterone (DHA), 16-hydroxy-DHA, androstenediol and androstenetriol, which were isolated from the sulfate fraction In vitro incubation of tumor tissue demonstrated its capacity to sulfurylate pregnenolone It is postulated that DHA-S is the principal precursor of the urinary metabolites isolated, and that its metabolism in this case (possibly a function of age) led to a relatively small contribution of androsterone and etiocholanolone, in contrast to other steroids measured, particularly 16-hydroxy-DHA
TL;DR: Results indicated that introduction of an oxygen atom from molecular oxygen at the 20α-position of cholesterol was essential for the side-chain cleavage, as the oxygen atom, in the form of the 20-oxo group of pregnenolone and also progesterone, originated from the oxygen of the hydroxy group introduced in the 20 α- position of cholesterol prior to the cleavage.
TL;DR: Evidence is presented indicating a new pathway of androgen biosynthesis exists parallel to the well established classical pathway, i.e. the direct conversion of cholesterol to dehydroepiandrosterone and 2-methylheptan-6-one by rat adrenal, ovarian and testicular tissue homogenates without intermediary formation of pregnenolone.
TL;DR: The findings suggest that the biosynthetic pathways in the aldosterone secreting tumor were directed both by the presence of 18-hydroxylase system and by the relative absence of alternate pathways leading to cortisol or androgen synthesis.
Abstract: The metabolism of pregnenolone-7α-3H has been studied in vitro in an aldosterone producing tumor and in the adrenal tissue adjacent to the tumor. The predominant products in the incubation of the tumor tissue were progesterone, and lesser amounts of deoxycorticosterone and aldosterone, with no cortisol. The adjacent adrenal tissue converted pregnenolone to androstenedione and cortisol, with no aldosterone. Although 17-oxygenated steroids were produced by both tissues, the tumor failed to convert 17-hydroxyprogesterone to 11-deoxycortisol or cortisol. Androstenedione was formed in the tumor incubation but was not converted to an 11-hydroxylated product. These findings suggest that the biosynthetic pathways in the aldosterone secreting tumor were directed both by the presence of 18-hydroxylase system and by the relative absence of alternate pathways leading to cortisol or androgen synthesis.
TL;DR: An enzyme system capable of cleaving the cholesterol side chain between carbon atoms 17 and 20 to yield dehydroepiandrosterone and 2-methylheptan-6-one has been identified in acetone powder obtained from calf testes, suggesting the existence of a new pathway of androgen biosynthesis.
TL;DR: Androgen producing activity of a transplanted tumor of mice, originating in the testicular interstitial cell, was biologically confirmed by the fact that the sex-accessory organs of the tumor-bearing mice did not show any decrease in weight even after removal of thetesticular glands.
Abstract: Androgen producing activity of a transplanted tumor of mice, originating in the testicular interstitial cell, was biologically confirmed by the fact that the sex-accessory organs of the tumor-bearing mice did not show any decrease in weight even after removal of the testicular glands. After slices of the testicular interstitial cell tumor which had been transplanted to the mice were incubated in vitro with relabeled pregnenolone and progesterone, respectively, as substrate, not only 17α-hydroxyprogesterone, androstenedione and testosterone, but also the corticosteroids such as deoxycorticosterone, 18-hydroxy-ll-deoxycorticosterone, corticosterone and 11-deoxycortisol were isolated as the metabolites and identified, suggesting that this tumor retained enzyme functions of producing adrenocorticoids as well as androgen. Furthermore, progesterone which was administered as a substrate was catabolized into 20α-and 20β-hydroxypregn-4-en-3-one and 6β-hydroxyprogesterone. The enzyme activities related to androgen ...
TL;DR: The results obtained seem to indicate that the biogenetic pathways leading from pregnenolone to testosterone are the same in fish as in mammals.
TL;DR: Feminizing testicular tissue was homogenized and incubated with a variety of labeled steroids as substrates and analysis of the products obtained suggest that in this tissue steroids were metabolized primarily by the Δ5-pregnenolone pathway rather than by theDelta4-progesterone pathway.
Abstract: Feminizing testicular tissue was homogenized and incubated with a variety of labeled steroids as substrates. Analysis of the products obtained suggest that in this tissue steroids were metabolized primarily by the Δ5-pregnenolone pathway rather than by the Δ4-progesterone pathway. No labeled estrogens could be isolated from these incubations. The 3β-hydroxysteroid dehydrogenase system in this tissue was much less active than that in the normal testis. Among the products of progesterone were androst-4-ene-3,17-dione, 20α-hydroxypregn-4-en-3-one, 17-hydroxyprogesterone, 16-hydroxyprogesterone, and testosterone. Incubation with pregnenolone gave dehydroepian-drosterone, androst-4-ene-3,17-dione, 17-hydroxyprogesterone, 17-hydroxypregnenolone, testosterone, progesterone, and androst-5-ene-3,17-diol. Incubations with dehydroepiandrosterone gave testosterone, androst-4-ene-3,17-dione and androst-5-ene-3,17-diol as products. Kinetic experiments with dehydroepiandrosterone as substrate suggest that dehyd...