Showing papers on "Pregnenolone published in 1975"

Follicle-stimulating hormone stimulates estradiol-17beta synthesis in cultured Sertoli cells.

Abstract: Sertoli cells isolated from testes of 20-day-old rats and maintained in primary culture synthesized estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol] (measured by specific radioimmunoassay) when testosterone (17beta-hydroxy-4-androsten-3-one) 0.5 muM, was added to the culture medium. No detectable estradiol synthesis occurred when cells were incubated in medium containing pregnenolone (3beta-hydroxypregn-5-en-20-one), 0.5 muM, or containing no added steroid substrate. Follicle-stimulating hormone (FSH) (NIH-FSH-S10, 5 mug/ml) stimulated estradiol synthesis 12- to 80-fold when added to medium containing testosterone, but not when added to medium containing pregnenolone or no exogenous steroid substrate. A highly purified FSH preparation, with FSH potency 50 times that of the NIH-FSH, caused a similar stimulation at a concentration of 0.25 mug/ml of medium, whereas luteinizing hormone (NIH-LH-S18, 5 MUG/ML) Caused only marginal stimulation. Dibutyryl-adenosine 3':5' cyclic monophosphate, 0.1 mM, caused a 30-fold increase in estradiol synthesis by Sertoli cells cultured in medium containing testosterone. These studies provide direct demonstration of estradiol-17beta production by Seroli cells from normal animals, and offer evidence that the synthesis of this steroid is regulated at the level of the aromatizing enzyme system by FSH and adenosine 3':5' cyclic monophosphate.

Mechanism of action of glucocorticoids in induction of ovine parturition: effect on placental steroid metabolism

Abstract: Maternal plasma progesterone levels in sheep may fall dramatically druing the last few days of gestation and following the administration of glucocorticoids to the foetus. To investigate the mechanism of the fall, metabolism of [3H] progesterone in vitro by ovin placental tissue was studied in five ewes before and after intra-foetal administration of dexamethasone in a dosage sufficient to induce parturition, and in one ewe after the spontaneous onset of labour at 143 days of gestation. Manual separation of maternal and foetal placental tissues showed that, in 11 out of 12 cases, the foetal and not the maternal placenta produced progesterone from pregnenolone in vitro. Total activities of cholesterol side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase in the foetal placenta were not influenced by intra-foetal dexamethasone. Befre administration of dexamethasone, homogenates of foetal placenta converted [3H] progesterone to 20alpha-hydroxy [3H]pregn-4-en-3-one in the presence of NADPH. Within 12 h of administration of dexamethasone, and after the natural onset of labour at 143 days, large amounts of 17alpha, 20alpha-dihydroxyI1pregn-4-en-3-one were formed form [3H]progesterone. Intra-foetal dexamethasone treatment also induced the formation of 17alpha, 20alpha-dihydroxy[3H]pregn-4-en-3-one by miced foetal placental tissue incubated with [3H]pregnenolone. This change in steroid metabolism did not occur in foetal placental tissue from a sham-operated animal receiving no dexamethasone. Assay of progesterone in foetal placentae showed that the increased fromation of 17alpha,20alpha-dihydroxypregn-4-en-3-one was unlikely to be caused by a change in the specific activity of added 3H-labelled precursor, although the production of 17alpha, 20alpha-dihydroxypregn-4-en-3-one in vitro increased at a time when both foetal placental and utero-ovarian venous levels of progesterone were decreasing in response to dexamethasone treatment. These observations indicate that intra-foetal dexamethasone treatment induces a placental 17alpha-hydroxylase enzyme, which is also present in foetal placental tissue after the spontaneous onset of labour at term.

Indices of gonadal function in the human male. II. Seminal plasma levels of steroids in normal and pathological conditions.

Abstract: A radioimmunoassay method developed previously for the measurement of unconjugated pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone and oestradiol in peripheral plasma was applied to the assay of these steroids in seminal plasma of normal, oligospermic and azoospermic males. It was not possible to use the plasma assay method for the determination of progesterone and oestrone in seminal plasma, because some of the reliability criteria were not fulfilled. A detailed analysis of these steroids in the peripheral plasma of the same subjects has been described previously. The levels of all steroids in seminal plasma were significantly lower than the corresponding blood levels. The ratios of blood plasma/seminal plasma levels of the various steroids varied from 37 (testosterone) to 1.7 (dihydrotestosterone). There was a positive correlation between the testosterone and dihydrotestosterone levels of the seminal plasma of normal and azoospermic subjects. The concentrations of dihydrotestosterone, pregnenolone and oestradiol were significantly lower in azoospermic subjects than in normals. The only pathological finding in the seminal plasma of oligospermic subjects was a diminished level of dihydrotestosterone. Enzymic hydrolysis of a seminal plasma pool resulted in a 3- to 8-fold increase in the concentration of pregnenolone, dehydroepiandrosterone, testosterone, dihydrotestosterone and oestradiol, indicating that human seminal plasma contains large amounts of steroid conjugates. It is suggested that the analysis of steroids in the seminal plasma in combination with determinations in peripheral plasma may be a valuable aid to the assessment of testicular function.

Indices of gonadal function in the human male. I. Plasma levels of unconjugated steroids and gonadotrophins under normal and pathological conditions.

Abstract: A radioimmunoassay technique for the simultaneous measurement of eight unconjugated steroids (progesterone, pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, oestrone and oestradiol) in the peripheral plasma of human males is described. Determinations of these steroids and of immunoreactive FSH and LH were carried out on the plasma of twenty-one normal individuals and the levels were compared to those of eleven and ten males exhibiting oligospermia and azoospermia, respectively. Mean values and tolerance limits for each hormone, based on a lognormal distribution of individual values, are presented for all groups. Oligospermia was associated with a significant reduction in plasma dihydrotestosterone and testosterone levels. Azoospermic subjects also exhibited decreased dihydrotestosterone levels but a normal range of testosterone concentrations. Mean peripheral plasma levels of FSH were significantly elevated in both pathological groups and this was paralleled in the azoospermic men by increased concentrations of plasma LH.

Cholesterol 7 α‐Hydroxylase

Abstract: 70% of the microsome-bound cholesterol is directly accessible to cholesterol 7α-hydroxylase in an assay in vitro. After 5 min of incubation this endogenous cholesterol makes a single pool with the exogenously added substrate and modifies its specific radioactivity. Thus, an accurate estimation of the enzymic activity should take the participation of endogenous cholesterol into account. Cholesterol 7α-hydroxylase activity is enhanced in vitro by thiol-containing substances like mercaptoethanol, dithiothreitol, or cysteamine. On the contrary, the enzymic activity is inhibited by heavy cations (Hg2+, Pb2+, Cu2+, Zn2+), or −SH-blocking agents (mersalic acid, p-chloro-mercuribenzoic acid). Several steroids are potent inhibitors (Ki < Km) of the enzyme, among them pregnenolone and its acetate derivative and the cholesterol closely related 7-oxocholesterol and 7-dehydrocholesterol. The cholesterol esters are neither substrates nor inhibitors of cholesterol 7α-hydroxylase. Only a high concentration (1 mM) of biliary acids or of their glyco or tauro derivatives inhibits cholesterol 7α-hydroxylase. The quantitatively less important lithocholic acid and deoxycholic acid are the strongest inhibitors; the most common cholic acid does not affect the enzymic activity even at 1 mM.

Inhibition of placental 3beta-hydroxy-steroid dehydrogenase by naturally occurring steroids. A potential mechanism regulating oestrogen synthesis from unconjugated precursors.

Abstract: We have proposed that inhibition of placental steroid 3-sulphatase by endogenous steroids may regulate oestrogen synthesis during human pregnancy. The possibility that an additional regulatory mechanism, involving the placental 3beta-hydroxy-steroid dehydrogenase (SDH), may also be operative has now been examined. Inhibitory effects of naturally occurring steroids on SDH activity were determined from the reduction in initial rate of conversion of 3H-dehydroepiandrosterone to non-digitonin precipitable products by 10 000 x g supernatant from homogenates of human term placentae. The apparent Km for dehydroepiandrosterone was 0.33 x 10(-6) M. delta4-3-Oxo products of SDH action (4-androstene-3,17-dione, app. Ki=0.60x10(-6) M; progesterone, app. Ki=1.5x10(-6) M) were the most potent inhibitors and appeared to act non-competitively. Delta5-3beta-Hydroxy alternative substrates were less inhibitory and in the case of pregnenolone (app. Ki=4.5x10(-6) M) behaved competitively. 11beta-, 16alpha-, 17alpha- or 21-hydroxylation and epimerization of 3beta- or 17beta-hydroxyl functions of inhibitors decreased their activity. It is concluded that inhibition of both sulphatase and SDH by endogenous steroids may provide complementary methods of regulating placental oestrogen synthesis in vivo. The SDH mechanism may regulate oestrogen synthesis from unconjugated precursors, either formed within the placenta or derived from the circulation. The major potential inhibitors appear to be delta4-3-ketones, acting non-competitively, and formed within the placenta. In the sulphatase mechanism alternative substrates of extraplacenta origin, acting competitively, are the major potential inhibitors controlling utilization of conjugated precursors.

Steroid entry into rete testis fluid and the blood-testis barrier.

Abstract: Evidence that steroids enter rete testis fluid (RTF) from the blood at varying rates was obtained during i.v. infusions into rats. Testosterone and dehydroepiandrosterone were readily transferred into the fluid, whereas cholesterol was excluded. Between these extremes, the appearance of radioactivity in the RTF suggested the following order of entry rate: progesterone greater than pregnenolone greater than 5-alpha-reduced androgens greater than oestrogens greater than corticosteroids. Preliminary identification of metabolites in RTF and blood suggested that testosterone and dehydroepiandrosterone were transferred largely unchanged. Androstenedione and progesterone, however, were largely metabolized during transfer into the RTF, the former being transformed to testosterone. The results are used to discuss the nature of the blood-testis barrier to steroids and the source of androgens in the RTF.

Maintenance of Rete Testis Fluid Testosterone and Dihydrotestosterone Levels by Pregnenolone and Other C21 Steroids in Hypophysectomized Rats1

Abstract: This study was undertaken to determine whether maintenance of spermatogenesis in hypophysectomized rats by pregnenolone and other C21steroid s may be due to in vivo conversion of these compounds to androgens. Hypophy-sectomized rats were treated SC with 2 mg of pregnenolone, 17–hydroxypregnenolone, proges-terone, 17–hydroxyprogesterone or testosterone propionate in 0.2 ml sesame oil daily for 14 days beginning 2 days after hypophysectomy. Rete testis fluid (RTF), peripheral blood, and testicular venous blood were collected on the day of the last in ection. Testosterone (T) and dihydrotestosterone (DHT) were measured by radioimmunoassay after chromatographic separation. Results demonstrate that T and DHT could befound in the RTF of C21 steroid-treated hypophysectomized rats at levels similar to those seen in the intact rat. Results imply that the maintenance of spermatogenesis by C21steroids is probably due to the conversion of these compounds to T in the testis. Relatively little T was released from the t...

Studies of the Human Testis. VII. Conversion of Pregnenolone to Progesterone

Abstract: Delta5-3beta-Hydroxysteroid dehydrogenase (EC.1.1.1.145) and steroid delta-isomerase (EC.5.3.3.1) were extracted from frozen human testicular tissue and co-precipitated by addition of ammonium sulfate. The activities of both enzymes were localized in the 0-40% (NH4)2SO4 fraction. The enzyme preparation catalyzed conversion of pregnenolone, 17alpha-hydroxypregnenolone, dehydroepiandrosterone, and androstenediol to the corresponding delta4-3-oxosteroid. Since isomerization appeared not to be the rate-limiting step of the overall reaction, measurement of activity of delta5-3beta-hydroxysteroid dehydrogenase was related to the amount of delta4-3-oxosteroid produced from the corresponding delta5-3beta-hydroxysteroid. Delta5-3beta-Hydroxysteroid dehydrogenase required NAD for maximal activity. The Michaelis constants (Km) for NAD were 50 muM, 33 muM and 14 muM, respectively for the dehydrogenation of pregnenolone, 17alpha-hydroxypregnenolone, androstenediol and dehydroepiandrosterone. Km values for each substrate were: pregnenolone 10 muM, 17alpha-hydroxypregnenolone and dehydroepiandrosterone 2.5 muM and androstenediol 3.0 muM. Human testicular delta5-3beta-hydroxysteroid dehydrogenase was inhibited by most of the steroids procued by the testis. The following steroids acted as competitive inhibitors with pregnenolone: 17alpha-hydroxypregenolone (Ki = 1.3 muM), androstenediol (Ki = 2.4 muM), dehydroepiandrosterone (Ki = 0.74 muM), 20alpha-dihydroprogesterone (Ki = 1.1 muM) estrone (Ki = 0.33 muM) and estradiol-17beta (Ki = 0.87 muM). 17alpha-Hydroxyprogesterone, testosterone and androstenedione showed mixed-type inhibition of the enzyme for pregnenolone. Progesterone and NADH were noncompetitive inhibitors of the enzyme for pregnenolone. Ki values, with respect to prenenolone, were 7.4 muM for progesterone and 150 muM for NADH. NADH, however, acted competitively with NAD and Ki value was 30 muM.

PLASMA PREGNENOLONE, PROGESTERONE, 17‐HYDROXYPROGESTERONE, TESTOSTERONE AND 5α‐DIHYDROTESTOSTERONE IN DIFFERENT TYPES OF CONGENITAL ADRENAL HYPERPLASIA

Abstract: SUMMARY Unconjugated pregnenolone, progesterone, 17-hydroxyprogesterone, testosterone and 5a-dihydrotestosterone were simultaneously measured by radioimmunoassay in plasma from children with congenital adrenal hyperplasia (CAH) due to a deficiency of 21-hydroxylase (21-OHase), 11 β-hydroxylase (11β-OHase) or 3β-hydroxysteroid dehydrogenase (3β-HSD). These steroids were also measured in a reference group of children of similar age. The following concentrations of these five steroids were observed in the prepubertal children aged 0-4-10-9 years: pregnenolone <0-2-0-85 ng/ml; progesterone 0-17-0-68 ng/ml; 17-hydroxyprogesterone 0-10-0-53 ng/ml; testosterone <0-05-0-14 ng/ml, and 5α-dihydrotestosterone <0-05 ng/ml (detection limit of the method). The concentrations were clearly elevated in the plasma of children with CAH. A very high plasma concentration of 17-hydroxyprogesterone differentiates a 21-OHase deficiency from the two other types: children with this defect had levels mostly in excess of 100-fold that of normal. Plasma progesterone concentrations in these patients were also high being in the range found during the luteal phase level in the adult. Their plasma testosterone concentrations showed a scatter from normal values to high adult male levels being mostly at the level of adult females. The concentrations of 5a-dihydrotestosterone were at or above those of adult males. A high plasma concentration of pregnenolone with at most slightly elevated progesterone and 17-hydroxyprogesterone levels differentiated the 3β-HSD defect from the other two forms of CAH. The plasma profile of the five steroids determined in a patient with an 11 β-OHase deficiency was close to normal, but slightly elevated pregnenolone, progesterone and 17-hydroxyprogesterone levels were found to be characteristic of this enzyme deficiency.

In Vitro Steroid Metabolic Studies in Testicular 17β-Reduction Deficiency

Abstract: In order to document testicular 17β-reduction deficiency (17RD) and to search for additional metabolic aberrations possibly associated with this disorder, the metabolism of 14C-labeled pregnenolone (Δ5P), 17-hydroxyprogesterone (170HP), dehydroepiandrosterone (DHEA), androstenedione (A), testosterone (T) and estrone (E1) was studied in testicular minces from a 46-year-old male pseudohermaphrodite (MPH) with highly elevated testicular A and minimal T secretion but normal extragonadal conversion of A to T. Testicular minces from a 20-year-old MPH with apparently normal testicular T biosynthesis served as a control. The results of this investigation show that the 17RD testes metabolized Δ5P along Δ5- and Δ4-pathways but, in contrast to the control, converted more 17OHP, metabolizing it predominantly to A rather than T, failed to reduce DHEA to androst-5-ene-3β,17β-diol, metabolized DHEA very efficiently to A and produced little T, and converted only minimal quantities of A and E1 to their 17β-reduced counter...