Showing papers on "Pregnenolone published in 1989"

Neurosteroids: Biosynthesis of Pregnenolone and Progesterone in Primary Cultures of Rat Glial Cells*

Abstract: Cells dissociated from newborn rat forebrains were established in long term primary cultures. The cultures were made up almost exclusively of oligodendrocytes and astrocytes, as confirmed by indirect immunofluorescence staining with monoclonal antibodies to galactocerebroside and glial fibrillary acidic protein, respectively. After 3 weeks of culture, the oligodendrocytes were also highly immunoreactive to monospecific polyclonal antibodies against cytochrome P450SCC, an enzyme involved in the conversion of cholesterol to pregnenolone (P). Biosynthesis of [3H]cholesterol, [3H]P, and [3H]Pregn-5- ene-3β,20α-diol was demonstrated in these primary cultures by incubating cells with [3H]mevalonolactone in the presence of mevinolin and trilostane. The activity of the 2',3'-cyclic nucleotide 3'-phosphodiesterase enzyme, a documented indicator of oligodendrocyte differentiation, increased rapidly after day 10 of culture, together with the onset of steroid biosynthetic activity. Both reached a maximum at ...

Effects of pure FSH and LH preparations on the number and function of Leydig cells in immature hypophysectomized rats.

Abstract: The effects of pure FSH and/or LH preparations on the number of Leydig cells and their function in immature hypophysectomized rats have been investigated. As a result of hypophysectomy at the age of 17-18 days, the number of recognizable Leydig cells per testis decreased, as did the steroidogenic capacity in vivo and in vitro. Treatment with 64 micrograms FSH on both 22 and 23 days of age, did not affect the number of recognizable Leydig cells. In contrast, two injections of LH (10 micrograms) caused a sixfold increase in the number of Leydig cells, but had a negative effect on spermatogenesis. These stimulatory and inhibitory effects of LH diminished when FSH was added. Treatment with FSH for 7 days caused a twofold increase in the number of Leydig cells when compared with hypophysectomized controls. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and esterase activity in Leydig cells also increased under the influence of FSH. The pregnenolone production per Leydig cell in the presence of 5-cholesten-3 beta,22(R)-diol (22R-hydroxycholesterol) as substrate showed a sevenfold increase. Plasma testosterone levels 2 h after injection of human chorionic gonadotrophin in intact rats and hypophysectomized FSH-treated rats were the same. Following LH treatment for 7 days, the number of Leydig cells proved to be 11 times higher, and 3 beta-HSD and esterase activity were not different from intact controls. The testicular pregnenolone production was four- to fivefold higher when compared with untreated hypophysectomized rats. However, pregnenolone production per Leydig cell in LH-treated rats was only slightly different from the hypophysectomized controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria

Abstract: In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.

Ovine placental steroid 17α-hydroxylase/C-17,20-lyase, aromatase and sulphatase in dexamethasone-induced and natural parturition

Abstract: Parturition in the sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to oestrogen production. This change is believed to be a consequence of the preparatum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17) alpha), steroid C-17,20-lyase, and possibly aromatase and steroid sulphatase. The activity levels have been determined of steroid 17 alpha-hydroxylase, aromatase and steroid sulphatase in placental microsomes in late pregnancy, dexamethasone-induced labour and in natural labour at term. Over the gestational period of 118-140 days, basal levels of placental aromatase were relatively constant (mean value (+/- S.E.M.) of 5.6 +/- 0.5 pmol/min per mg microsomal protein (n = 10]. Pregnenolone and progesterone 17 alpha-hydroxylase activities were undetectable (less than 0.5 pmol/min per mg microsomal protein (n = 7]. In six animals in labour induced with infusion of dexamethasone into the fetus, placental aromatase activity increased to a value of 14.0 +/- 1.0 pmol/min per mg protein; placental pregnenolone 17 alpha-hydroxylase, measured in four of the animals, also increased to 453 +/- 77 pmol/min per mg microsomal protein. In five animals in natural spontaneous labour with vaginal delivery, aromatase activity was 26.7 +/- 5.2 pmol/min per mg microsomal protein and pregnenolone 17 alpha-hydroxylase activity was 141 +/- 14 pmol/min per mg microsomal protein. Steroid sulphatase activity was barely detectable (less than 1.5 pmol/min per mg microsomal protein) during late pregnancy, dexamethasone-induced labour or natural parturition.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cholesterol side-chain cleavage and 17α-hydroxylase/lyase activities in proliferating human theca interna cells in long term monolayer culture

Abstract: In this report we describe the development and characterization of a long term culture system to study regulation of the expression of 17 alpha-hydroxylase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase in human theca interna cells. Conditions have been established for the dispersal, growth, freezing, and storage of functional human theca interna cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Theca interna cells grown under these conditions have a doubling rate of 28-32 h and are morphologically distinct from human granulosa cells grown under the same conditions. Theca interna cells were grown, passed for successive passages, and transferred into serum-free medium containing forskolin, hCG, LH, or cAMP analogs. There was a time- and dose-dependent increase in 17 alpha-hydroxylase activity and progesterone synthesis from endogenous precursors. Added pregnenolone was converted to 17 alpha-hydroxypregnenolone, which was further converted primarily to dehydroepiandrosterone and, to a much lesser extent, androstenedione. Progesterone was converted to 17 alpha-hydroxyprogesterone and 16 alpha-hydroxyprogesterone. In studies using 17 alpha-hydroxyprogesterone as substrate, no metabolism to androstenedione or any other product was detectable. Similarly, 4-pregnen-20 alpha-ol-one (20 alpha-dihydroprogesterone) was not metabolized to any detectable products. Northern analysis performed on total RNA obtained from forskolin-stimulated theca interna cultures verified that the increase in 17 alpha-hydroxylase activity was associated with a corresponding increase in levels of mRNA specific for 17 alpha-hydroxylase cytochrome P-450. Message levels for cholesterol side-chain cleavage P-450 were similarly increased in cells treated with forskolin. No detectable mRNA encoding aromatase cytochrome P-450 was discerned. This procedure for the preparation and study of proliferating human theca internal cells provides an opportunity to study regulation of the expression of steroidogenic enzymes and other cellular processes unique to human ovarian cells.

Dexamethasone regulates bile acid synthesis in monolayer cultures of rat hepatocytes by induction of cholesterol 7 alpha-hydroxylase

Abstract: To study the effect of steroid hormones on bile acid synthesis by cultured rat hepatocytes, cells were incubated with various amounts of these compounds during 72 h and conversion of [4-14C]cholesterol into bile acids was measured. Bile acid synthesis was stimulated in a dose-dependent way by glucocorticoids, but not by sex steroid hormones, pregnenolone or the mineralocorticoid aldosterone in concentrations up to 10 microM. Dexamethasone proved to be the most efficacious inducer, giving 3-fold and 7-fold increases in bile acid synthesis during the second and third 24 h incubation periods respectively, at a concentration of 50 nM. Mass production of bile acids as measured by g.l.c. during the second day of culture (28-52 h) was 2.2-fold enhanced by 1 microM-dexamethasone. No change in the ratio of bile acids produced was observed during this period in the presence of dexamethasone. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of the bile acid pathway, to bile acids was not affected by dexamethasone. Measurement of cholesterol 7 alpha-hydroxylase activity in homogenates of hepatocytes, incubated with 1 microM-dexamethasone, showed 10-fold and 90-fold increases after 48 and 72 h respectively, as compared with control cells. As with bile acid synthesis from [14C]cholesterol, no change in enzyme activity was found in hepatocytes cultured in the presence of 10 microM steroid hormones other than glucocorticoids. Addition of inhibitors of protein and mRNA synthesis lowered bile acid production and cholesterol 7 alpha-hydroxylase activity and prevented the rise of both parameters with dexamethasone, suggesting regulation at the mRNA level. We conclude that glucocorticoids regulate bile acid synthesis in rat hepatocytes by induction of enzyme activity of cholesterol 7 alpha-hydroxylase.

Pregnenolone sulfate antagonizes barbiturate-induced hypnosis

Abstract: The potential influence of the neurosteroid pregnenolone sulfate (PrS) on barbiturate-induced hypnosis was tested in rats. PrS, when injected intracerebroventricularly or intraperitoneally, significantly shortened the sleep-time produced by pentobarbital. The results suggest an important physiological and pharmacological role for PrS in the regulation of CNS excitability.

Regulation of progesterone production in the rabbit corpus luteum.

Abstract: Substantial evidence accumulated over six decades has established that estradiol exerts a dominant stimulatory influence on the production of progesterone by luteal tissue in pseudopregnant or pregnant rabbits, beginning approximately five days after ovulation. The direct steroidogenic action of estradiol on the luteal cell is mediated by the estrogen-receptor protein complex at the nuclear level. Major effects of estradiol lie distal to cholesterol ester and the formation of lipid droplets, and proximal to cholesterol availability for translocation into cytochrome P-450 cholesterol side-chain cleavage enzyme (P-450scc). Structure-function studies corroborate this as an estrogen-sensitive segment of the steroidogenic pathway in the rabbit corpus luteum. Estradiol increases the amount of precursor available for pregnenolone production in rabbit luteal mitochondria. Whether this is because of enhanced precursor storage in the mitochondria or because of effects on intramitochondrial movement of precursor, or both, is unclear. There is a void in knowledge between events at the nuclear level in response to the estrogen stimulus and known post-translational effects at the level of cellular and subcellular organelles. Studies to determine estrogen-sensitive transcriptional and translational events associated with steroidogenesis in the rabbit luteal cell model offer a novel perspective for an improved understanding of the regulatory processes governing steroidogenesis.

Insulin and Insulin-Like Growth Factor-I Stimulate the 3α-Hydroxysteroid Dehydrogenase Activity of Human Placental Cytotrophoblasts*

Abstract: The placenta is the primary source of progesterone during pregnancy. Because pregnant diabetic women are reported to have higher serum progesterone levels than nondiabetic pregnant women, we studied the roles of insulin and insulin-like growth factor-I (IGF-I) in the regulation of human cytotrophoblastic 3β-hydroxysteroid dehydrogenase (3βHSD) activity. Incubation of cytotrophoblasts with insulin or IGF-I for 24 h significantly increased the ability of these cells to convert pregnenolone to progesterone by 75.8 ± 16.5% (±SE) and 65.4±12.7%, respectively. Treatment with either insulin or IGF-I did not alter cytotrophoblastic production of 20α-hydroxypregn- 4-en-3-one (the primary metabolite of progesterone), thus demonstrating that these peptides increased progesterone synthesis (i.e. 3βHSD activity) rather than decreased progesterone catabolism. Insulin and IGF-I stimulated 3βHSD activity at concentrations as low as 50 and 10 ng/ml, respectively. Insulinand IGF-I-stimulated 3βHSD activities were ...

An endogenous digitalis-like factor derived from the adrenal gland: studies of adrenal tissue from various sources.

Abstract: We have previously reported that the adrenal gland is the probable origin of the digitalis-like immunoreactive material (DLI) present in the plasma of rats and other species which have never received cardiac glycoside drugs. The present study demonstrates that adrenal glands removed from rats and then chopped release an immunoreactive digitalis-like material into a serum-free minimal incubation medium. HPLC studies indicated that this immunoreactivity was not homogeneous. Since such material may be a mammalian steroidal ligand for the glycoside receptor on the sodium pump, we investigated whether release of this material could be inhibited by antagonizing the conversion of cholesterol to pregnenolone through the addition of aminoglutethimide (AG) to the incubation medium. Our observations indicate that this manipulation successfully inhibited pregnenolone production during both of our 2-h serial incubation periods. However, in neither incubation period was AG able to inhibit the release of DLI in...

3 Beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase from bovine adrenals: mechanism of inhibition by 3-oxo-4-aza steroids and kinetic mechanism of the dehydrogenase.

Abstract: Several 3-oxo-4-aza steroids (1) have been identified as inhibitors of the 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase catalyzed conversion of pregnenolone to progesterone. By kinetically decoupling the two enzyme activities isolated from bovine adrenal cortex, it has been demonstrated that inhibition by 1 occurs through interference of both activities. A preferred ordered association of substrates to the 3 beta-hydroxy-delta 5-steroid dehydrogenase in which the cofactor binds prior to steroid was determined by isotope exchange at equilibrium. With this result, the dead-end inhibition patterns of 1 with the dehydrogenase were interpreted to originate from a preferred association of inhibitor within an enzyme ternate containing NADH; this proposal is supported by data from multiple inhibition analysis indicating synergistic binding of NADH and 1. Similarly, inhibition of the 3-keto-delta 5-steroid isomerase by the 3-oxo-4-aza steroids was enhanced in the presence of the positive effector NADH. On the basis of pH profiles upon Vm, Vm/Km, and 1/Ki for both enzyme activities, inhibition is proposed to result from the structural similarity of 1 to intermediate states formed upon enzyme catalysis.

Molecular characterization of a Leydig cell tumor presenting as congenital adrenal hyperplasia.

Abstract: We present an unusual patient with a Leydig cell tumor to show that greatly elevated serum concentrations of 17-hydroxyprogesterone (17OHP) may not be diagnostic of congenital adrenal hyperplasia (CAH). A 3.5-yr-old boy had a small testicular mass and plasma 17OHP concentrations of 147-333 nmol/L (4,850-11,000 ng/dL), suggesting CAH with adrenal rests. However, normal plasma cortisol values and the unresponsiveness of the 17OHP concentration to dexamethasone suppression or ACTH stimulation suggested a diagnosis of Leydig cell tumor. A 4-fold elevation in plasma 21-deoxycortisol compared with a 200-fold elevation in 17OHP suggested that the elevated 17OHP derived from the normal pathway of testosterone synthesis in the testis. This was proven by normalization of all hormonal values after tumor resection. Compared to the abundance of mRNA for P450c17, the tumor contained unusually large amounts of mRNA for P450scc, the cholesterol side-chain cleavage enzyme, which is the rate-limiting step in steroid hormone synthesis. Increased P450scc activity, which increased the conversion of cholesterol to pregnenolone, apparently permitted the 17,20-lyase activity of P450c17 to become rate limiting, thus accounting for the increased secretion of 17OHP. Thus, Leydig cell tumors can produce quantities of 17OHP previously reported only in CAH due to 21-hydroxylase deficiency. The molecular characterization of steroidogenic mRNAs in this tumor indicates an unusual ratio in the expression of the genes for the steroidogenic enzymes, probably accounting for the unusual pattern of serum steroids.

Insulin enhances luteinizing hormone-stimulated steroidogenesis by porcine theca cells.

Abstract: It has been shown recently that insulin enhances differentiation of rat, pig, and human granulosa cells. The present studies were done to determine if insulin also plays a role in the regulation of theca cell steroidogenesis. Theca cells were obtained from prepubertal gilts and cultured under serum-free conditions for 48 h. Theca cell androstenedione production under basal and luteinizing hormone (LH)-stimulated conditions was significantly increased by adding insulin (1 microgram/ml) to the culture medium. Treatment of basal and LH-stimulated cultures with increasing concentrations of insulin (0.001-10 micrograms/ml) caused dose- and time-dependent increments in androstenedione production, but the effect was independent of the dose of LH employed. The ability of insulin to enhance thecal cell androstenedione production was mimicked by somatomedin C, but not by relaxin. Studies to determine the mechanism(s) of action of insulin showed that insulin action is exerted, at least in part, at a site(s) proximal to cyclic adenosine 3'5'-monophosphate (cAMP) generation, since insulin enhanced both basal and LH-stimulated accumulation of extracellular cAMP in addition to increasing androstenedione production. This effect was further enhanced by 3-isobutyl-1-methyl xanthine, an inhibitor of phosphodiesterase activity. Insulin treatment also caused dose-dependent increments in forskolin- and prostaglandin E2-stimulated accumulation of extracellular cAMP and androstenedione. Insulin also increased both the basal and LH-stimulated production of progesterone and its precursor pregnenolone, in addition to the increases in androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effect of insulin and insulin-like growth factor-I on cell multiplication and adrenocorticotropin responsiveness of fetal adrenal cells.

Abstract: The present study examined the effects of both insulin and insulin-like growth factor-I (JGF-I) on cell division and specific functions of cultured adrenocortical cells from 100- to 122-day-old ovine fetuses. When culture was performed in a serum-free medium containing transferrin and ascorbic acid, the number of cells increased only slightly (1.2-fold) over a 4-day period. Addition of insulin or IGF-I in the culture medium enhanced the number of cells counted on Day 5. The effect of both peptides was dose-dependent, but 10 ng/ ml IGF-I was as potent as 10 jig/mI insulin. The acute cyclic adenosine 3’, 5’-monophosphate (cAMP) and steroidogenic responses to adrenocorticotropin (ACTH1_24) decreased in fetal cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute ACTH1_24-induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells/or 4 days with increasing concentrations of insulin or IGF-I enhanced the acute cAMP and steroidogenic responses (3- to 4-fold) to ACTH1_24 over that of control cells. The ED50 of IGF-I was about 3 ng/ml (� 0.4 nM) whereas that of insulin was about 10 ng/ml (1.7 nM). However, a second plateau was apparent at concentrations of insulin above 1 jig/mI. The acute cholera toxin stimulation of cAMP production of cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute AC TH1 �24 -induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells for 4 days with response to forskolin did not change during culture, irrespective of the culture conditions. The positive effect of both insulin and IGF-I on the acute ACTHJ_24 stimulation of steroidogenesis was also observed when cells were stimulated by 8Br-cAMP. By contrast, cells cultured in the presence of 10 pg/mI insulin or 10 ng/ml IGF-I resulted in lower amounts of corticosteroids produced when cells were incubated with 22R-hydroxycholesterol or exogenous pregnenolone. The acute cAMP and steroidogenic responses to ACTH1..24 of cells cultured in the continuous presence of 10_11 M ACTHI_� in insulin-free medium increased during the culture period. Insulin and IGF-I were able to enhance both the cAMP and steroidogenic response of these cells to ACTH1_24. The present data suggest that both insulin and IGF-I are involved in the maintenance and the maturation of ovine fetal adrenocortical cell differentiated functions, whereas, at physiological concentrations only, IGF-I has a growth-promoting effect on these cells.

Pregnenolone synthesis from cholesterol and hydroxycholesterols by mitochondria from ovaries following the stimulation of immature rats with pregnant mare's serum gonadotropin and human choriogonadotropin.

Abstract: The rate of pregnenolone synthesis by cytochrome P-450scc was measured in mitochondria isolated from ovaries of immature rats treated with pregnant mare's serum gonadotropin and human choriogonadotropin. Using cholesterol, 25-hydroxycholesterol, 20 alpha-hydroxycholesterol, (22R)-22-hydroxycholesterol and (22R)-20 alpha,22-dihydroxycholesterol as substrates, we have determined that the first hydroxylation of cholesterol, in the 22R position, is rate limiting in pregnenolone synthesis. It proceeds at only 22% of the rate of either of the subsequent two hydroxylations. 25-Hydroxycholesterol proved to be a suitable substrate for determining the maximum rate of pregnenolone synthesis by cytochrome P-450scc in isolated mitochondria. The maximum rate was 13 mol steroid.min-1.mol cytochrome P-450scc-1 and did not change after the follicles in the immature ovary had been stimulated to mature and luteinize with gonadotropin. Using endogenous cholesterol in isolated mitochondria as substrate, the time course of pregnenolone synthesis was the same during the follicular phase as in the luteal stage of gonadotropin-induced development. We conclude that during the artificial induced development of follicles in the immature ovary, the major cause of the increase in the rate of pregnenolone synthesis is the increase in the cytochrome P-450scc content of the mitochondria, rather than changes in the catalytic activity of cytochrome P-450scc or the cholesterol availability to the cytochrome.

The influence of exogenous free cholesterol on steroid synthesis in cultured adrenal cells.

Abstract: Previous studies showed that Y-l cells in which the concentration of cholesterol in plasma membranes (expressed relative to phospholipid as a molar ratio, C/P) was raised by incubation with cholesterol produced more 20α-dihydroprogesterone than cells with normal or low plasma membrane C/P values. This difference was not associated with any difference in the production of cAMP. The cells respond to (Bu)2cAMP in the order high > normal > low C/P. High C/P is associated with higher levels of cholesterol in the cytosol and higher concentrations of this steroid in the inner mitochondrial membrane. The rate of transport of cholesterol to the inner membrane differed in the three groups of cells: high > normal > low C/P. Production of pregnenolone by mitoplasts was no different in the three groups, and the same applies to the production of pregnenolone by mitochondria from cells incubated with and those incubated without (Bu)2cAMP. 25-Hydroxycholesterol increased the production of 20α-dihydroprogesterone by cells...