Showing papers on "Pregnenolone published in 1992"

Memory-enhancing effects in male mice of pregnenolone and steroids metabolically derived from it.

Abstract: Immediate post-training intracerebroventricular administration to male mice of pregnenolone (P), pregnenolone sulfate (PS), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, testosterone, dihydrotestosterone, or aldosterone caused improvement of retention for footshock active avoidance training, while estrone, estradiol, progesterone, or 16 beta-bromoepiandrosterone did not. Dose-response curves were obtained for P, PS, DHEA, and testosterone. P and PS were the most potent, PS showing significant effects at 3.5 fmol per mouse. The active steroids did not show discernible structural features or known membrane or biochemical effects that correlated with their memory-enhancing capacity. The above, together with the findings that DHEA acted even when given at 1 hr after training and that P, PS, and DHEA improved retention over a much wider dose range than do excitatory memory enhancers, led to the suggestion that the effects of the active steroids converge at the facilitation of transcription of immediate-early genes. P and PS, for which receptors have not yet been demonstrated, may exert their effects by serving as precursors for the formation of a panoply of different steroids, ensuring near-optimal modulation of transcription of immediate-early genes required for achieving the plastic changes of memory processes. Low serum levels of P in aging and the increases of cancer and behavioral disorders in individuals receiving drugs that block synthesis of cholesterol, the immediate precursor of P, suggest possible clinical utility for P.

Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: Regulation by a mitochondrial diazepam binding inhibitor receptor

Abstract: The C6-2B glioma cell line, rich in mitochondrial receptors that bind with high affinity to benzodiazepines, imidazopyridines, and isoquinolinecarboxamides (previously called peripheral-type benzodiazepine receptors), was investigated as a model to study the significance of the polypeptide diazepam binding inhibitor (DBI) and the putative DBI processing products on mitochondrial receptor-regulated steroidogenesis DBI and its naturally occurring fragments have been found to be present in high concentrations in C6-2B glioma cells, to compete against specific isoquinolinecarboxamide or 4'-chlorodiazepam binding to mitochondrial recognition sites with high affinity, and to stimulate mitochondrial pregnenolone formation These data suggest that this cell type may express both the receptor and the putative agonist ligand to regulate steroidogenesis Therefore, we propose to term this mitochondrial receptor MDR (mitochondrial DBI receptor) to indicate its responsiveness to DBI in steroid biosynthesis In the present work, we show that mitochondria of C6-2B cells convert (22R)-22-hydroxycholesterol to pregnenolone by a mechanism blocked by aminoglutethimide Immunoblotting confirmed the presence of relatively high levels of cytochrome P-450 cholesterol side-chain-cleavage enzyme in C6-2B cell mitochondria Furthermore, isoquinolinecarboxamide binding sites associated with the 18-kDa mitochondrial polypeptide subunit of the MDR are abundant in C6-2B glioma cell mitochondria (Bmax approximately 30 pmol/mg protein) and are coupled to the regulation of steroid biosynthesis Occupancy of MDRs with nanomolar concentrations of the naturally occurring polypeptide, DBI, as well as its naturally occurring processing product tetratriacontaneuropeptide [DBI-(17-50)] increases pregnenolone formation Clonazepam and octadecaneuropeptide [DBI-(33-50)], which exhibit a higher affinity for gamma-aminobutyric acid type A receptors but a low affinity for MDR, were ineffective in stimulating pregnenolone synthesis These findings provide evidence that C6-2B cells exhibit a significant steroidogenic activity which resembles that found in peripheral endocrine organs and they suggest that MDRs and DBI are involved in the regulation of glial cell steroidogenesis

Neurosteroid metabolism. 7α-Hydroxylation of dehydroepiandrosterone and pregnenolone by rat brain microsomes

Abstract: Two 'neurosteroids', dehydroepiandrosterone (DHEA) and pregnenolone (PREG), are converted by rat brain microsomes into polar metabolites, identified as the respective 7 alpha-hydroxylated (7 alpha-OH) derivatives by the 'twin ion' technique of glc-ms with deuterated substrates The enzymic reaction requires NADPH and is stimulated 2-4-fold by EDTA Under optimal conditions (pH 74, 05 mM-NADPH, 1 mM-EDTA), the Km values for DHEA and PREG are 138 and 44 microM respectively, and the Vmax values are 322 and 388 pmol/min per mg of microsomal protein respectively Trace amounts of putative 7 beta-OH derivatives of DHEA and PREG are detected Oestradiol, at a pharmacological concentration of 5 microM, inhibits DHEA and PREG 7 alpha-hydroxylation Formation of 7 alpha-hydroxylated metabolites is low in prepubertal rats and increases 5-fold in adults Derivatives of PREG and DHEA, such as PREG sulphate, DHEA sulphate, progesterone and 3 alpha-hydroxy-5 alpha-pregnan-20-one, are known to be neuroactive Therefore the quantitatively important metabolism to 7 alpha-OH compounds may contribute to the control of neurosteroid activity in brain

Regulation of pregnenolone synthesis in C6-2B glioma cells by 4'-chlorodiazepam.

Abstract: An experimental model to study synthesis of cholesterol and pregnenolone from the precursor mevalonolactone (MVA) was developed in C6-2B glioma cells. The steroidogenic capability of this cell line and the regulation of pregnenolone production by 4'-chlorodiazepam (4'CD), a specific ligand for the mitochondrial diazepam binding inhibitor (DBI) receptor (MDR), were investigated. Cells maintained in serum-free media were incubated with lovastatin (20 microM) and two inhibitors of pregnenolone metabolism, trilostane (25 microM) and 1,2,3,4-tetrahydro-4-oxo-7-chloro-2-naphthylpyridine (10 microM). Under these conditions the incorporation of [3H]MVA into cholesterol and pregnenolone formation was biphasic, with an initial rapid phase (within 1 min) followed by a slower phase. Cholesterol and pregnenolone were identified by coelution with authentic steroids from a Si 60 Lichrosorb column and gas chromatography/mass spectrometry. Pregnenolone synthesis in intact C6-2B glioma cells was stimulated by nanomolar concentrations of 4'CD after 5 min of incubation with MVA. The stimulatory effect was dependent on drug concentration and the maximal effect was achieved at 10 nM. The time course showed that the incorporation of MVA into pregnenolone is accelerated by the MDR ligand. Cholesterol synthesis is only slightly and not significantly affected by 4'CD. These results support the view that steroid synthesis occurs in a glioma cell line. Moreover, we provide evidence for a rapid steroid synthesis in C6-2B glioma cells, which in turn appears to be accelerated by 1-100 nM 4'CD, a MDR ligand.

Paradoxical transcriptional activation of rat liver cytochrome P-450 3A1 by dexamethasone and the antiglucocorticoid pregnenolone 16 alpha-carbonitrile: analysis by transient transfection into primary monolayer cultures of adult rat hepatocytes.

Abstract: The family 3A cytochromes P-450, among the most abundant members of this supergene family of microsomal hemoproteins expressed in animal and human liver, are inducible by glucocorticoids but also by such antiglucocorticoids as pregnenolone 16 alpha-carbonitrile (PCN). To investigate the mechanism for this nonclassical glucocorticoid effect, we analyzed the ability of 1.5 kilobases of DNA or of its successive subsegments isolated from the 5' flanking region of the rat CYP3A1 structural gene to modulate transcription of a reporter gene consisting of a viral promoter coupled to the chloramphenicol acetyltransferase (CAT) structural gene (expression vector pBLCAT2) and transiently expressed in a homologous cell system consisting of primary monolayer cultures of adult rat hepatocytes in which CYP3A1 mRNA and protein are inducible. The CAT activity measured after chimeric gene constructions were transferred into the cultured rat hepatocytes by lipofection increased as much as 7.2-fold if the cells were treated with dexamethasone (DEX). One CYP3A1 fragment (positions -220 to -56; 164 base pairs), which does not contain a traditional glucocorticoid responsive element, conferred dose-dependent DEX responsiveness independent of its orientation but not its position in pBLCAT2. This construction was activated by addition of PCN to the cultures and was synergistically induced by PCN plus DEX. In contrast, induction of CAT activity in cultures containing MMTVCAT, a plasmid containing the CAT gene controlled by the mouse mammary tumor virus long terminal repeat, was unaffected by PCN treatment, required lower concentrations of DEX for a maximal response, and was inhibited by treatment with DEX plus PCN. We conclude that a primary mechanism for induction of CYP3A1 is stimulated transcription through a pathway activated by steroid hormones.

Dose-dependent effects of human prolactin on the immature hypophysectomized rat testis.

Abstract: We have studied dose-dependent effects of highly purified human PRL (39 IU/mg) on the testis of immature (22-day-old) hypophysectomized rats daily supplemented for 7 days with 2, 10, or 30 micrograms hormone. Dose-dependent stimulation was observed for all parameters: testis weight (1.6- and 2-fold above control for 10 and 30 micrograms PRL), basal and hCG-stimulated testosterone (14- and 21-fold), intratesticular testosterone (7- and 21-fold) and estradiol (1.2- and 1.5-fold), LH receptor concentration (1.8- and 2.5-fold), in vitro pregnenolone production by cholesterol side-chain cleavage enzyme (3-, 5- and 7-fold), and aromatase activity (2.1- and 2.4-fold). The number of Leydig cells exhibiting immunoreactivity toward anti-P450scc, anti-P450(17 alpha), and anti-3 beta-hydroxysteroid dehydrogenase antibodies also underwent a dose-dependent increase (under conditions revealing many immunopositive cells in hypox control animals). The respective increases were 8- to 14-fold for anti-P450scc and P450(17 alpha) and 1.5- to 2-fold for anti-3 beta-hydroxysteroid dehydrogenase. The number of germ cells and the percentage of tubular sections containing late pachytene spermatocytes as most advanced stages of spermatogenesis were subject to similar dose-dependent effects. These results suggest that during puberty PRL stimulates testicular function by promoting multiplication and differentiation of Leydig cells (acting at various steps of steroidogenesis and on tissue responsiveness to LH) and germ cells.

Neurosteroids: pregnenolone in human sciatic nerves.

Abstract: The characterization and quantification of pregnenolone in human sciatic nerves were undertaken, following previous demonstration of the synthesis of this steroid in rat brain oligodendrocytes, to explore the hypothesis that Schwann cells may demonstrate the same biosynthetic activity. Pregnenolone was definitively identified by mass spectrometry and quantified by specific radioimmunoassay. Its concentration (mean +/- SD, 63.9 +/- 45.9 ng/g of wet tissue, n = 12) was greater than or equal to 100 times the plasma level and concentration found in tendons and muscle. No correlation was found with sex or age. Free dehydroepiandrosterone as well as sulfate and fatty acid esters of pregnenolone and dehydroepiandrosterone were also measured. Results are discussed in terms of the concept that these "neurosteroids" may be synthesized in the peripheral nervous system.

Diazepam-binding inhibitor (DBI)-processing products, acting at the mitochondrial DBI receptor, mediate adrenocorticotropic hormone-induced steroidogenesis in rat adrenal gland.

Abstract: Diazepam-binding inhibitor (DBI) is a 9-kDa polypeptide that colocalizes in glial, adrenocortical, and Leydig cells with the mitochondrial DBI receptor (MDR). By binding with high affinity to the MDR, DBI and one of its processing products--DBI-(17-50)--regulate pregnenolone synthesis and have been suggested to participate in the immediate activation of adrenal steroidogenesis by adrenocorticotropic hormone (ACTH). In adrenals of hypophysectomized rats (1 day after surgery), ACTH failed to acutely affect the amount of adrenal DBI and the density of MDR but increased the rate of DBI processing, as determined by the HPLC profile of DBI-(17-50)-like immunoreactivity. The similar latency times for this effect and for ACTH stimulation of adrenal steroidogenesis suggest that the two processes are related. The ACTH-induced increase in both adrenal steroidogenesis and rate of DBI processing were completely inhibited by cycloheximide; this result suggests the requirement for the de novo synthesis of a protein with a short half-life, probably an endopeptidase. This enzyme, under the influence of ACTH, may activate formation of a DBI-processing product that stimulates steroidogenesis via the MDR. In support of this hypothesis is the demonstration that in hypophysectomized rats the MDR antagonist PK 11195 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide completely inhibited the adrenal steroidogenesis stimulated by ACTH and by the high-affinity MDR ligand 4'-chlorodiazepam.

Endothelin-1 as a luteinization inhibitor: inhibition of rat granulosa cell progesterone accumulation via selective modulation of key steroidogenic steps affecting both progesterone formation and degradation.

Abstract: Endothelin (ET)-1, is a 21 amino acid vasoactive peptide subject to regulation by cellular oxygen tension. However, an increasing body of information now suggests that ET-1 is a multifunctional peptidergic regulator the actions of which are not limited to the vascular system. Although ET-1 has been shown to inhibit the gonadotropin-supported accumulation of progesterone by cultured granulosa cells, the precise cellular mechanism(s) involved remain unknown. It was therefore the objective of this study to examine in greater detail the effects of ET-1 on progestin economy in cultured granulosa cells from immature rats. Treatment with ET-1 was inhibitory to the FSH-supported accumulation of progesterone in a dose-dependent manner, an action characterized by a median inhibitory dose of 2 x 10(-11) M and a maximal inhibitory effect of 90%. This inhibitory action of ET-1 was reversible following extensive washing and could not be accounted for by a decrease in the viable cell mass. Evaluation of the activities of progesterone-forming enzymes revealed ET-1 to be a potent (P < 0.01) inhibitor of cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase (HSD)/isomerase (76.1 +/- 1.2% and 47.3 +/- 8.6% inhibition, respectively). Cellular radiolabeling with [3H]pregnenolone confirmed an ET-1-induced inhibition of the FSH-supported accumulation of radiolabeled progesterone. However, this effect was concomitant with enhancement of the accumulation of more distal metabolites, i.e. 20 alpha-dihydroprogesterone, 5 alpha-pregnane-3 alpha, 20 alpha-diol, and 5 alpha-pregnane-3 alpha-ol-20-one. Analysis of the FSH-supported activities of the progesterone-degrading enzymes revealed ET-1 as a potent (P < 0.05) stimulator of 20 alpha-HSD and 5 alpha-reductase (3.6 +/- 1.0 and 1.7 +/- 0.3-fold, stimulation respectively). In contrast, no significant changes were observed in 3 alpha-HSD activity. Taken together, our findings demonstrate that the ET-1 induced inhibition of gonadotropin-supported progesterone accumulation constitutes a complex phenomenon wherein ET-1 inhibits the activities of steroidogenic enzymes concerned with progesterone formation while enhancing the activities of enzymes concerned with progesterone degradation. We speculate that ET-1, possibly of intraovarian origin, acts as a luteinization-inhibitor to suppress premature luteinization at a time when continued preovulatory expression of ET-1 (in the intact but not ruptured follicle) may be contingent upon relative intrafollicular hypoxia.

Interaction of pregnanolone and pregnenolone sulfate with ethanol and pentobarbital.

Abstract: 3-α-Hydroxy-5-β-pregnan-20-one [pregnanolone (Pa)] and 3-β-hydroxy-5-pregnen-20-one 3-sulfate [pregnenolone sulfate (PS)] are steroids that have been shown in biochemical studies to be active at the GABA-benzodiazepine-chloride receptor complex, Pa as a “barbiturate-like” agonist and PS as a “picrotoxin-like” antagonist. Since other compounds that are active at this site interact with the effects of pentobarbital and ethanol, the behavioral effects of these steroids alone and in combination with pentobarbital and ethanol were tested. Pa blocks the convulsions caused by pentylenetetrazole (PTZ) and increases motor activity when given alone in low doses. In combination with either pentobarbital or ethanol, it enhances the depression in motor activity, hypothermia, and hypnosis. In contrast, PS has no effect on PTZ convulsions and depresses motor activity by itself. With pentobarbital, PS enhances the depression in motor activity but has no effect on hypothermia or hypnosis. With ethanol, PS enhances the hypothermia but does not affect motor activity or hypnosis. Therefore, Pa and PS show different but no opposite effects in interacting with compounds active at the GABA-benzodiazepine-chloride receptor complex.

The effects of ageing on the morphology and function of the zonae fasciculata and reticularis of the rat adrenal cortex.

Abstract: The morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.

Normative data for the steroidogenic response of mineralocorticoids and their precursors to adrenocorticotropin in a healthy pediatric population

Abstract: The responses of mineralocorticoids and their precursors 1 h after a 0.25-mg bolus of ACTH has not previously been established in infancy or childhood. We report the steroidogenic responses of pregnenolone, progesterone (Prog), deoxycorticosterone (DOC), corticosterone (B), 18-hydroxycorticosterone (18OHB), and aldosterone (A) measured 1 h after a 0.25-mg bolus of ACTH in 102 healthy children who were divided into 5 age groups: group 1 (< 1 yr; n = 22), group 2 (1-5 yr; n = 22), group 3 (6-12 yr; n = 15), group 4 (early to midpuberty; n = 21), and group 5 (late puberty; n = 22). Baseline pregnenolone levels were constant throughout childhood; however, there was a significant fall in the stimulated level after the first year of life (group 1 vs. 2, P < 0.0125). Baseline Prog levels rose significantly with the onset of puberty (group 3 vs. 4, P < 0.0125), but levels did not increase after ACTH stimulation during puberty. Both baseline and stimulated levels of Prog, DOC, and 18OHB were significantly higher i...

Elevated levels of endogenous pregnenolone fatty acid esters in follicular fluid high density lipoproteins support progesterone synthesis in porcine granulosa cells.

Abstract: Fatty acid esters of pregnenolone (PREG-FA) are present at high levels within ovarian follicular fluid (FF), although no physiological function has yet been attributed to these pregnenolone metabolites. We demonstrate in the following study that PREG-FA are predominantly localized in association with high density lipoproteins (HDL) within FF at levels that surpass those found in plasma HDL by approximately 200-fold. Extensive treatment with dextran-coated charcoal revealed that although nonconjugated steroids could be removed very efficiently from whole fluid and HDL, little effect could be demonstrated on the PREG-FA levels present within the fluid or its HDL component. This observed resistance to charcoal stripping may reflect a steroid-protein interaction that is altogether different from the non-conjugated steroid-protein association. Both plasma and FF-derived HDL were equally effective in supporting PROG biosynthesis in porcine granulosa cell cultures under normal culture conditions, but when PROG synthesis was dramatically blocked by inhibiting the P450 side-chain cleavage enzyme complex with the P450 enzyme inhibitor ketoconazole, only HDL of FF origin was capable of restoring PROG synthesis. Similar cultures that received HDL of plasma origin, containing similar levels of total cholesterol but far less PREG-FA, showed no significant difference in PROG accumulation over control cultures under these conditions. Several reports have described the presence of an entity within FF that was shown to resist charcoal stripping and enhanced PROG synthesis in granulosa cell cultures. Our data suggest that this entity within FF may indeed be the high levels of endogenous PREG-FA. We, therefore, propose a role for PREG-FA as a potential substrate in the biosynthesis of PROG in porcine granulosa cells.

Affinity labeling of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase: evidence for bifunctional catalysis by a different conformation of the same protein for each enzyme activity.

Abstract: 3 beta-Hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase copurify from human placental microsomes as a single enzyme protein. The affinity-alkylating secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivates the dehydrogenase and isomerase reactions in a time-dependent manner, but which of the two activities is targeted depends on the concentration of secosteroid. At 2-5 microM secosteroid, the dehydrogenase activity is alkylated in a site-specific manner (pregnenolone slows inactivation) that follows first-order inactivation kinetics (KI = 4.2 microM, k3 = 1.31 x 10(-2) min-1). As the secosteroid level increases from 11 to 30 microM, dehydrogenase is paradoxically inactivated at progressively slower rates, and pregnenolone no longer protects against the alkylator. The inactivation of isomerase exhibits the expected first-order kinetics (KI = 31.3 microM, k3 = 6.42 x 10(-2) min-1) at 11-30 microM secosteroid. 5-Androstene-3,17-dione protects isomerase from inactivation by 15 microM secosteroid, but the substrate steroid unexpectedly fails to slow the inactivation of isomerase by a lower concentration of alkylator (5 microM). A shift from a dehydrogenase to an isomerase conformation in response to rising secosteroid levels explains these results. Analysis of the ligand-induced conformational change along with cofactor protection data suggests that the enzyme expresses both activities at a bifunctional catalytic site. According to this model, the protein begins the reaction sequence as 3 beta-hydroxysteroid dehydrogenase. The products of the first step (principally NADH) promote a change in protein conformation that triggers the isomerase reaction.

Pregnenolone fatty acid esters incorporated into lipoproteins: substrates in adrenal steroidogenesis.

Abstract: The presence of fatty acid ester derivatives of pregnenolone (PFA) has been recently reported in guinea pig plasma and adrenals. Moreover, it has equally been demonstrated that PFA is present in plasma lipoproteins, including high density lipoproteins (HDL) and low density lipoproteins (LDL). We report here the conversion of LDL- and HDL-incorporated [3H]PFA in guinea pig and bovine adrenocortical cells into nonconjugated steroids, thus indicating that PFA can be used as substrate for steroid production. When guinea pig glomerulosa-fasciculata (FG) cells were incubated for 48 h in the presence of either LDL-incorporated [3H]PFA or HDL-incorporated [3H]PFA, 66 +/- 2% and 47 +/- 1% of the added radioactivity were converted to nonconjugated steroids, respectively. The transformation of LDL-incorporated [3H]PFA into nonconjugated steroids was further increased in FG cells when 10 nmol/liter ACTH were present in the culture medium. This ACTH-induced liberation of nonconjugated steroids from LDL-incorporated [3...

Androgenic 17β-hydroxysteroid dehydrogenase activity of expressed rat type I 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase

Abstract: Transient expression in nonsteroidogenic mammalian cells of the rat wild type I and type II 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β- HSD) cDNAs shows that the encoded proteins, in addition to being able to catalyze the oxidation and isomerization of Δ5-3β-hydroxysteroid precursors into the corresponding Δ4-3-ketosteroids, interconvert 5α- dihydrotestosterone (DHT) and 5α-androstane-3β,17β-diol (3β-diol). When homogenate from cells transfected with a plasmid vector containing type I 3β-HSD is incubated in the presence of DHT using NAD+ as cofactor, a somewhat unexpected metabolite is formed, namely 5α-androstanedione (A- dione), thus indicating an intrinsic androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) activity of this 3β-HSD isoform. Although the relative Vmax of 17β-HSD activity is 14.9-fold lower than that of 3β-HSD activity, the Km value for the 17β-HSD activity of type I 3β-HSD is 7.97 μM, a value which is in the same range as the conversion of DHT into 3β- diol which shows a Km value of 4.02 μM. Interestingly, this 17β-HSD activity is highly predominant in unbroken cells in culture, thus supporting the physiological relevance of this 'secondary' activity. Such 17β-HSD activity is inhibited by the classical substrates of 3β-HSD, namely pregnenolone (PREG), dehydroepiandrosterone (DHEA), Δ5-androstene-3β,17β- diol (Δ5-diol), 5α-androstane-3β,17β-diol (3β-diol) and DHT, with IC50 values of 2.7, 1.0, 3.2, 6.2, and 6.3 μM, respectively. Although dual enzymatic activities have been previously reported for purified preparations of other steroidogenic enzymes, the present data demonstrate the multifunctional enzymatic activities associated with a recombinant oxidoreductase enzyme. In addition to its well known 3β-HSD activity, this enzyme possesses the ability to catalyze DHT into A-dione thus potentially controlling the level of the active androgen DHT in classical steroidogenic as well as peripheral intracrine tissues.

Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist RU486.

Abstract: Since administration of the antiprogesterone RU486 to cyclic female rats at metestrus and diestrus results in increased serum levels of LH, estradiol, and testosterone at proestrus, we investigated whether RU486 affects follicular steroidogenesis. Female rats with a 4-day estrous cycle, induced experimentally by a single injection of bromocriptine on the morning of estrus, were given RU486 (2 mg) twice daily (0900 and 1700 h) on metestrus and diestrus. At proestrus the preovulatory follicles were isolated and incubated for 4 h in the absence and presence of LH. In the absence of LH, accumulation of estradiol, testosterone, and progesterone in the medium was not different for RU486-treated rats and oil-treated controls. In contrast, LH-stimulated estradiol, testosterone, and progesterone secretions were significantly lower in RU486-treated rats compared with controls. Addition of pregnenolone to the incubation medium resulted in a significantly lower increase of progesterone in follicles from RU486-treated rats compared with those from oil-treated controls. This suggests that 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is decreased by administration of RU486 in vivo. Aromatase and 17 alpha-hydroxylase/C17-20 lyase activities were not affected: addition of substrate (androstenedione and progesterone respectively) did not affect differently the amount of product formed (estradiol and testosterone) in RU486- and oil-treated rats. However, LH-stimulated pregnenolone secretion was lower in follicles from RU486-treated rats compared with follicles from oil-treated controls, suggesting that either cholesterol side-chain cleavage activity or LH responsiveness is decreased. At proestrus the preovulatory follicles from RU486- and oil-treated rats were not morphologically different.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms by which calcium ionophore and phorbol ester modulate steroid production by goldfish preovulatory ovarian follicles

Abstract: Recent studies have shown that agents that increase intracellular calcium content (calcium ionophore A23187) and activate protein kinase C (phorbol 12-myristate 13-acetate; PMA) modulate basal and cAMP-stimulated steroid production by goldfish preovulatory ovarian follicles. This report describes studies evaluating sites in the steroidogenic cascade (pre- and postadenylyl cyclase) influenced by calcium and protein kinase C. PMA (12.5–400 nM) inhibits hCG stimulated cAMP production, whereas phorbol 12, 13-dideconate, an inactive phorbol ester that does not activate protein kinase C, was without effect. Earlier studies suggested that PMA also acts post cAMP formation, but this action was shown to be exerted prior to cholesterol side-chain cleavage as metabolism of 25-hydroxycholesterol to testosterone was not inhibited by PMA. A high dosage of A23187 (4000 nM) inhibited the stimulatory action of hCG on cAMP and testosterone production; lower dosages of A23187 were without effect. In contrast, a wide range of A23187 dosages (250–4,000 nM) enhanced conversion of 25-hydroxycholesterol to testosterone. This effect appears to be exerted at the level of the cholesterol side-chain cleavage enzyme as A23187 did not affect conversion of exogenous pregnenolone to testosterone. These data demonstrate specific regulatory actions of calcium and protein kinase C at multiple sites in the steroidogenic cascade both proximal and distal to cAMP generation. © 1992 Wiley-Liss, Inc.