Showing papers on "Pregnenolone published in 1986"
TL;DR: By expression of bovine adrenocortical P-450(17 alpha) in COS 1 (transformed monkey kidney) cells, it is established in situ that a single polypeptide chain does catalyze both the 17 alpha-hydroxylase and the 17,20-lyase reactions.
Abstract: Cortisol production requires the activity of only 17 alpha-hydroxylase, whereas the formation of sex steroids requires both 17 alpha-hydroxylase and 17,20-lyase activities. Studies in reconstituted enzyme systems have suggested that a single steroid hydroxylase, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha), catalyzes both activities. By expression of bovine adrenocortical P-450(17 alpha) in COS 1 (transformed monkey kidney) cells, which normally contain no detectable P-450(17) alpha, it has now been established in situ that a single polypeptide chain does catalyze both the 17 alpha-hydroxylase and the 17,20-lyase reactions. This heterologous system supports 17 alpha-hydroxylation of pregnenolone and progesterone with equal efficiency, but catalyzes about five times as much 17,20-lyase activity when 17 alpha-hydroxypregnenolone is the substrate than when 17 alpha-hydroxyprogesterone is the substrate. For these activities to be observed in COS 1 cells, newly synthesized apocytochrome P-450(17) alpha must bind heme and insert into the endoplasmic reticulum such that endogenous cytochrome P-450 reductase can support hydroxylation. Thus, COS 1 cells are a useful system for expression and study of various forms of cytochrome P-450.
388 citations
TL;DR: The data demonstrate that P450PCN1 and P450 PCN2 genes are differentially regulated during development and after administration of inducing compounds and furthermore suggest that both enzymes possess testosterone 6 beta-hydroxylase activity.
Abstract: A cytochrome P-450 cDNA clone, designated pP450PCN2, homologous to the previously characterized pregnenolone 16 alpha-carbonitrile (PCN)-induced P-450 cDNA (pP450PCN1; F. J. Gonzalez, D. W. Nebert, J. P. Hardwick, and C. B. Kasper, J. Biol. Chem. 260:7435-7441), was isolated from a rat liver cDNA expression library by use of a polyclonal anti-P450PCN1 antibody. This P-450 cDNA contains 2,014 base pairs and yields an open reading frame of a protein consisting of 504 amino acids (Mr = 57,760). P450PCN2 cDNA and protein shared 90% nucleotide and 89% amino acid similarity with P450PCN1 cDNA and protein, respectively. The 5' untranslated, coding, and 3' untranslated regions between the two cDNAs share 94, 93, and 79% similarities, respectively. Nucleotide differences in the coding regions, however, are not evenly distributed. Complete homology exists between the two mRNAs for 425 nucleotides (positions 346 through 771). Other regions of 93 nucleotides containing only one difference and 147 nucleotides containing two differences exist toward the 3' end of the coding regions. These data suggest the possibility that a gene conversion event(s) have occurred subsequent to duplication of the ancestral P450PCN gene. Oligonucleotide probes unique for P450PCN1 and P450PCN2 cDNAs were used to examine the levels of their respective mRNAs in noninduced and PCN-induced liver cells and in male and female rats of various ages. P450PCN1 mRNA was not detectable in either male or female rats at any ages. In contrast, P450PCN2 mRNA was present at a low level in newborn rats and became elevated in both males and females at 1 week of age. Levels of p450PCN2 mRNA continued to increase in males until 12 weeks, whereas the mRNA in females reached peak levels at 2 weeks of age but declined continuously at the onset of puberty (between 4 and 12 weeks). These levels of P45PCN2 mRNA closely parallel the increases in testosterone 6 beta-hydroxylase activity and P450PCN2 protein level, as analyzed by Western blots. P450PCN1 mRNA was induced by PCN, dexamethasone, and phenobarbital in both male and female rats. P450PCN2 mRNA was not significantly induced by PCN or dexamethasone but was readily induced by phenobarbital. Testosterone 6 beta-hydroxylase activity was also induced severalfold by PCN, dexamethasone, and phenobarbital. These data demonstrate that P450PCN1 and P450PCN2 genes are differentially regulated during development and after administration of inducing compounds and furthermore suggest that both enzymes possess testosterone 6 beta-hydroxylase activity.
288 citations
TL;DR: Gonadotropin-specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P 450scc gene transcription, strongly support the two-cell theory of human ovarian estrogen synthesis.
Abstract: Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to 32P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0-100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02-2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490% and 240% of control values, respectively, with hCG, to 166% and 168% with FSH, and to 495% and 380% with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10(-6) M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin-specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription. Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with 32P-labeled human P450c17 cDNA (P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450c17 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis.
244 citations
TL;DR: There was a definite trend for the free dehydroepiandrosterone values to be higher in women, and the possible significance of these observations is discussed.
179 citations
TL;DR: A clear coupling could be demonstrated between the morphological and functional maturation of the steroidogenic mitochondrion in the ovary: from a few lamella cristae devoid of P-450scc in the unstimulated granulosa mitochondria, to numerous tubulovesicular inner membranes, heavily loaded with the enzyme, in the mitochondria of the interstitial cells.
Abstract: Rabbit antiserum to rat cytochrome P-450 cholesterol side chain cleavage (P-450scc) was produced without a previous biochemical purification of the enzyme. Instead, for immunization we used a single protein band of mol wt 53,000, which was isolated from sodium dodecyl sulfate polyacrylamide gel electrophoresis of rat steroidogenic mitochondrial membranes. The resulting antiserum cross-reacted in a proteinblotting test with affinity purified and biologically active bovine adrenocortical P-450scc enzyme. The antiserum to the rat P-450scc also substantially blocked the conversion of cholesterol to pregnenolone in sonicated steroidogenic mitochondria, suggesting a successful cross-reactivity with the native form of the enzyme, despite the fact that the immunizing antigen was sodium dodecyl sulfate-denatured protein. The antiserum was applied for ultrastructural immunocytochemical visualization of the P-450scc in thin sections of adrenal cortex and immature ovary. Immunoreactive enzyme was identified by the pr...
154 citations
TL;DR: Pieces of theca interna or follicle wall, obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione, suggesting that the delta 5 (delta 5) pathway is the preferred pathway for androstensione synthesis by bovines theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone.
Abstract: Pieces of theca interna or follicle wall (theca interna + attached granulosa cells), obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione. Luteinizing hormone, but not follicle-stimulating hormone (FSH), increased production of androstenedione 3 to 4-fold. In both the presence and absence of LH, follicle wall preparations secreted about 4-fold more androstenedione than did equivalent amounts of theca interna tissue. Isolated granulosa cells produced only negligible quantities of androstenedione, which suggests that they may contribute to the greater production of androstenedione by follicle wall by supplying progestin precursor to the theca cells. The addition of pregnenolone or progesterone to isolated theca interna increased the secretion of androstenedione, but pregnenolone was by far the more effective precursor. This suggested that the delta 5 (delta 5) pathway is the preferred pathway for androstenedione synthesis by bovine theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone. Follicle wall and granulosa cell cultures secreted 2 and 7 times more pregnenolone, respectively, than did theca cultures. Luteinizing hormone, but not FSH, increased production of pregnenolone by the follicle wall, whereas the gonadotropins had no effect on secretion by either granulosa or theca cells. Since exogenous testosterone enhanced the production of pregnenolone by granulosa cells, thecal androgen (which is stimulated by LH) may increase the ability of granulosa cells to make pregnenolone and explain the stimulatory effect of LH on pregnenolone secretion by follicle wall.(ABSTRACT TRUNCATED AT 250 WORDS)
140 citations
TL;DR: Patterns of 17 beta-estradiol 2- and 4-hydroxylation activities both increased rapidly during puberty in male rats and were induced by treatment of rats with phenobarbital or pregnenolone 16 alpha-carbonitrile.
Abstract: The participation of rat hepatic P-450 in the conversion of 17β-estradiol to catechol estrogens was examined by means of enzyme reconstitution and immunoinhibition studies. It was thus demonstrated that three rat liver microsomal cytochrome P-450 forms, designated P-450UT-A, P-450PCN-E, and P-450ISF-G, each contribute to the 2- and 4-hydroxylation of 17β-estradiol catalyzed by hepatic microsomal preparations. Two of these enzymes, P-450UT-A and P-450PCN-E, are expressed constitutively, are male-specific, and are regulated by testosterone as well as influenced by the administration of various chemicals. Consistent with these observations, 17β-estradiol 2- and 4-hydroxylation activities both increased rapidly during puberty in male rats and were induced by treatment of rats with phenobarbital or pregnenolone 16α-carbonitrile. Castration of male rats at birth or at 5 weeks of age suppressed the levels of 17β-estradiol 2- and 4-hydroxylase activities measured at 10 weeks of age. This suppression of activity w...
126 citations
TL;DR: The results suggest that in the adrenal, T GF-beta or TGF-beta-like proteins may be playing an important role in modifying the differentiated state of the adrenocortical cell.
Abstract: Transforming growth factor type beta (TGF-beta) suppresses basal as well as corticotropin (ACTH)-stimulated steroid formation by bovine adrenocortical cells in culture. The effect is dose dependent and is not accompanied by any change in adrenocortical cell growth. The minimum effective dose of TGF-beta is 4 X 10(-13) M (10 pg/ml), and maximal inhibition is observed at a concentration of 4 X 10(-11) M (1 ng/ml). A 16- to 20-hr incubation with TGF-beta is required to decrease steroidogenesis, and 12-18 hr are required before cells treated with TGF-beta recover complete responsiveness to corticotropin. Increases in cAMP mediated by corticotropin, forskolin, and isobutylmethylxanthine are not modified by the addition of TGF-beta; thus adenylate cyclase activity is unaffected by TGF-beta. Although TGF-beta inhibits the formation of all of the delta 4-steroids measured (including cortisol, corticosterone, aldosterone, and androstenedione), its effect can be completely reversed by the addition of 25-hydroxycholesterol, pregnenolone, or progesterone to the cells. In contrast, the addition of low density lipoprotein has no effect suggesting that TGF-beta targets the conversion of cholesterol precursors to cholesterol. The results demonstrate a highly potent effect of TGF-beta on the differentiated function of the adrenocortical cell. The inhibition of steroidogenesis can be dissociated from any effect on cell proliferation, and it occurs distal to the formation of cAMP but proximal to the formation of cholesterol. The results suggest that in the adrenal, TGF-beta or TGF-beta-like proteins may be playing an important role in modifying the differentiated state of the adrenocortical cell.
101 citations
TL;DR: In rats with testosterone implants that suppressed LH levels to less than 2 ng/ml and maintained normal FSH levels, as well as in hypophysectomized rats, no repopulation of Leydig cells could be observed until 35 days after EDS treatment, which indicates that some Leydigs cells from sterile rats, unlike LeydIG cells from normal rats, were resistant to EDS.
Abstract: After selective destruction of Leydig cells in mature rats with ethylene dimethane sulfonate (EDS), repopulation of Leydig cells occurs. This repopulation process was studied in normal and sterile (prenatally irradiated) rats using morphological and histochemical techniques and by measuring hormone concentrations. Three days after administration of EDS to normal rats, extensive Leydig cell degeneration had occurred, testosterone concentrations were decreased to less than 10% of the normal value, and no 3 beta-hydroxysteroid dehydrogenase activity or pregnenolone production could be detected in isolated interstitial cells. Seven days after EDS administration, no cells with the appearance of Leydig cells were observed, and steroidogenic activities were still absent. After 14 days, single or paired Leydig cells were present again in the interstitium, but only after 21 days an increase in the plasma testosterone concentration and LH-dependent pregnenolone production was observed. On day 35, numerous Leydig cells were present, and testosterone levels were restored to normal. The depletion and repopulation of Leydig cells after administration of EDS to sterile rats showed a somewhat different pattern. Three days after administration of EDS, testosterone concentrations were decreased to less than 10% of the normal value, and isolated interstitial cells showed no steroidogenic activities as in normal rats, but a small number of Leydig cells was still present. A similar picture was observed between 4 and 9 days after EDS administration. This indicates that some Leydig cells from sterile rats, unlike Leydig cells from normal rats, were resistant to EDS. The repopulation of Leydig cells in sterile rats was faster than in normal rats. After 14 days, many groups of Leydig cells were present in the interstitium, and the plasma testosterone concentration and pregnenolone production in vitro were significantly increased. Normal plasma testosterone levels were restored on day 21. Serum LH and FSH were decreased immediately after EDS administration, but during the next days a sharp rise was observed in both normal and sterile rats. The rise in LH correlated with the decrease in testosterone, and restoration of LH levels took place when testosterone levels increased. FSH levels changed similarly, but were delayed, in comparison to LH. In rats with testosterone implants that suppressed LH levels to less than 2 ng/ml and maintained normal FSH levels, ranging from 150-340 ng/ml, as well as in hypophysectomized rats, no repopulation of Leydig cells could be observed until 35 days after EDS treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
93 citations
TL;DR: The results demonstrate that an insulin-like growth factor, somatomedin C, exerts discrete differentiating effects on ovarian cells characterized by increased synthesis of immunospecific cytochrome P-450scc and adrenodoxin.
91 citations
TL;DR: The volume of milt that could be stripped from male goldfish, Carassius auratus, increased dramatically when fish were exposed overnight to water with concentrations of 17,20β-dihydroxy-4-pregnen-3-one (17,20P) as low as 10−10 M.
Abstract: The volume of milt that could be stripped from male goldfish, Carassius auratus, increased dramatically when fish were exposed overnight to water with concentrations of 17α,20β-dihydroxy-4-pregnen-3-one (17,20P) as low as 10−10 M. A variety of free steroids (pregnenolone, androstenedione, testosterone, 11-ketotestosterone, 17β-estradiol) and glucuronated steroids (etiocholanolone glucuronide, testosterone glucuronide, 17β-estradiol glucuronide), suggested by others to function as pheromones in fish, failed to increase milt volume at a concentration of 5 × 10−9 M. The milt response appears to be specific to 17,20P and progesterone precursors (17α-hydroxyprogesterone, progesterone), but is most sensitive to 17.20P. Because bilateral section of the olfactory tracts completely blocked the milt response to 17.20P, it is believed to be a pheromone. Selective sectioning of the lateral and medial subdivisions of the olfactory tracts demonstrated that the milt response to 17,20P is dependent on the medial tract. I...
TL;DR: Results are consistent with several previous studies involving salmonids and various other teleosts, and suggest a possible physiological role for a 20 beta-dihydroprogestin in the resumption of meiotic maturation in F. heteroclitus.
TL;DR: Steroid metabolism in the seminal vesicles of Clarias gariepinus, collected in the Hula nature reserve and in a fish pond in North Israel during the spawning season, was studied in vitro by homogenate and tissue incubations with [3H]pregnenolone, [3h]dehydroepiandrosterone, or [3 H]androstenedione as precursor.
TL;DR: It is concluded that estradiol and somatomedin-C interact synergistically in a time- and dose-dependent manner to enhance the biosynthesis of pregnenolone and progesterone by swine granulosa cells.
Abstract: Estradiol amplified synergistically the dose- and time-dependent stimulatory actions of human somatomedin-C on progesterone biosynthesis by cultured swine granulosa cells. This facilitative interaction was not attributable to inhibition of the catabolism of progesterone to 20α-hydroxypregn-4-en-3-one, but, rather, reflected time-dependent stimulation of pregnenolone synthesis measured in the presence of exogenous soluble sterol substrate for cholesterol side-chain cleavage. Moreover, treatment with somatomedin-C was accompanied by increased synthesis of two immunoprecipitable cholesterol side-chain cleavage constituents, viz. cytochrome P-4508CC and adrenodoxin. The synergism between estradiol and somatomedin-C was associated with significantly greater specific binding of somatomedin- C in estrogen-treated than control cultures, with no change in apparent receptor affinity. In vitro synergism occurred at somatbmedin-C concentrations estimated by sequence-specific immunoassay to be attainable in ovarian fo...
Journal Article•
TL;DR: Findings are consistent with the view that cytochrome P-450 isozymes that catalyze 6 beta-hydroxylation of steroids in rat liver microsomes also catalyze the dehydrogenation of testosterone to form a double bond between the C-6 and C-7 positions.
Abstract: A testosterone metabolite, 17 beta-hydroxy-4,6-androstadiene-3-one, possessing an absorbance maximum at 284 nm, was formed during incubation of testosterone with liver microsomes from dexamethasone-treated rats. The metabolite was identified by HPLC, UV spectroscopy, and thermospray liquid chromatography/mass spectrometry. The formation of this metabolite by rat liver microsomes required NADPH and oxygen and was inhibited markedly by SKF 525-A, 2,4-dichloro-6-phenylphenoxyethylamine, or CO/O2 (8:2, v/v), but not by cyanide, an inhibitor for stearyl-CoA desaturase. Pretreatment of rats with phenobarbital, pregnenolone 16 alpha-carbonitrile, and dexamethasone enhanced the formation of this metabolite in parallel with the increase in formation of 6 beta-hydroxytestosterone (r2 = 0.99). Although 16-methylprogesterone, a known 6 beta-hydroxylase inhibitor, competitively inhibited the formation of the metabolite and 6 beta-hydroxytestosterone by liver microsomes from dexamethasone-treated rats, the metabolite was not formed from either 6 beta-hydroxytestosterone or 7-hydroxytestosterone during incubation with liver microsomes. These findings are consistent with the view that cytochrome P-450 isozymes that catalyze 6 beta-hydroxylation of steroids in rat liver microsomes also catalyze the dehydrogenation of testosterone to form a double bond between the C-6 and C-7 positions.
TL;DR: TGF-beta appears as a potent modulator of differentiated adrenocortical cell functions in vitro and may play a significant role in the development and the regulation of adrenal cortex in vivo.
TL;DR: Kinetic analyses of 17-hydroxylase and 17,20-desmolase activities have been performed on human adrenal microsomes, compatible with the suggestion that increased enzyme synthesis mediated by ACTH and differential inhibition by endogenous steroids may, in part, account for developmental changes in adrenal hormone secretion.
Abstract: Kinetic analyses of 17-hydroxylase and 17,20-desmolase activities have been performed on human adrenal microsomes from 12 individuals, aged 1-60 yr. The median Michaelis constant of 17-hydroxylase for the substrate pregnenolone was 0.09 microM, and that of 17,20-desmolase for the substrate 17-hydroxypregnenolone was 0.12 microM. The median maximum velocity of 17-hydroxylase (0.25 nmol/mg X min) was significantly greater than that of the desmolase (0.13 nmol/mg X min). There was no significant correlation between the age of the adrenal donor and the Michaelis constant, but the maximum velocity for both activities in the single infant donor was lower than the values in older individuals. The inhibitory effects of various steroids on both enzyme activities also were studied. All steroids examined, except cortisol, competitively inhibited both enzymes. In each case the inhibition constant was higher for 17-hydroxylase than for 17,20-desmolase, indicating that C21 side-chain cleavage would be more sensitive to inhibition by endogenous steroids. The results, taken together with those of similar studies of 3 beta-hydroxysteroid dehydrogenase kinetics, are compatible with the suggestion that increased enzyme synthesis mediated by ACTH and differential inhibition by endogenous steroids may, in part, account for developmental changes in adrenal hormone secretion.
TL;DR: Results of this investigation demonstrate that the rat placenta secretes significantly more androstenedione that testosterone both in vivo and in vitro, produces more androgen than the corpus luteum because of its greater mass, and forms its androgen primarily via the delta 4-st
Abstract: In contrast to the human placenta, which does not secrete androgens, the rat placenta synthesizes significant amounts of these steroids. The purpose of this study was to determine why the rat placenta does not secrete androgens before day 12 of pregnancy, to ascertain whether the rat placenta secretes more androstenedione than testosterone, to compare the capacity of luteal and placental tissue to secrete androgen, and to determine whether the rat placental produces androstenedione via the delta 4- or delta 5-steroidogenic pathway. To determine whether the inability of the rat placenta to produce androstenedione before midpregnancy was due to the absence of active 17 alpha-hydroxylase and 17,20-lyase enzymes and also to investigate the ontogeny of both placental production of androstenedione and enzyme activities, placentas were isolated from rats between days 8-21 of pregnancy and either incubated or used to determine the activities of 17 alpha-hydroxylase and 17,20-lyase. Before day 11, enzyme activity was not detectable. From day 11, both enzyme activities and placental secretion of androstenedione steadily increased to peak values by day 18 and declined just before parturition. To investigate the principal aromatizable androgen secreted both in vivo and in vitro approaches were used. Levels of androstenedione and testosterone found in the uterine vein as well as those produced by placental tissue were determined. Rat placentas secreted markedly more androstenedione than testosterone, both in vivo and in vitro. When placental and luteal secretion of androstenedione and testosterone were compared, it was found that luteal tissue had a higher capacity for androgen synthesis than did the placenta. Yet, because of its greater mass, each placenta secreted 15 times more androstenedione and 4.5 times more testosterone than each corpus luteum. To determine the preferential usage of progesterone or pregnenolone as substrate by the placenta, [14C] progesterone and [3H]pregnenolone were added in equimolar concentrations. The resulting 14C to 3H ratio of the androgen produced indicates that the preferred substrate is progesterone. In summary, results of this investigation describe, for the first time, the development of 17 alpha-hydroxylase and 17,20-lyase activities in the rat placenta and demonstrate that the placenta does not produce androgen before day 11 due to the absence of active enzymes. The results further demonstrate that the rat placenta secretes significantly more androstenedione that testosterone both in vivo and in vitro, produces more androgen than the corpus luteum because of its greater mass, and forms its androgen primarily via the delta 4-st
TL;DR: It is concluded that the liver enzymes controlled by the postulated PCN recognition system include not only P-450p but also one or more UDP-glucuronosyltransferases.
TL;DR: With the technique used, dehydroepiandrosterone, progesterone, 17 alpha-hydroxyprogesterone, and estrogens could not be detected in male or female hemolymph extracts.
TL;DR: In this article, it is shown that the electron transfer reaction from NADPH to P-450 scc is mediated rapidly by adrenodoxin reductase and p-450scc.
TL;DR: Comparison of the calculated Ki values with available data concerning changes in intra-adrenal steroid concentrations during childhood suggests that these changes would be sufficient to cause a relative decline in 3 beta-HSD activity during adrenarche.
Abstract: The inhibitory effects of varying concentrations of steroids upon 3β-hydroxysteroiddehydrogenase/Δ5-Δ4 isomerase (3β–HSD) kinetics were studied in human adrenal microsomes. Each enzyme assay was conducted in triplicate at five different concentrations of three substrates (dehydroepiandrosterone, pregnenolone, and 17OH-pregnenolone), using microsomes from at least three donors. Each steroid was screened for possible inhibition at concentrations of 10-−8 and 10−6M and thenstudied in more detail at five different concentrations. The type of inhibition and the inhibition constant (Ki) were determined by analysis of Lineweaver-Burk and Dixon plots,together with replots of the slopes from the Dixon plots. The mean Km (Michaelis-Menten constant)for the three substrates was 0.42 ± 0.04 (SE) μmol/liter (n = 73). Each steroidtested, including Δ5-3β-hydroxysteroids, estrogens, and several Δ4–3–ketosteroids, with the exception of cortisol, caused significant inhibition of 3β-HSD activity, and in each case the steroid...
TL;DR: It is interpreted to indicate that the continuous presence of extracellular calcium is essential for hormonal regulation of steroidogenesis and is important for events both proximal and distal to cyclic AMP generation up to pregnenolone synthesis.
TL;DR: In contrast to the biphasic effect of PRL on production of androgen, PRL treatment enhanced hCG-stimulated production of progesterone in a dose-related manner without exerting an inhibitory effect.
Abstract: Prolactin (PRL) exerts both stimulatory and inhibitory effects upon testicular steroidogenesis in vivo. The direct effects of PRL on biosynthesis of testicular androgen were studied in primary cultures of testicular cells obtained from adult, hypophysectomized or neonatal, intact rats. In cells from adult animals, treatment with human chorionic gonadotropin (hCG) (10 ng/ml) significantly increased testosterone and progesterone production relative to their respective controls. In contrast, neither steroid was increased by treatment with rat PRL (rPRL) or ovine PRL (oPRL) alone. Upon addition of 0.1-3 ng/ml of either rPRL or oPRL to the hCG-treated cultures, testosterone production was progressively increased up to a maximum of 70% greater than with hCG alone. However, when PRL exceeded 3 ng/ml, the testosterone response began to decline and was 39 or 24% less than from cells treated with hCG alone at 300 ng/ml of rPRL or oPRL, respectively. A similar biphasic response pattern was observed in cells from neonatal animals. In contrast to the biphasic effect of PRL on production of androgen, PRL treatment enhanced hCG-stimulated production of progesterone in a dose-related manner without exerting an inhibitory effect. At 3 and 300 ng/ml, rPRL augmented hCG action by 2.5- and 8-fold, respectively. Similarly, in the presence of inhibitors of pregnenolone metabolism, rPRL also enhanced hCG-stimulated production of pregnenolone. Quantitation of steroid intermediates in the testosterone biosynthetic pathway revealed that the stimulatory effect of 3 ng/ml rPRL on testosterone production was associated with 1.3- and 2.8-fold increases in accumulation of androstenedione and 17 alpha-hydroxyprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Ketoconazole inhibits the conversion of cholesterol to pregnenolone in a dose-dependent fashion, and is a more potent inhibitor than metyrapone in the P 450scc-catalyzed reaction system; other inhibitors fail to show any inhibitory effects in this system.
TL;DR: It is shown that LH action involves calcium fluxes through the plasma membrane, in addition to activation of protein kinase A, which indicates that cyclic AMP is not involved in the stimulation of steroid production by an LH-releasing hormone agonist.
Abstract: The stimulation of steroid production in Leydig cells by LH is accompanied by increased cyclic AMP levels, activation of protein kinase A, increased phosphorylation of at least six phosphoproteins and requires protein synthesis. However, an LH-releasing hormone agonist (LHRH-A) can stimulate steroid production without stimulation of cyclic AMP levels. In the present study we have shown that LH action involves calcium fluxes through the plasma membrane, in addition to activation of protein kinase A. The action of LHRH-A, in contrast, does not require calcium fluxes and is not potentiated by 1-methyl-3-isobutylxanthine, indicating that cyclic AMP is not involved. Extracellular calcium is required for the action of both LH and LHRH-A. An increase in intracellular calcium concentration due to the effect of ionophore A23187 did not stimulate steroidogenesis and had deleterious effects on intracellular adenosinetriphosphate levels. LH and 4 beta-phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C, both stimulated phosphorylation of proteins of 17 000 and 33 000 mol. wt, whereas LHRH-A had no effect. However, compared with the effect of LH, PMA had a much smaller effect on steroid production, indicating that even if protein kinase C may be activated by LH its role in the regulation of steroid production may be less important than the role of protein kinase A. Action of LHRH-A does not appear to be mediated by calcium fluxes, protein kinase C activation or active protein phosphorylation.
TL;DR: Vasoactive intestinal peptide stimulated androgen production in an LH-responsive subpopulation of testis cells but failed to affect steroid production in non-LH-responsive cells.
TL;DR: The intratesticular steroid pattern in elderly men was essentially characterized by a decrease of the 5-ene steroid concentration, whereas the authors did not observe a decrease in the 4-ene steroids, progesterone concentration being even significantly higher in the aged testes.
TL;DR: Rat testicular interstitial fluid and hydroxycholesterol both stimulated testosterone production by isolated Leydig cells in vitro in a dose-dependent manner, but the dose-response lines were not parallel.
Abstract: Rat testicular interstitial fluid and hydroxycholesterol both stimulated testosterone production by isolated Leydig cells in vitro in a dose-dependent manner, but the dose-response lines were not parallel. The addition of cycloheximide blocked the stimulation by interstitial fluid but not that of hydroxycholesterol. Use of the compounds SU 10603 and cyanoketone (which inhibit 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase respectively) or aminoglutethimide (which acts on the cholesterol side-chain cleavage enzyme) showed that the stimulatory factor(s) in interstitial fluid stimulated steroidogenesis at the cholesterol side-chain cleavage enzyme, before the conversion of pregnenolone. This enzyme is rate-limiting in the synthesis of testosterone by Leydig cells and a site of action of LH; therefore, these results support the view that an interstitial fluid factor may be involved in the paracrine regulation of testicular steroidogenesis.
TL;DR: It is hypothesized that some of the psychotropic actions of progestational agents may involve an enhancement of ‘purinergic’ tone in the central nervous system.
Abstract: The effects of four progestational agents pregnenolone sulphate, cyproterone acetate, norethindrone acetate and progesterone, on adenosine-evoked depression of the firing of rat cerebral cortical neurones have been studied. When applied iontophoretically, pregnenolone sulphate, cyproterone, and norethindrone enhanced the actions of iontophoretically applied adenosine and failed to potentiate the depressant effects of adenosine 5'-N-ethylcarboxamide and gamma-aminobutyric acid. Cyproterone acetate (50 micrograms kg-1) and progesterone (200 micrograms kg-1) administered intravenously enhanced the depressant actions of iontophoretically applied adenosine. When applied by large currents, cyproterone, and less frequently norethindrone, depressed the firing of cerebral cortical neurones. The depressant effects of cyproterone were antagonized by caffeine. Pregnenolone sulphate tended to excite cortical neurones but neither this action, nor its potentiation of adenosine were reproduced by application of sulphate ions. It is hypothesized that some of the psychotropic actions of progestational agents may involve an enhancement of 'purinergic' tone in the central nervous system.