Developmental expression of genes for the stereoidogenic enzymes P450scc (20,22-desmolase), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase) in the human fetus

Fetal adrenal steroidogenesis is required for the production of placental estrogen, and fetal testicular steroidogenesis is required for the development of male external genitalia. We studied the ontogeny and tissue specificity of expression of the genes for three steroidogenic enzymes: P450scc (the cholesterol side-chain cleavage enzyme), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase) in the human fetus. RNA from fetal tissues was probed with homologous human P450scc, P450c17, and P450c21 cDNAs cloned in our laboratory. At 20-21 weeks gestation, P450scc mRNA was most abundant in the adrenal, followed by testis, placenta, and ovary. P450c17 mRNA was also most abundant in the adrenal, followed by testis and ovary, but was undetectable in the placenta. P450c21 mRNA was detected only in the adrenal. None of these mRNAs was detected in kidney, liver, spleen, intestine, or muscle. Twenty-two fetal testis samples (13-25.8 weeks gestation) were studied. P450scc and P450c17 mRNAs were most abundant at 14-16 weeks and diminished to 35 and 19% of their peak values, respectively, by 20-25.8 weeks. Ovarian P450scc and P450c17 mRNAs were present, respectively, in only 6.2% and 1.8% of the maximum amount in the testis and did not vary detectably from 14.9 to 21.5 weeks gestation. The testicular and ovarian steroidogenic enzyme mRNA data correlate well with previously reported changes in gonadal steroidogenesis with gestational age. The presence of P450scc mRNA, but not P450c17 mRNA, in the placenta indicates that the placenta is able to initiate the synthesis of some steroid hormones, but is not able to synthesize estrogen de novo. Since P450c21 was found only in the adrenal, the extraadrenal 21-hydroxylation of progesterone to deoxycorticosterone, a common event in the fetus, is probably mediated by an enzyme(s) other than P450c21.     

Interaction of phorbol ester and adrenocorticotropin in the regulation of steroidogenic P450 genes in human fetal and adult adrenal cell cultures.

The role of protein kinase-C-dependent mechanisms in steroidogenic enzyme gene expression was studied in primary cultures of human fetal and adult adrenals. Cells were first cultured for 7-10 days and then stimulated with ACTH or 12-O-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase-C activator, for 1-2 days. Cytoplasmic RNA was extracted and analyzed by Northern and dot blotting with 32P-labeled cDNA probes for P450scc (cholesterol side-chain cleavage enzyme/20,22-desmolase), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase); for P450c11 (11 beta-hydroxylase/18-hydroxylase/18-methyl oxidase), a 30-mer oligonucleotide was used as a probe. ACTH (200 ng/ml) increased the accumulation of all of the studied steroidogenic enzyme mRNAs in both fetal and adult cultures by several-fold. TPA inhibited this accumulation in a dose-dependent manner (0.01-100 ng/ml), whereas the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate was without effect. On the other hand, in the absence of ACTH, TPA slightly increased all steroidogenic P450 mRNAs in adult cultures. In fetal cultures TPA slightly increased P450scc, P450c11, and P450c21 mRNA levels, whereas it decreased P450c17 mRNA. (Bu)2cAMP and cholera toxin increased steroidogenic enzyme mRNAs such as ACTH. TPA down-regulated (Bu)2cAMP- and cholera toxin-induced P450mRNAs in the same way as ACTH-induced mRNAs. The secretion of ACTH-stimulated cortisol, dehydroepiandrosterone sulfate, and aldosterone was decreased by TPA in both fetal and adult cultures. The basal steroid production was slightly increased by TPA in both culture types. The changes in steroid production correlated well with the alterations in the steroidogenic enzyme gene expression. Our results show that the inhibitory effect of TPA on ACTH-stimulated adrenal steroidogenesis is mediated at the mRNA level of steroidogenic enzymes. Thus, it seems likely that both protein kinase-C- and cAMP-dependent mechanisms are involved in the long term maintenance of steroidogenic enzymes and hormone production in adrenocortical cells.     

Regulation of adrenal steroidogenesis by adrenaline: expression of cytochrome P450 genes.

The effect of adrenaline on the secretion of cortisol and cyclic AMP (cAMP) and on the accumulation of four different mRNAs encoding cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450(17 alpha)), 21-hydroxylase cytochrome P450 (P450c21) and 11 beta-hydroxylase cytochrome P450 (P450(11 beta)) was studied in bovine adrenocortical cells in primary culture and compared with the effects of ACTH. Treatment of cultured cells with adrenaline (1-100 mumol/l) showed a biphasic response in cortisol release over 1-24 h. Concentration of cAMP in the culture media increased from a basal level of < 0.06 pmol/dish to a maximal level of 40.14 +/- 8.9 pmol/dish with a half-maximal release of 20.07 pmol cAMP/dish in the medium reached 1.2 h after treatment with 10 mumol adrenaline/l. This stimulation resulted in an uniform increase in the levels of all four P450 mRNAs as revealed by Northern blot analysis. Increasing doses of adrenaline produced a maximal mRNA accumulation at a concentration of 10 mumol adrenaline/l. Incubation of the cells with 10 mumol adrenaline/l for 1-24 h produced a biphasic time-course with a half-maximal stimulation after about 5-6 h. Maximal stimulation with ACTH (100 nmol/l) caused different accumulations of the four mRNAs: P450sec mRNA increased twice as much and P450(17 alpha) mRNA six times as much as the accumulation of P450c21 mRNA and P450(11 beta) mRNA, which was about ten-fold over basal values. Propranolol totally blocked the stimulatory effect of adrenaline but not the effect of ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased expression of cytochrome P450 17 alpha-hydroxylase mRNA in senescent bovine adrenal gland.

To investigate the mechanisms of age-related decline in synthesis of adrenal androgen, we studied the contents of cortisol (F) and dehydroepiandrosterone (DHEA) and the amounts of cytochrome P450 17 alpha-hydroxylase (P450c17) mRNA and cytochrome P450 11 beta-hydroxylase (P450c11) mRNA in young (1 year old) and senescent (10-12 years old) bovine adrenal glands. We also examined effects of ACTH (10(-7) M) on the secretion of F and DHEA and on the induction of P450c17 and P450c11 mRNA expression in cultured adrenal cells from young and aged cows. The content of DHEA in adrenal glands and the secretion of DHEA in response to ACTH in cultured adrenal cells from senescent cow were lower than those from young cow, while the content and ACTH-stimulated secretion of F in senescent adrenals were of a similar level to those in young adrenals. The adrenal gland from aged cow showed a significantly lower level of P450c17 mRNA compared with young bovine adrenal. However, P450c11 mRNA was expressed in senescent adrenal glands at the same level as that in young adrenals. The induction of P450c17 mRNA by ACTH (10(-7) M for 24 h) in cultured adrenal cells from aged cow also showed a decline compared with that from young cow, although there was no difference the ACTH-induced accumulation of P450c11 mRNA in cultured adrenal cells between young and senescent cow. These results suggested that the expression of P450c17 mRNA decreased in aged bovine adrenal, which may cause the age-associated decline in biosynthesis of adrenal androgen.     

Regulation of testicular P-450 cholesterol side-chain cleavage and P-450 C17-20 lyase/C17 hydroxylase enzymes in the neonatal and adult rat

Adult Leydig cells respond to LH or hCG with an initial stimulation of testosterone secretion followed by LH receptor down-regulation and blockade of androgen biosynthesis. In contrast, fetal Leydig cells respond with increased LH receptor number and enhanced steroidogenesis. In this study, the molecular mechanisms of high-dose hCG treatment on steroidogenesis in adult and neonatal testes (containing predominantly the fetal generation of Leydig cells) were examined using two recombinant DNA clones specific for enzymes of the rat steroidogenic pathway (P-450 cholesterol side-chain cleavage enzyme, P-450scc and P-450 17 alpha-hydroxylase/C17-20 lyase, P-450c17). We treated adult (60 days of age) and neonatal (2 days of age) rats with a single high dose of hCG (600 IU/kg), sc. The high dose of hCG caused neonatal testicular P450scc and P450c17 mRNA levels to increase, and stimulated adult testicular P450scc mRNA levels, but caused a decrease in adult P450c17 mRNA levels. These studies suggest that high doses of hCG regulate testosterone production differently in adult and fetal Leydig cells at a pretranslational level of the P450c17 enzyme, while mRNA for P450scc is stimulated in both the adult and fetal Leydig cell.     

Multiple mechanisms for regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5----delta 4-isomerase, 17 alpha-hydroxylase/C17-20 lyase cytochrome P450, and cholesterol side-chain cleavage cytochrome P450 messenger ribonucleic acid levels in primary cultures of mouse Leydig cells

The regulation of mRNA levels for delta 5-3 beta-hydroxysteroid dehydrogenase/delta 5----delta 4-isomerase (3 beta HSD), 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 (P450(17 alpha] and cholesterol side-chain cleavage cytochrome P450 (P450scc) was studied in primary cultures of mouse Leydig cells. Treatment of Leydig cells with 8-bromo-cAMP (cAMP) was essential for expression of P450(17 alpha) mRNA, but not for 3 beta HSD. Treatment with cAMP caused a decrease in basal levels of 3 beta HSD mRNA. The addition of aminoglutethimide (AG), an inhibitor of cholesterol metabolism, to the cAMP-treated cultures resulted in increased expression of both 3 beta HSD and P450(17 alpha) mRNA levels. The addition of testosterone or the androgen agonist mibolerone to cAMP- plus AG-treated cultures reduced 3 beta HSD and P450(17 alpha) mRNA to levels comparable to those observed when cells were treated with cAMP only. The glucocorticoid dexamethasone reduced both basal and cAMP- plus AG-induced increases in 3 beta HSD mRNA, but not in P450(17 alpha) mRNA. Estradiol at a concentration of 1 microM had no effect on cAMP- plus AG-induced 3 beta HSD or P450(17 alpha) mRNA levels. The role of protein synthesis in mediating the cAMP induction of 3 beta HSD, P450(17 alpha), and P450scc was investigated. The addition of cycloheximide (10 micrograms/ml) to cAMP-treated cultures for 24 h completely suppressed both constitutive and cAMP-induced 3 beta HSD mRNA levels. Cycloheximide also repressed cAMP-induced levels of P450(17 alpha) to 12% of levels observed in the absence of cycloheximide. In sharp contrast, 24-h treatment with cycloheximide did not suppress cAMP induction of P450scc mRNA, but reduced basal levels by approximately 50%. A time course of induction by cAMP (50 microM) of P450(17 alpha) and P450scc mRNA showed very similar rates of increase in P450(17 alpha) and P450scc mRNA, with the greatest increase occurring between 12 and 24 h of treatment. The results of the study demonstrate that in normal mouse Leydig cells steady state levels of mRNA for 3 beta HSD, P450(17 alpha), and P450scc are differentially regulated. cAMP is required for maximal levels of all three mRNAs. There is high constitutive expression of 3 beta HSD and P450scc mRNA, while expression of P450(17 alpha) mRNA is absolutely dependent on cAMP stimulation. Endogenously produced testosterone negatively regulates the expression of cAMP-induced P450(17 alpha) and 3 beta HSD, while the glucocorticoid dexamethasone negatively regulates 3 beta HSD and P450scc.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins essential for adrenal steroidogenesis in the late-gestation ovine fetus

To further understand the relative roles of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins that are essential for adrenal development, we investigated the effects of, first, an ACTH infusion and labour in intact fetuses and, secondly, the effect of an ACTH infusion to fetuses with and without a pituitary gland, on the relative abundance of the mRNA encoding for the ACTH receptor (MC2R), steroidogenic factor 1 (SF-1), cholesterol side-chain cleavage enzyme (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase (P450(C17)) in the fetal adrenal gland. ACTH(1-24) infusion (14.7 pmol/kg per h) to intact fetuses was without effect on the abundance of mRNA encoding MC2R and SF-1, irrespective of whether the infusion was given for 18 (115-132 days of gestation) or 32 days (115 days to term (147 days of gestation)). Hypophysectomy (HX) did not alter the expression of MC2R mRNA; however, the abundance of SF-1 mRNA fell by approximately 50% following the removal of the pituitary gland. ACTH(1-24) infusion to HX fetuses failed to restore levels of SF-1 mRNA to that seen in intact animals. P450(scc) and 3betaHSD mRNAs were increased by ACTH(1-24) infusion for 18 days in intact animals, although no effects of the infusion were seen on P450(C17) mRNA levels. For all three of these mRNAs, there was a significant increase in their abundance between 132 days of gestation and term in intact fetuses. By term, ACTH(1-24) infusion was without any additional effect on their abundance. HX decreased the expression of P450(scc), 3betaHSD and P450(C17) mRNAs, while ACTH(1-24) infusion to HX fetuses increased the expression of these mRNAs to levels seen in intact animals. There were significant correlations between the abundance of the mRNA for P450(scc), 3betaHSD and P450(C17), but not MC2R and SF-1, and premortem plasma cortisol concentrations. These results emphasise the importance of the pituitary gland and ACTH in the regulation of the enzymes involved in adrenal steroidogenesis. Factors in addition to ACTH may also play some role, as the infusion was not always effective in increasing the abundance of the mRNAs. Surprisingly, the mRNA for MC2R and SF-1 did not appear to be regulated by ACTH in the late-gestation ovine fetus, though a pituitary-dependent factor may be involved in the regulation of SF-1 mRNA abundance.     

Cytochrome P450c17 (steroid 17 alpha-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues.

P450c17 is the single enzyme mediating both 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. We sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, lambda hac17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17. Comparison of the amino acid sequence of P450c17 with two other human steroidogenic cytochromes P450 show much greater homology with P450c21 (28.9%), another microsomal enzyme, than with P450scc (12.3%), a mitochondrial enzyme.     

Adrenaline stimulates cholesterol side-chain cleavage cytochrome P450 mRNA accumulation in bovine adrenocortical cells.

The effect of adrenaline on the accumulation of mRNA encoding cholesterol side-chain cleavage cytochrome P450 (P450scc) and cortisol secretion was studied in bovine adrenocortical cells in primary culture. Treatment of cultured cells with adrenaline resulted in a 2-fold increase in mRNA encoding P-450scc, as revealed by Northern blot analysis. Under these conditions the maximal stimulation with ACTH resulted in a 6-fold accumulation of mRNA encoding P450scc. The effect of adrenaline on the expression of P450scc was abolished by the beta-blocker propranolol, while propranolol had no effect on ACTH-induced P450scc mRNA accumulation. Adrenaline stimulated the secretion of cortisol in a dose-dependent manner with a median effective dose of 0.5 mumol/l. The adrenaline-stimulated cortisol secretion amounted to 42% of the effect of ACTH (0.1 nmol/l). Upon adrenaline treatment, cAMP concentration in the culture medium increased about 50-fold over the basal value. It is concluded that the stimulatory action of adrenaline upon cortisol formation requires beta-adrenergic receptors and is due, at least in part, to a cAMP-mediated increases in the accumulation of mRNA encoding P450scc.     

Markedly increased expression of cytochrome P-450 17 alpha-hydroxylase (P-450c17) mRNA in adrenocortical adenomas from patients with Cushing's syndrome.

We studied the contents of cortisol (F) and dehydroepiandrosterone (DHEA) and the expression of mRNA of cytochrome P-450 for side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450c17), 21 alpha-hydroxylase (P-450c21) and 11 beta-hydroxylase (P-450c11) in adrenocortical adenomas from three patients with Cushing's syndrome. The F content was significantly higher in adrenocortical adenomas than in normal adrenal glands, while the DHEA level was similar to that in normal adrenal glands. The adrenal adenomas showed a markedly higher level of P-450c17 mRNA, and a slightly but not significantly increased level of P-450c21 mRNA, compared with normal adrenal glands. The expression of P-450scc and P-450c11 mRNA in the adenomas was similar to that in normal adrenal glands. These results suggest that the overproduction of cortisol in adrenocortical adenomas associated with Cushing's syndrome results from an increased expression of P-450c17 and P-450c21 mRNA.     

Interleukin-1 inhibits Leydig cell steroidogenesis primarily by decreasing 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 expression.

Macrophage-secreted cytokines, such as interleukin-1 (IL-1), have been shown to modulate Leydig cell function. The present study examined the effect of recombinant murine IL-1 alpha on Leydig cell steroidogenesis and its mechanism of action. Addition of IL-1 to macrophage-depleted primary cultures of mouse Leydig cells caused a dose-dependent decrease in cAMP-dependent testosterone production and 17 alpha-hydroxylase/C17-20 lyase (P450c17) mRNA levels. Chronic treatment (48 h) of Leydig cells in culture with 50 microM 8-bromo-cAMP (8-Br-cAMP) resulted in a 60-fold increase in testosterone production. Treatment with 8-Br-cAMP plus 0.2 and 2 U/ml IL-1 decreased testosterone production to 63 +/- 14% and 41 +/- 19%, respectively, while 5, 10, and 20 U/ml IL-1 decreased testosterone production to less than 5% that of cells treated with 8-Br-cAMP alone. Chronic treatment with IL-1 plus 8-Br-cAMP caused a shift in steroid production from testosterone to progesterone, but total steroid production (the sum of testosterone plus progesterone) was unaffected by IL-1 treatment. Treatment with 8-Br-cAMP alone caused a marked increase in P450c17 mRNA levels compared to that in control cultures, where P450c17 mRNA was undetectable. IL-1 caused a dose-dependent decrease in 8-Br-cAMP-stimulated P450c17 levels (0.2 U/ml by 31 +/- 9%, 2 U/ml by 82 +/- 12%, and 10 or 20 U/ml by 100% compared to that in cells treated with 8-Br-cAMP alone). In contrast to the effect on P450c17 mRNA, only the highest concentrations of IL-1 (10 and 20 U/ml) had any effect on cholesterol side-chain cleavage enzyme (P450scc) mRNA levels (53 +/- 16% and 38 +/- 20% decreases, respectively). The inhibitory effect of IL-1 on 8-Br-cAMP-stimulated P450c17 expression was reversible. Within 12 h after the removal of IL-1, P450c17 mRNA was restored to 24%; after 24 h, to 36%; after 36 h, to 65%; and after 48 h, to 84% of that with 8-Br-cAMP alone. P450c17 expression was more sensitive to IL-1-mediated inhibition than P450scc; therefore, inhibition of P450c17 is most likely primarily responsible for the observed inhibitory effects of IL-1 on Leydig cell testosterone production.     

Inherited congenital adrenal hyperplasia in the rabbit: absent cholesterol side-chain cleavage cytochrome P450 gene expression.

We investigated adrenal steroidogenic enzymes, their activity and mRNA expression, and in vitro biosynthesis of an enzyme in rabbits with congenital adrenal hyperplasia (CAH; weight: CAH, 19 +/- 5 mg/adrenal; normal, 2.7 +/- 1.0 mg/adrenal). Serum pregnenolone (delta 5-P) levels in CAH newborn rabbits (12-36 h) were normal (mean/range, 438/51-2191 ng/dl), but corticosterone levels were low [0.05 +/- 0.05 microgram/dl; P less than 0.001 vs. normal (0.66 +/- 0.57)]. Serum Na+ levels in CAH newborn rabbits were in the normal range (143 +/- 30 meq/liter), but K+ levels were elevated [7 +/- 1.1 meq/liter; P less than 0.05 vs. normal (5.9 +/- 0.6 meq/liter)]. Minced normal adrenal tissue incubated with [3H] cholesterol (30-100 pmol/flask) and ACTH (100 mU/flask) produced [3H]delta 5-P (newborn, 21 and 45 fmol/100 mg; adult, 3 and 5 fmol/100 mg) and [3H]corticosterone (newborn, 23 fmol/100 mg; adult, 11.3 fmol/100 mg), but CAH adrenals produced no product (less than 1.3 fmol/100 mg). Adrenal mitochondria from normal newborn rabbits produced delta 5-P (4.4-7 nmol/mg protein), but CAH adrenals did not, while CAH adrenal mitochondria demonstrated over 4 times greater 11 beta-hydroxylase activity. A Western blot of adrenal homogenate from normal newborn rabbits revealed a cholesterol side-chain cleavage cytochrome P450 (P450scc)-immunoreactive species (mol wt, 53 x 10(3), but this species was absent in CAH adrenals; CAH adrenals had a normal adrenodoxin and intensified 17 alpha-hydroxylase cytochrome P450 (P450(17)alpha) band compared to normal adrenals. In vitro translation of RNA in a cell-free rabbit reticulocyte lysate system containing [35S] methionine yielded a precursor P450scc protein (mol wt, 58.5 x 10(3)) with normal adrenal RNA, but not with CAH adrenal RNA. P450scc mRNA was detected in all normal adrenals, but was not detected in all CAH adrenals. 21-Hydroxylase cytochrome P450 mRNA expression was detected at a similar level in both normal and CAH adrenals. We conclude that CAH in the rabbit is caused by inherited absent P450scc gene expression. The clinical, pathological, and biochemical manifestations of P450scc deficiency in the rabbit are nearly identical to the human disorder. Increased 11 beta-hydroxylase activity and increased P450(17)alpha on Western blot of CAH adrenals indicate altered gene expression of other steroidogenic enzymes due to CAH. Further molecular analysis of the P450scc gene in this animal CAH model will facilitate understanding of P450scc deficiency CAH.     

Immunoelectron microscopic localization of three key steroidogenic enzymes (cytochrome P450(scc), 3 beta-hydroxysteroid dehydrogenase and cytochrome P450(c17)) in rat adrenal cortex and gonads.

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) which catalyzes the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450(c17) which is responsible for the transformation of C(21) into C(19) steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450(scc) and 3 beta-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450(scc) was exclusively found in large mitochondria. In contrast, 3 beta-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450(c17) could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450(scc) and 3 beta-HSD was restricted to mitochondria, while P450(c17) immunoreactivity was exclusively observed in ER. In the ovary, P450(scc) and 3 beta-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450(c17) could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450(scc), 3 beta-HSD and P450(c17) have the same association with cytoplasmic organelles (with the exception of 3 beta-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.     

Corticotropin-releasing hormone stimulates P450 17alpha-hydroxylase/17,20-lyase in human fetal adrenal cells via protein kinase C.

CRH directly stimulates dehydroepiandrosterone sulfate (DHEAS) production in human fetal adrenal cells. In the human fetal and adult pituitary, CRH acts via protein kinase A (PKA). We determined the CRH signal transduction pathway in fetal adrenal cells, i.e. whether CRH modulates human fetal adrenal steroidogenesis via PKA and/or protein kinase C (PKC). In primary cultures, CRH increased inositol trisphosphate. After CRH treatment, inositol tris-, bis-, and monophosphates increased within 1 min, reaching maximal levels at 5 min. In contrast, PGF2alpha, known to act via PKC, induced a sustained response for up to 20 min. The response to CRH was dose dependent, maximal at 1 micromol/L at both 1 and 5 min. CRH increased DHEAS production, with a much lesser effect on cortisol. CRH did not stimulate inositol phospholipid in adult adrenal glands, suggesting that this pathway is unique to the fetal adrenal. CRH increased messenger ribonucleic acid encoding 17alpha-hydroxylase/17,20 lyase (P450c17), but not 3beta-hydroxysteroid dehydrogenase/delta(4-5) isomerase. However, 3betaHSD expression was stimulated by ACTH. PKC, but not PKA, inhibitors blocked CRH-stimulated P450c17 induction, whereas PKA inhibitors blocked ACTH-stimulated cortisol. Thus, CRH is coupled to the phospholipase C-inositol phosphate second messenger system and preferentially induces the expression of P450c17 and DHEAS, suggesting a unique role of CRH regulating human fetal adrenal function via PKC.