Effect of bilateral lesions of the ovine fetal hypothalamic paraventricular nuclei at 118-122 days of gestation on subsequent adrenocortical steroidogenic enzyme gene expression.

Fetal adrenal steroid hydroxylase activity and messenger RNA (mRNA) expression increases concurrent with the preterm rise in fetal plasma cortisol during late gestation in sheep. By placing bilateral lesions of the fetal paraventricular nuclei (PVN) we have previously demonstrated that the fetal PVN is necessary for the initiation of parturition, the late gestation preparturient increase in fetal plasma cortisol and ACTH, and ACTH secretion in response to fetal hypoxemia and hypotension. The purpose of this study was to determine the role of the fetal PVN in the late gestation increase in expression of mRNA for 17 alpha-hydroxylase (P-450(17)alpha), side-chain cleavage (P-450SCC), 11 beta-hydroxylase (P-450(11)beta), 21 hydroxylase (P-450C21), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the fetal adrenal. Ovine fetuses were subjected to bilateral lesions of the PVN (Lx; n = 4) or sham lesions (Sh; n = 4) at 118-122 days gestational age (dGA). Lx fetuses were recovered by cesarean section at greater than or equal to 157 dGA; Sh fetuses were recovered immediately postbirth at normal term (146.5 +/- 0.9 dGA). In addition, uninstrumented fetuses were obtained at 145-147 dGA by cesarean section (n = 3). RNA obtained from individual fetal adrenals was subjected to Northern analysis. Lx of the fetal PVN decreased (P less than or equal to 0.05) mRNA for P-450(17)alpha and P-450SCC but did not affect adrenocortical mRNA for P-450C21, P-450(11)beta, or 3 beta-HSD compared to Sh. To determine if the differences observed between Lx and Sh for P-450(17)alpha and P-450SCC mRNA were due to the process of labor, we compared uninstrumented 145-147 dGA to Sh. No differences in adrenal mRNA content were observed for P-450(17)alpha or P-450SCC between these groups. We conclude that in late gestation fetal sheep an intact fetal PVN is necessary for normal gene expression of adrenocortical P-450(17)alpha and P-450SCC while P-450(11)beta, P-450C21, and 3 beta-HSD may be primarily regulated by factors not dependent upon a functional PVN.     

Effect of implantation of dexamethasone adjacent to the paraventricular nucleus on messenger ribonucleic acid for corticotropin-releasing hormone and proopiomelanocortin during late gestation in fetal sheep.

Glucocorticoids act upon the hypothalamic paraventricular nucleus (PVN) and anterior pituitary in a classic negative feedback loop to regulate ACTH biosynthesis and secretion. Evidence exists to indicate that glucocorticoid feedback may be attenuated during late gestation in the sheep fetus to allow the preterm rise in fetal plasma cortisol necessary for parturition in this species. The present studies were undertaken to determine the effect of glucocorticoids placed adjacent to the fetal PVN on messenger RNA (mRNA) for CRH in the PVN and mRNA for POMC in the anterior pituitary during late gestation. We performed our studies at two critical stages during late gestation to determine if gestational age related changes occur in the efficacy of negative feedback regulation of expression of CRH and subsequently POMC. Dexamethasone (DEX) implants were placed bilaterally 2 mm lateral to the fetal PVN at 105 to 107 days gestational age (dGA; group I, n = 4) and 121-123 dGA (group II; n = 4). Gestational-age matched, sham implanted fetuses were used as controls (CONT) for both groups (n = 4 per group). Fetuses were recovered at 126-128 (group I) and 136 dGA (group II). Fetal PVN were isolated by micropunching, and the anterior pituitary was separated from neurointermediate and posterior lobes after necropsy. Total RNA was subjected to Northern analysis using specific complementary DNA probes to CRH and POMC, and specific message was normalized to actin mRNA content in each individual sample. Anterior pituitary POMC mRNA was not different in DEX fetuses compared to CONT for either group I (78 +/- 26% of CONT; mean +/- SEM) or group II (84 +/- 17% of CONT). PVN CRH mRNA content was lower in DEX fetuses in group I (28 +/- 14% of CONT; P less than or equal to 0.01) and group II (65 +/- 12% of CONT; P less than or equal to 0.01). The degree to which DEX suppressed mRNA for CRH was greater in group I compared to group II (P less than or equal to 0.05). We conclude that 1) CRH expression in the PVN of fetal sheep is suppressible by glucocorticoids; 2) suppression can occur directly at the level of the PVN and 3) that the efficacy of negative feedback decreases with increasing gestational age. Furthermore, the lack of effect of hypothalamic administration of DEX on anterior pituitary POMC mRNA indicates that basal expression of POMC in fetal sheep may be independent from support from the PVN at this stage of gestation.     

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)     

Tissue distribution and levels of gene expression of three steroid hydroxylases in ovine fetal adrenal glands

Northern blotting and hybridization histochemistry were used to evaluate the ontogeny and cellular distribution of the mRNAs of the cytochrome P-450 enzymes: cholesterol side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450(17 alpha] and 21-hydroxylase (P-450c21) in 40 ovine fetal adrenals from 42 days of gestation until term (151 days). The genes for P-450(17 alpha) and P-450scc were expressed strongly in tissue from young (40-60 days) and old fetuses (120 days to term), but to a very minor degree in 90-120 day fetuses. P-450c21 showed a steady increase throughout gestation. In the morphologically immature an unzoned adrenal of the 40-50 day fetus there was some differentiation in gene expression, all cells containing P-450scc and P-450c21 but a few lacking P-450(17 alpha). Once morphological zonation had occurred (80 days), P-450(17 alpha) was confined to the fasciculata. After 120 days there was a radial maturation pattern of the fasciculata cells morphologically, adult-type cells first appearing at the medullary border. However, P-450(17 alpha) and P-450scc mRNAs were equally well expressed in all sections of the fasciculata. The conclusions were: 1) the previously demonstrated triphasic cortisol biosynthetic capacity of ovine fetal adrenals was correlated with the presence, absence, and reappearance of mRNAs P-450(17 alpha) and P-450scc; 2) morphological appearance of fetal adrenocortical cells and expression of three major steroidogenic enzyme genes were not correlated.     

Temporal and spatial distribution of corticosteroidogenic enzymes immunoreactivity in developing human adrenal

Steroid hormones secreted by fetal adrenocortical cells are considered to be a requirement for a fetus to maintain intrauterine life, but, to date, the regulation of steroid hormone secretion has not been studied in detail in the human fetal adrenal gland. In this study, we examined the immunolocalization of steroidogenic enzymes and their local regulation, including adrenal 4-binding protein (Ad4BP or NR5A1), steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc or CYP11A1), P450 17alpha-hydroxylase/17,20-lyase (P450c17 or CYP17), 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD), P450 21 hydroxylase (P450c21 or CYP21), dehydroepiandrosterone sulfotransferase (DHEA-ST), P450 oxidoreductase and cytochrome b5, in the human fetal adrenal gland (n=31) obtained from fetuses ranging in ages from 14 to 40 weeks of gestation. Ad4BP immunoreactivity was detected in all adrenocortical zones throughout gestation, suggesting that this nuclear protein is likely to be essential in the development of the human adrenal. Immunoreactivity for StAR, P450scc, P450c21, P450 oxidoreductase and cytochrome b5 was detected only in fetal and transitional zone between 14 and 22 weeks of gestation, but was detected in all three zones after 23 gestational weeks. 3beta-HSD immunoreactivity was not detected in any of the three cortical zones prior to 22 weeks of gestation, but became discernible in the transitional zone and definitive zone after 23 weeks. Immunoreactivity for P450c17 and DHEA-ST was detected in the transitional and fetal zones throughout gestation, but not in the definitive zone. These results suggest that the human adrenal cortex may produce dehydroepiandrosterone (DHEA) in the transitional and fetal zones throughout gestation, and cortisol in the transitional zone after the 23rd week of gestation.     

Transforming growth factor-beta inhibits steroid 17 alpha-hydroxylase cytochrome P-450 expression in ovine adrenocortical cells

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of ACTH to promote the synthesis of the various steroid-metabolizing cytochrome P-450 enzymes. In the steroidogenic pathway, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) is a key enzyme controlling the formation of cortisol and androgens. Recently, we demonstrated that transforming growth factor-beta (TGF beta) is a potent inhibitor of steroid production in ovine adrenocortical cells. In the present study we used a polyclonal antibody to P450(17) alpha to determine adrenal cell P-450(17) alpha enzyme content by Western analysis. In addition, we used a cDNA probe encoding for bovine P-450(17) alpha mRNA to determine levels of P-450(17) alpha mRNA in sheep ovarian adrenocortical cells in primary culture. When cells were cultured in a serum-free medium in the presence of ACTH for 48 h, P-450(17) alpha activity, enzyme content, and mRNA levels for P-450(17) alpha increased by 3- to more than 10-fold. TGF beta decreased the basal level and completely blocked the stimulatory action of ACTH on P-450(17) alpha enzyme activity. The effects of TGF beta on P-450(17) alpha enzyme content and mRNA levels were manifested in a dose-dependent manner, with maximal inhibition observed using 1 ng/ml TGF beta. Importantly, the inhibitory effects of TGF beta on P-450(17) alpha were not overcome by (Bu)2cAMP. These findings indicate that TGF beta is a potent negative regulator of P-450, and the inhibitory action appears to be at the level of P-450(17) alpha gene expression. The ability of TGF beta to suppress the positive stimulatory action of ACTH suggests that TGF beta could play a role in determining the pathway of steroidogenesis and, thereby, the specific steroids secreted by 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)     

Effect of fetal adrenalectomy on messenger ribonucleic acid for proopiomelanocortin in the anterior pituitary and for corticotropin-releasing hormone in the paraventricular nucleus of the ovine fetus

In the ovine fetus, adrenalectomy at 90-120 days gestational age (dGA) results in a gradual increase in basal concentrations of fetal plasma ACTH beginning at approximately 122 dGA. Bilateral adrenalectomy at 116-119 dGA also results in an increase in POMC mRNA in the fetal pituitary. It is not known whether both the paraventricular nuclei (PVN) of the hypothalamus and the anterior pituitary of the ovine fetus are responsive in late gestation to the removal of cortisol negative feedback. The purpose of this study was to determine the subsequent effect of fetal adrenalectomy at 118-121 dGA on the CRH mRNA content in fetal PVN and on POMC mRNA in the fetal anterior pituitary at 134 dGA. Mature Rambouellet-Columbia cross-bred ewes (n = 10), bred on a single occasion only and carrying fetuses of known gestational ages, were used. Both fetal adrenal glands were exposed via a retroperitoneal approach and removed [adrenalectomized (ADX); n = 5]. In control fetuses (CONT; n = 5) adrenal glands were exposed and isolated, but not removed. At 134 dGA, fetal plasma cortisol concentrations were significantly greater in CONT fetuses (7.2 +/- 2.5 ng/ml) than in ADX fetuses (mean +/- SD, 1.97 +/- 0.9 ng/ml; P less than 0.025). At 134 dGA the fetal PVN was removed by micropunching, and the anterior pituitary was separated from neurointermediate and posterior lobes after necropsy. Total RNA was prepared by the guanidium isothiocyanate-cesium chloride method and subjected to Northern analysis using specific cDNA probes to CRH and POMC. After autoradiography, quantification of mRNA was performed by scanning densitometry. Quantities of specific hybridization signal for POMC and CRH were normalized to the content of actin mRNA in each individual sample. RNA prepared from PVN exhibited a single specifically hybridizing band for CRH of approximately 1300 nucleotides. RNA prepared from anterior pituitary exhibited a single specifically hybridizing band for POMC at approximately 1300 nucleotides. Anterior pituitary POMC mRNA was significantly increased (P less than 0.025) in ADX fetuses (236 +/- 32% of CONT). CRH mRNA in PVN was greater in ADX fetuses than in CONT fetuses (P less than 0.05; mean +/- SEM, 179 +/- 21% of CONT). Adrenalectomy in fetal sheep significantly increased expression of CRH and POMC. We conclude that the increased levels of mRNA for CRH and POMC indicate that both the fetal PVN (CRH) and the anterior pituitary (POMC) are responsive to removal of the primary source of circulating glucocorticoid at this gestational age.(ABSTRACT TRUNCATED AT 400 WORDS)     

Steroidogenic acute regulatory protein mRNA expression in adrenal tumours

The rate limiting step in steroidogenesis is cholesterol transport through the outer to the inner mitochondrial membrane and the cytochrome P450 side chain cleavage (P450scc) complex. The protein factor responsible for this transport, and as such necessary for regulating the acute production of steroids, has been identified and named the steroidogenic acute regulatory protein (StAR). We investigated the expression of StAR in functional and non-functional adrenal neoplasms and compared the expression with that of P450scc. Poly A RNA was extracted from normal adrenal glands (NAG, n=5), aldosterone producing adenomas (APA, n=4), cortisol producing adenomas (CPA, n=5), adrenocortical carcinomas (ACC, n=6) and non-functional adenomas (NFA, n=3), electrophoresed through a 1% agarose gel, blotted and hybridised with a PCR-generated cDNA labelled with [(32)P]CTP. The blots were stripped and re-hybridised with a P450scc cDNA and a mouse beta-actin probe. Compared with P450scc, StAR mRNA expression showed little variability in the magnitude of expression and did not correlate with the endocrine profiles (NAG: StAR 100+/-16%, P450scc 100+/-14%; APA: StAR 80+/-3%, P450scc 94+/-13%; CPA: StAR 71+/-10%, P450scc 109+/-15%; NFA: StAR 64+/-9.5%, P450scc 18+/-5%; means+/-s.e.m.). ACC expressed low levels of both genes probably as a result of dedifferentiation (StAR 29+/-9%, P450scc 46+/-18%). Incubation of the NCI-h295 tumour cell line with 10nmol ACTH and 10micromol forskolin induced an increase in the abundance of StAR and P450scc mRNA, demonstrating gene regulation by the cAMP protein kinase A pathway. Furthermore, we incubated the NCI-h295 tumour cell line with the adrenostatic compounds, aminoglutethimide and metyrapone. We could not detect an effect on the expression of StAR mRNA, whereas the expression of P450scc mRNA was significantly reduced. We conclude that, in contrast to P450scc, StAR seems to be evenly expressed in adrenocortical adenomas. Therefore, the endocrine activity of a given tumour cannot be explained by the abundance of StAR expression. In ACC, both StAR and P450scc expression is low, explaining the relatively inefficient steroid production of these tumours.     

Expression of messenger ribonucleic acid species encoding steroidogenic enzymes in human follicles and corpora lutea throughout the menstrual cycle

The levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450(17 alpha], aromatase cytochrome P-450 (P-450AROM), and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were examined in human follicles and corpora lutea (CL) throughout the menstrual cycle. Tissues were obtained from women undergoing hysterectomy and oophorectomy. The largest follicle or the CL was dissected from the ovary depending on whether the surgery was performed in the follicular or luteal phase. The day of the cycle was determined by onset of last menstrual period and was confirmed by endometrial histology. Total RNA was examined by Northern blot analysis, using as probes specific 32P-labeled cDNA inserts encoding each human enzyme. Early follicles demonstrated detectable mRNA for both P450scc and P450(17 alpha), but not for P450AROM or 3 beta HSD. P450AROM was detectable late in the follicular phase and appeared markedly induced in the CL. 3 beta HSD was detectable only in the CL. Levels of P450(17 alpha) mRNA remained relatively unchanged throughout the cycle, whereas P450scc mRNA levels were greatly increased in the CL. The presence of P450(17 alpha) mRNA in the human CL is of interest, since it is absent from the bovine CL, and this is consistent with the ability of the human, but not the bovine, CL to synthesize 17 alpha-hydroxyprogesterone and estrogens. The fact that P450AROM expression is highest in CL is surprising, since plasma estrogen levels are highest during the late follicular phase of the cycle, and may suggest that CL estrogen biosynthesis is limited by 17 alpha-hydroxylase or 17,20-lyase activities.     

Steroid hydroxylase gene expression in the ovine fetal adrenal gland following ACTH infusion

Between 90 and 120 days of gestation (term = 147 +/- 5), when plasma cortisol concentrations in the fetus are at a minimum, levels of mRNA encoding the steroidogenic enzymes 17 alpha-hydroxylase (P-450(17 alpha] and cholesterol side-chain cleavage (P-450scc) are also very low. Over the following 30 days, P-450(17 alpha) and P-450scc gene expression increases concurrent with increasing fetal cortisol concentration. The hypothesis tested in this study was that cortisol biosynthesis is minimal in the period 90-120 days because of insufficient ACTH. Fetuses were cannulated between 98-102 days of gestation. Following recovery, 7 fetuses received 24-h ACTH infusions (12 micrograms/24 h) and 5 fetuses received 24-h vehicle infusions; 4 ACTH-infused and 4-vehicle-infused fetuses were then sacrificed immediately after cessation of the infusion. The other fetuses were left in utero for 3 days prior to sacrifice. Fetal blood samples were analysed for ACTH and cortisol and the adrenals processed for hybridization histochemistry and Northern blot analysis. ACTH, but not vehicle, induced significant increases in the width of the adrenal cortex and in the levels of P-450(17 alpha) and P-450scc mRNA. Concurrently, fetal plasma ACTH and cortisol concentrations also increased significantly. In adrenals from fetuses left in utero for 3 days after cessation of the ACTH infusion, P-450(17 alpha) and P-450scc mRNA levels returned to control levels. Plasma ACTH and cortisol levels also approximated basal values. P-450c21 mRNA levels did not vary significantly at any time with the treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of 3 beta-hydroxysteroid dehydrogenase in adrenocortical cells: effects of angiotensin-II and transforming growth factor beta.

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of ACTH to promote the synthesis of the various steroid metabolizing enzymes. In the steroidogenic pathway, the ratio of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) to 17 alpha-hydroxylase cytochrome P450 (P-450(17 alpha)) plays a key role in determining the final steroid products released by adrenal cells. The differences in these enzymes are particularly important when one considers the adrenal zones and the secretion of the zone-specific steroids. In the present study we have investigated the regulation of 3 beta HSD with regard to its enzyme activity, levels of protein and changes in specific mRNA encoding for this enzyme. Following eight days in primary culture, bovine adrenocortical (BAC) cells were found to respond to both ACTH and Bu2 cAMP by increased cortisol production. In addition, 3 beta HSD activity, enzyme protein and mRNA levels were increased in response to both factors. The increases varied from 2-fold for activity to 5-7 fold for mRNA. ACTH and Bu2cAMP also greatly increased P-450(17 alpha) from the near undetectable levels in control cells. In order to examine the possibility of differential regulation of these adrenal steroidogenic enzymes we determined the effects of angiotensin II (A-II) and transforming growth factor beta (TGF beta) on the levels of these enzymes. Both of these factors decreased the ACTH-stimulated levels of P-450(17 alpha) enzyme and mRNA to near nondetectable levels observed within control cells. In addition, these compounds inhibited the ACTH induction of 3 beta HSD. While the mechanism of TGF beta action is not clear, A-II probably is acting through protein kinase C. Indeed the protein kinase C activating phorbol ester, TPA, mimicked the inhibitory effects of A-II on 3 beta HSD and P450(17 alpha). It is important to point out, however, that the effects of A-II and TGF beta on P450(17 alpha) activity appeared more pronounced than their action of 3 beta HSD. This observation may relate to the relative stability of 3 beta HSD as compared to P450(17 alpha). Taken together these data indicate that, while A-II and TGF beta each decrease the levels of steroid-metabolizing enzymes, a differential regulation is observed in that P-450(17 alpha) protein and activity levels are much more sensitive to treatment with these factors.     

Expression of steroidogenic enzymes in the bovine placenta and fetal adrenal glands throughout gestation.

The expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P45017 alpha), and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was studied in bovine placenta and fetal adrenal glands throughout gestation. The levels of expression of these enzymes were much lower in the placenta than in the adrenals by Western and Northern analyses. The levels of P450scc, however, remained relatively constant in bovine placenta and fetal adrenal glands at all gestational stages studied. In contrast, P45017 alpha expression was higher in both the placenta and the fetal adrenal glands during the early stages of pregnancy, but declined markedly in both tissues through the period of midgestation. The expression of P45017 alpha increased markedly in the fetal adrenal glands in late gestation. The levels of 3 beta HSD were extremely low in placental tissues, but were higher in the fetal adrenals, where they were found to be slightly elevated in early and late gestation compared to those in midgestational stages. Immunocytochemical examination of the levels of P45017 alpha and 3 beta HSD in the fetal adrenal glands correlated with the results of Western and Northern analyses. In addition, the morphology and distribution of these two enzymes in the developing bovine fetal adrenal glands indicated that while the early activated gland is functional relative to the ability to secrete steroids, structural and functional organization more typical of mature adrenal glands is not achieved until the time of activation of the fetal adrenals in late gestation.     

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.     

Alteration in the expression of genes for cholesterol side-chain cleavage enzyme and 21-hydroxylase by hypophysectomy and ACTH administration in the rat adrenal

The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.     

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.     

Developmental changes in steroidogenic enzymes in human postnatal adrenal cortex: immunohistochemical studies

Adrenarche is considered to occur as a result of intra-adrenal changes in steroidogenic enzymes involved in C19 steroid production. The present study was conducted because developmental changes in steroidogenic enzymes have not been examined well in human postnatal adrenal. Twenty-four specimens of nonpathological human adrenals from 7 months to 62 years retrieved from autopsy files. Immunohistochemistry for P450 side-chain cleavage (P450scc), 17alpha hydroxylase (P450c17), dehydroepiandrosterone sulfotransferase (DHEA-ST), P450 oxidoreductase, cytochrome b5, and 3beta-hydroxysteroid dehydrogenase (3betaHSD) was per-formed in these specimens, and the immuno-intensity was evaluated using CAS 200 computed image analysis system. Immunoreactivity of P450scc was marked in the zona glomerulosa, fasciculata and reticularis in the adrenal glands of all the cases examined. P450c17 and DHEA-ST immunoreactivity was weak in the zona fasciculata and reticularis in the adrenals of age 7 months to 5 years, but thereafter became prominent in the zona reticularis. Immunoreactivity of P450 oxidoreductase and cytochrome b5, components of the electron transfer system hypothesized to regulate the 17-20 lyase activity of P450c17, was weak in all three zones of adrenal cortex from 7 months to 5 years, and became more marked in the zona reticularis after age 5 years. 3betaHSD immunoreactivity was marked in all three zones of the adrenal cortex from 7 months to 8 years but thereafter decreased in the zona reticularis. These data suggest that the human adrenal zona reticularis markedly begins to develop morphologically and functionally at around 5 years of age. The increased level of P450c17, DHEA-ST, P450 oxidoreductase, and cytochrome b5, and the decreased level of 3betaHSD in the reticularis is likely to contribute to increased C19 steroid production during adrenarche.     

Hypothalamic paraventricular nuclear lesions delay corticotroph maturation in the fetal sheep anterior pituitary.

The purpose of this study was to determine whether normal morphological development occurs in pituitary corticotrophs deprived of products of the hypothalamic paraventricular nucleus (PVN), e.g. corticotropin releasing hormone and arginine vasopressin (AVP), after PVN lesions. In addition, we have attempted to ascertain if the neurophysin/AVP-positive fibers innervating the fetal sheep anterior pituitary are affected by PVN lesions. The experimental groups consisted of fetal sheep in which 1) hypothalamic PVN lesions were placed at 118-122 days gestation (dGA) and the fetuses subsequently harvested while still in utero at 157 dGA or more (PVNX; n = 5); 2) sham PVN lesions were placed at 118-122 dGA and subsequently harvested as newborn lambs immediately after birth at 146.5 +/- 0.9 (mean +/- SEM) dGA combined with two uninstrumented fetuses harvested at 144 dGA or more but not in labor (perinatal; n = 6); and 3) no instrumentation was placed, and the fetuses were harvested at 120 dGA (control; n = 4). Two ACTH-immunoreactive cell types were seen in the anterior pituitary: 1) fetal cells: large and variably stained, often columnar, occurring in clusters and arranged in palisades; and 2) adult cells: smaller, darkly staining, and angular, occurring singly or in small groups. Quantification of the distribution of the two ACTH cell types was performed by scanning sections from a one in six series from each pituitary and estimating the percent area of each section in the well that showed adult type staining only. The observer was blind to the treatment group assignment of the sections. The estimated percentages of the portion of the pituitaries of each group that contained adult-type cells only were as follows: PVNX, 42.8 +/- 10.0%; perinatal, 90.9 +/- 2.1%; and control, 3.7 +/- 1.1% (mean +/- SEM; P less than 0.05 for all comparisons). There were no qualitative differences between all groups in the appearance of neurophysin-positive fibers innervating the anterior pituitary. AVP staining was strong in the internal zone of the median eminence in all groups, but was absent in the external zone of PVNX fetuses only. The intermediate pituitary lobes stained darkly in all groups. We conclude that lesions of the PVN at 120 dGA delay development of fetal pituitary corticotrophs, but have no effect on the presence of neurophysin-positive nerve fibers in the anterior pituitary.