Differential actions of metyrapone on the fetal pituitary-adrenal axis in the sheep fetus in late gestation

It is not clear if an increase in intra-adrenal cortisol is required to mediate the actions of adrenocorticotropic hormone (ACTH) on adrenal growth and steroidogenesis during the prepartum stimulation of the fetal pituitary-adrenal axis. We infused metyrapone, a competitive inhibitor of cortisol biosynthesis, into fetal sheep between 125 and 140 days of gestation (term = 147 +/- 3 days) and measured fetal plasma cortisol, 11-desoxycortisol, and ACTH; pituitary pro-opiomelanocortin mRNA and adrenal expression of ACTH receptor (melanocortin type 2 receptor), steroidogenic acute regulatory protein (StAR), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), cytochrome P450 17-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase, and cytochrome P450 21-hydroxylase mRNA; and StAR protein in the fetal adrenal gland. Plasma ACTH and 11-desoxycortisol concentrations were higher (P < 0.05), whereas plasma cortisol concentrations were not significantly different in metyrapone- compared with vehicle-infused fetuses. The ratio of plasma cortisol to ACTH concentrations was higher (P < 0.0001) between 136 and 140 days than between 120 and 135 days of gestation in both metyrapone- and vehicle-infused fetuses. The combined adrenal weight and adrenocortical thickness were greater (P < 0.001), and cell density was lower (P < 0.01), in the zona fasciculata of adrenals from the metyrapone-infused group. Adrenal StAR mRNA expression was lower (P < 0.05), whereas the levels of mature StAR protein (30 kDa) were higher (P < 0.05), in the metyrapone-infused fetuses. In addition, adrenal mRNA expression of 11betaHSD2, CYP11A1, and CYP17 were higher (P < 0.05) in the metyrapone-infused fetuses. Thus, metyrapone administration may represent a unique model that allows the investigation of dissociation of the relative actions of ACTH and cortisol on fetal adrenal steroidogenesis and growth during late gestation.     

11beta-hydroxysteroid dehydrogenase and glucocorticoid receptor messenger RNA expression in porcine placentae: effects of stage of gestation,breed, and uterine environment

Glucocorticoids are known to influence many aspects of prenatal development. Three important regulators of glucocorticoid actions at the cellular level are the enzymes 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD-1), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD-2), and glucocorticoid receptors (GR). The present study was conducted to determine the presence of these regulators in porcine placentae during early gestation (Days 24-40; term = 114 days) and to examine the influence of breed and uterine environment. Three pig models differing in uterine environment as reflected by embryonic survival from Days 24 to 40 were used: intact white cross-bred gilts (WC-INT); white cross-bred gilts that had been unilaterally hysterectomized-ovariectomized before puberty (WC-UHO); and intact Meishan gilts (ME). Porcine-specific partial cDNAs for 11betaHSD-1 and 11betaHSD-2 and a cRNA for GRalpha were developed and used to produce 32P-labeled probes for Northern blot analyses. The 11betaHSD dehydrogenase activity was measured in vitro at saturating concentrations of substrate and coenzyme. At Day 24 of gestation, 11betaHSD-2 mRNA, dehydrogenase activity, and GR mRNA were present, but 11betaHSD-1 mRNA was absent. All three mRNAs and dehydrogenase activity increased (P < 0.01) by Day 40. On Day 30, placental 11betaHSD-2 mRNA was decreased (P = 0.03) by 47% in WC-UHO versus WC-INT. Placental 11betaHSD dehydrogenase activity was 2-fold greater (P < 0.01) in ME versus WC-INT on Day 24 of gestation. These results demonstrate, to our knowledge for the first time, the presence of 11betaHSD-1, 11betaHSD-2, and GR mRNA as well as 11betaHSD dehydrogenase activity in the porcine placenta during early pregnancy. Moreover, a role for glucocorticoids in porcine embryonic development is suggested.     

Activation of adrenal function in fetal sheep by the infusion of adrenocorticotropin to the fetus in utero

We determined whether ACTH1-24, infused into fetal lambs at a rate that is known to cause premature labor, elicits changes in the responsiveness of the fetal adrenal glands, and alters the pattern of corticosteroid output. Plasma cortisol (F), corticosterone (B) and progesterone (P4) were measured during 72 h of infusion of saline or ACTH (10 micrograms/h) beginning on Day 127 of pregnancy. Adrenals were then dispersed into isolated cells, and the output of F, B and P4 after exogenous ACTH determined in vitro. Plasma concentrations of F and B were higher in ACTH-treated fetuses. The increment in F (5-to 7-fold) was greater than that in B (2-fold) such that the F:B ratio in plasma of ACTH-treated fetuses on Days 2 and 3 of infusion was 2.5 times higher than in controls. After 72 h of infusion, the adrenal weights in ACTH-treated fetuses (741 +/- 38 mg, +/- SEM; n = 4) were greater than in the control animals (349 +/- 11 mg). There was a significant effect of ACTH pretreatment in vivo on F output by isolated adrenal cells in vitro. Mean increments in F output after addition of ACTH1-24 (5000 pg/ml) in vitro rose from 368 +/- 235 pg/50,000 cells in controls, to 64,639 +/- 19,875 pg/50,000 cells after ACTH in vivo. There was no significant effect of ACTH in vivo on B output in vitro; the ratio of F:B output, either in the absence or presence of ACTH in vitro, was significantly higher in cells from ACTH-pretreated fetuses. There was a significant effect of in vivo ACTH on in vitro P4 output. After ACTH treatment in vivo there was an increase in the vitro output ratio of F:P4, but no change in the output ratio of B:P4. We conclude that ACTH treatment of the fetal lamb in vivo results in activation of fetal adrenal function, increased fetal adrenal responsiveness to ACTH, and directed corticosteroid biosynthesis towards cortisol. Our results are consistent with an increase in fetal adrenal 17 alpha-hydroxylase activity after ACTH treatment.     

Antenatal glucocorticoids reset the level of baseline and hypoxemia-induced pituitary-adrenal activity in the sheep fetus during late gestation

This study examined the effects of dexamethasone treatment on basal hypothalamo-pituitary-adrenal (HPA) axis function and HPA responses to subsequent acute hypoxemia in the ovine fetus during late gestation. Between 117 and 120 days (term: approximately 145 days), 12 fetal sheep and their mothers were catheterized under halothane anesthesia. From 124 days, 6 fetuses were continuously infused intravenously with dexamethasone (1.80 +/- 0.15 microg.kg(-1).h(-1) in 0.9% saline at 0.5 ml/h) for 48 h, while the remaining 6 fetuses received saline at the same rate. Two days after infusion, when dexamethasone had cleared from the fetal circulation, acute hypoxemia was induced in both groups for 1 h by reducing the maternal fraction of inspired O2. Fetal dexamethasone treatment transiently lowered fetal basal plasma cortisol, but not ACTH, concentrations. However, 2 days after treatment, fetal basal plasma cortisol concentration was elevated without changes in basal ACTH concentration. Despite elevated basal plasma cortisol concentration, the ACTH response to acute hypoxemia was enhanced, and the increment in plasma cortisol levels was maintained, in dexamethasone-treated fetuses. Correlation of fetal plasma ACTH and cortisol concentrations indicated enhanced cortisol output without a change in adrenocortical sensitivity. The enhancements in basal cortisol concentration and the HPA axis responses to acute hypoxemia after dexamethasone treatment were associated with reductions in pituitary and adrenal glucocorticoid receptor mRNA contents, which persisted at 3-4 days after the end of treatment. These data show that prenatal glucocorticoids alter the basal set point of the HPA axis and enhance HPA axis responses to acute stress in the ovine fetus during late gestation.     

Marked Cortisol Production by Intracrine ACTH in GIP-Treated Cultured Adrenal Cells in Which the GIP Receptor Was Exogenously Introduced

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing’s syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (–) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.     

Changes of AT(2) receptor levels in the rat adrenal cortex and medulla induced by bilateral nephrectomy and its modulation by circulating ANG II.

We studied regulation of the AT(2) receptor by investigating the effect of bilateral nephrectomy (bNX) in Sprague-Dawley rats. The expression of aldosterone synthase (CYP11B2) and AT(2) receptor mRNA was detected by nonradioactive in situ hybridization. AT(2) receptor mRNA was detected in cells of the first two or three subcapsular cell layers of the zona glomerulosa (ZG) and in the medulla of sham-operated animals. After bNX, the number and area of distribution of AT(2) receptor-positive cells increased in the ZG. This was associated with an enlargement of the steroidogenic active ZG and with reduced proliferation rate (sham 5.9 +/- 0.9%; bNX 2.4 +/- 0.2%; p<0.02). Infusion of angiotensin II (ANG II; 200 ng/kg/min SC for 56 hr) to bNX rats did not reverse the effect of nephrectomy on the distribution of AT(2) receptor expression, although mRNA levels per cell were reduced compared to NX alone. ANG II infusion decreased proliferation rate further (0.4 +/- 0.07%; p<0.001). In the adrenal medulla after bNX, decreased expression of the AT(2) receptor was associated with increased proliferation (2.6 +/- 0.2% vs 6.6 +/- 0.5%). These results demonstrate differential regulation of the AT(2) receptor in the adrenal gland and suggest that expression of the AT(2) receptor is involved in regulating proliferation and differentiation in the ZG and medulla. (J Histochem Cytochem 49:649-656, 2001)     

Ontogeny of angiotensin II type 1 receptor and cytochrome P450(c11) in the sheep adrenal gland

In the present study we investigated the ontogeny of the expression of the type 1 angiotensin receptor (AT(1)R mRNA) and the zonal localization of AT(1)R immunoreactivity (AT(1)R-ir) and cytochrome P450(c11) (CYP11B-ir) in the sheep adrenal gland. In the adult sheep and in the fetus from as early as 90 days gestation, intense AT(1)R-ir was observed predominantly in the zona glomerulosa and to a lesser extent in the zona fasciculata, and it was not detectable in the adrenal medulla. AT(1)R mRNA decreased 4-fold between 105 days and 120 days, whereas AT(1)R mRNA levels remained relatively constant between 120 days and the newborn period. In contrast, both in the adult sheep and in the fetal sheep from as early as 90 days gestation, intense CYP11B-ir was consistently detected throughout the adrenal cortex and in steroidogenic cells that surround the central adrenal vein. In conclusion, we speculate that the presence of AT(1)R in the zona fasciculata, and the higher levels of expression of AT(1)R at around 100 days gestation, may suggest that suppression of CYP17 is mediated via AT(1)R at this time. The abundant expression of AT(1)R-ir and CYP11B-ir in the zona glomerulosa of the fetal sheep adrenal gland would also suggest that lack of angiotensin II stimulation of aldosterone secretion is not due to an absence of AT(1)R or CYP11B in the zona glomerulosa.     

Ovine fetal adrenal maturation at term and during fetal ACTH administration: evidence that the modulating effect of cortisol may involve cAMP

Exogenous ACTH1-24 promotes adrenal maturation in fetal sheep, and this effect appears to be modulated in part by cortisol (Challis et al. 1985). We have examined whether similar changes in adrenal metabolism of progesterone occur with ACTH-induced labour as at spontaneous term and whether the site of cortisol modulation is on adrenal steroidogenesis or at the level of cAMP generation. Chronically catheterized fetal sheep were infused in utero for 100 h between days 127 and 131 of pregnancy with P-ACTH, P-ACTH + metopirone, P-ACTH + metopirone + cortisol, or saline. After 100 h the metabolism of [3H]progesterone was measured in adrenal homogenates. Similar incubations were performed with adrenal tissue from fetal sheep at day 130 of pregnancy and at spontaneous labour. In the treatment groups of sheep, cAMP output by dispersed adrenal cells in response to ACTH added in vitro was also determined. Similar qualitative patterns of [3H]progesterone metabolism were found in adrenal homogenates after in vivo ACTH or at term. At both times there was an increase in cortisol and in total 17 alpha-hydroxycorticosteroid accumulation and also evidence for increased activity of 11 beta-hydroxylase enzyme. The formation of total 17 alpha-hydroxycorticosteroids was not affected significantly by concurrent treatment in vivo with metopirone +/- cortisol. The accumulation of cAMP in vitro was increased after in vivo ACTH, attenuated after ACTH + metopirone, but statistical significance over controls was restored after ACTH + metopirone + cortisol treatment. We conclude that ACTH-induced labour and spontaneous parturition in sheep is associated with qualitatively similar changes in progesterone metabolism by the fetal adrenal gland.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroidogenic acute regulatory protein expression is decreased in the adrenal gland of the growth-restricted sheep fetus during late gestation

Functional development of the adrenal cortex is critical for fetal maturation and postnatal survival. In the present study, we have determined the developmental profile of expression of the mRNA and protein of an essential cholesterol-transporting protein, steroidogenic acute regulatory protein (StAR), in the adrenal of the sheep fetus. We have also investigated the effect of placental restriction (PR) on the expression of StAR mRNA and protein in the growth-restricted fetus. Adrenal glands were collected from fetal sheep at 82-91 days (n = 10), 125-133 days (n = 10), and 140-144 days (n = 9) and from PR fetuses at 141-145 days gestation (n = 9) (term = 147 +/- 3 days gestation). The adrenal StAR mRNA:18S rRNA increased (P < 0.05) between 125 days (7.44 +/- 1.61) and 141-144 days gestation (13.76 +/- 1.88). There was also a 13-fold increase (P < 0.05) in the amount of adrenal StAR protein between 133 and 144 days gestation in these fetuses. However, the amount of StAR protein (6.9 +/- 1.7 arbitrary densitometric units [AU]/microg adrenal protein) in the adrenal of the growth-restricted fetal sheep was significantly reduced, when compared with the expression of StAR protein (17.1 +/- 1.9 AU/microg adrenal protein) in adrenals from the age-matched control group. In summary, there is a developmental increase in the expression of StAR mRNA and protein in the fetal sheep adrenal during the prepartum period when adrenal growth and steroidogenesis is dependent on ACTH stimulation. We have found that, while the level of expression of StAR protein is decreased in the adrenal gland of the growth-restricted fetus during late gestation, this does not impair adrenal steroidogenesis. Our data also suggest that the stimulation of adrenal growth and steroidogenesis in the growth-restricted fetus may not be ACTH dependent.     

Effects of leptin on fetal plasma adrenocorticotropic hormone and cortisol concentrations and the timing of parturition in the sheep

We investigated whether leptin can suppress the prepartum activation of the fetal hypothalamus-pituitary-adrenal (HPA) axis and delay the timing of parturition in the sheep. First, we investigated the effects of a 4-day intravascular infusion of recombinant ovine leptin (n = 7) or saline (n = 6) on fetal plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations, starting from 136 days gestation (i.e., at the onset of the prepartum activation of the fetal HPA axis. The effects of a continuous intrafetal infusion of leptin (n = 7) or saline (n = 5) from 144 days gestation on fetal plasma ACTH and cortisol concentrations and the timing of delivery were also determined in a separate study. There was an increase in fetal plasma ACTH (P < 0.01) and cortisol (P < 0.001) concentrations when saline was infused between 136-137 and 140-141 days gestation. Plasma ACTH and cortisol concentrations did not rise, however, when leptin was infused during this period of gestation. When leptin was infused after 144 days gestation, there was no effect of a 4- to 5-fold increase in circulating leptin on fetal ACTH concentrations. In contrast, leptin infusion from 144 days gestation suppressed (P < 0.05) fetal plasma cortisol concentrations by around 40% between 90 and 42 h before delivery. There was no difference, however, in the length of gestation between the saline- and leptin-infused groups (saline infused, 150.2 +/- 0.5 days; leptin infused, 149.8 +/- 1.0 days). In saline-infused fetuses, there was a significant negative relationship between the plasma concentrations of cortisol (y) and leptin (x) between 138 and 146 days gestation (y = 81.4 - 7.7x, r = 0.38, P < 0.005). This study provides evidence for an endocrine negative feedback loop between leptin and the HPA axis in fetal life.     

Congenital adrenal hyperplasia: biochemical and molecular perspectives

Congenital adrenal hyperplasia is a disorder occurring in both sexes and is the commonest cause of ambiguous genitalia. It is a group of autosomal recessive disorders in which, on the basis of an enzyme defect the bulk of steroid hormone production by adrenal cortex shifts from corticosteroids to androgens. Autosomal recessive mutations in the CYP21, CYP17, CYP11B1 and 3betaHSD genes that encode steroidogenic enzymes, in addition to mutations in the gene encoding the intracellular cholesterol transport protein steroidogenic acute regulatory protein StAR can cause CAH. Each of the defects causes different biochemical consequences and clinical features. Deficiencies in 21 hydroxylase (21-OH) and 11beta-Hydroxylase (11beta-OH) are the two most frequent causes of CAH. All the biochemical defects impair cortisol secretion, resulting into compensatory hypersecretion of ACTH and consequent hyperplasia of the adrenal cortex. Research in recent years has clarified clinical, biochemical and genetic problems in diagnosis and treatment of the disorders. Expanding knowledge of the gene mutations associated with each of these disorders is providing valuable diagnostic tools in addition to the biochemical profile and phenotype. Genotyping is useful in selecting instances to provide genetic counseling and to clarify ambiguous cases.     

8-Phenylthio-adenines stimulate the expression of steroid hydroxylases, Cav3.2 Ca²⁺ channels, and cortisol synthesis by a cAMP-independent mechanism

The regulation of cortisol synthesis and the expression of genes coding for steroidogenic proteins by 8-substituted cAMP and 8-substituted adenine derivatives were studied in bovine adrenal zona fasciculata (AZF) cells. At concentrations of 10-50 μM, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP), but not the poorly hydrolyzable Sp-8CPT-cAMP, stimulated large increases in cortisol synthesis and CYP17 mRNA expression. Of the three Epac (exchange protein activated by cAMP)-specific cAMP analogs, 8CPT-2'-OMe-cAMP, but not 8HPT-2'-OMe-cAMP or 8MeOPT-2'-OMe-cAMP, induced mRNAs for CYP17 and CYP11a1 steroid hydroxylases and stimulated cortisol synthesis. 8-Substituted adenine derivatives (10-200 μM), including 8PT-adenine, 8MeOPT-adenine, and 8CPT-adenine, stimulated similar large, concentration-dependent, and reversible increases in cortisol synthesis and steroid hydroxylase gene expression, whereas 8Br-adenine was ineffective. The phenylthio-adenine derivatives produced additive effects on cortisol synthesis when applied to AZF cells in combination with 8Br-cAMP. In contrast, no additivity was observed for these three compounds when used in combination with ACTH. 8PT-adenine did not activate PKA or inhibit DNA synthesis by AZF cells. 8PT-adenine-stimulated cortisol secretion and CYP17 steroid hydroxylase mRNA expression were potently inhibited by diphenyl-butylpiperidine T-type Ca(2+) antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Ca(v)3.2 Ca(2+) current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Ca(v)3.2 Ca(2+) channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism.     

Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11beta HSD) activity in follicular fluid from bovine and porcine large antral follicles and spontaneous ovarian cysts

In the ovary, cortisol is oxidized to cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). The present study investigated whether follicular fluid (FF) from large antral follicles and spontaneous ovarian cysts, isolated from bovine and porcine ovaries, contained modulators of 11betaHSD activity. Whereas FF from antral follicles had no significant effect over 1 h on NADP+-dependent 11betaHSD activity in rat kidney homogenates, enzyme activity was inhibited by FF from bovine and porcine ovarian cysts (80.5% +/- 2.3% and 72.8% +/- 3.4% of control, respectively). Following C18 reverse-phase chromatography, the hydrophilic fractions of FF from bovine and porcine antral follicles stimulated NADP+-dependent 11betaHSD activities (111.5% +/- 21.6% and 55.2% +/- 5.7% respectively). Hydrophobic compounds inhibited NADP+-dependent cortisol oxidation by 58.2% +/- 5.1% (bovine) and 45.7% +/- 2.0% (porcine). In both species, FF from ovarian cysts appeared to contain less of the hydrophilic stimuli to 11betaHSD activity and more of the hydrophobic inhibitors. The FF from antral follicles and ovarian cysts, and the C18 fractions thereof, had no significant effect on NAD+-dependent cortisol oxidation. The ovarian modulators of NADP+-dependent 11betaHSD activities did not coelute with cortisol, cortisone, estradiol, testosterone, progesterone, pregnenolone, and cholesterol. However, the 11betaHSD stimuli in porcine FF from both antral follicles and cysts coeluted with prostaglandin (PG) E2 and PGF2alpha. We conclude that large antral follicles and spontaneous ovarian cysts, in both the cow and the pig, contain ovarian modulators of the NADP+-dependent 11betaHSD activity. Moreover, FF from spontaneous ovarian cysts, because of decreased content of the 11betaHSD stimulus accompanied by increased content of the 11betaHSD inhibitors, exerts a net inhibitory effect on 11betaHSD activity.     

Synergistic effect of cortisol and thyrotrophin releasing hormone on lung maturation in the fetal sheep entails connective tissue maturation.

Pressure-volume relationships and collagen and elastin contents were measured in the lungs of fetal sheep infused either with saline (n = 4), thyrotrophin-releasing hormone (TRH; n = 6), cortisol (n = 9) or TRH plus cortisol (n = 10) at 128 days of gestation (term = 149 days) for 7 days. Lung distensibility (V40 = 1.8 +/- 0.1 ml/g wet wt; mean +/- SD) and stability (V5 = 0.6 +/- 0.1) increased along with collagen (C) (10.1 +/- 2.7 micrograms/mg) and elastin (E) contents (128 +/- 35 ng/mg) in the animals infused with TRH plus cortisol and were significantly higher (p < 0.05) than those observed in TRH (V40 0.62 +/- 0.07; V5 0.32 +/- 0.04; C 3.53 +/- 1.3; E 38.2 +/- 8.3), cortisol (V4 0.66 +/- 0.6; V5 0.27 +/- 0.03; C 4.27 +/- 0.8; E 41.02 +/- 12.7) or saline infused fetuses (V40 0.40 +/- 0.1; V5 0.20 +/- 0.06; C 3.28 +/- 0.9; E 31.5 +/- 9.2). Plasma concentrations of prolactin (PRL), triiodothyronine (T3) and cortisol (F) were also higher in the group of fetuses infused with both hormones in comparison with the other groups. In fetuses treated with TRH plus cortisol, PRL (32 +/- 8.3 ng/ml) and T3 (308.3 +/- 36 micrograms/dl) were significantly higher than in those infused with cortisol alone (PRL 3.7 +/- 2.3; T3 128 +/- 30) or with saline (PRL 4.2 +/- 1.6; T3 < 5 micrograms/dl). In the group treated with TRH alone, PRL also increased significantly (37 +/- 6.4), but T3 increased only slightly (18 +/- 3.4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of premature delivery in sheep following infusion of cortisol to the fetus. I. The effect of maternal administration of progestagens

Premature induction of delivery in fetuses infused with graded doses of cortisol was brought about in 123.5 +/- 7.7 h (mean +/- SEM, n = 6) after the start of cortisol infusion. This treatment caused a rise in fetal plasma cortisol similar to that observed at normal delivery. Maternal and fetal progesterone and 20 alpha-dihydroprogesterone concentrations decreased to basal levels during infusion of cortisol to the fetus. Induction of premature delivery was delayed or prevented by concomitant treatment of the ewe with progestagen. Maternal intramuscular injection of 100 mg progesterone, 2 times daily, prevented delivery in four of four ewes treated during the time that cortisol was infused into the fetus (11-13 days). Maternal plasma progesterone and 20 alpha-dihydroprogesterone concentrations were maintained during this period, but fetal plasma progesterone concentrations decreased to the same extent as in the fetuses infused with cortisol alone. A single intramuscular injection of 250 mg of medroxyprogesterone acetate to ewes on the day before commencement of infusion of cortisol to the fetus prevented delivery in four of six ewes during the time that cortisol was infused for 9, 13, 14, and 15 days, respectively. One ewe delivered a live lamb at 133.5 h and another at 147.7 h after the start of infusion of cortisol to the fetus. Maternal and fetal plasma cortisol, progesterone, and 20 alpha-dihydroprogesterone concentrations were similar to those observed during infusion of cortisol alone to the fetus. Although fetal cortisol concentrations rose in a similar fashion, and to a similar extent, in all three groups during infusion of cortisol to the fetus, fetal 11-desoxycortisol concentrations only rose above basal levels close to the time of delivery in cortisol-infused fetuses or, in the progestagen-treated groups, when the fetus showed signs of being stressed.     

Interleukins 1α and 1β as regulators of steroidogenesis in human NCI-H295R adrenocortical cells

Inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) regulate the activity of the hypothalamo-pituitary-adrenal (HPA) axis at several levels. Although hypothalamic CRH secretion may be the primary mechanism by which these cytokines activate the HPA axis, IL-1 expression is increased within the adrenal glands in models for systemic inflammation, and IL-1 may augment adrenal glucocorticoid production. Our aim was to investigate the direct effects of IL-1α and IL-1β on adrenal steroidogenesis and expression of three key steroidogenic genes in human adrenocortical cells using the NCI-H295R cell line as a model. mRNAs encoding receptors for IL-1, TNF-α, and leukemia inhibitory factor (LIF) were detectable in the cell line (Affymetrix microarray analysis). Both IL-1α and IL-1β increased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate production, and the accumulation of mRNAs for steroidogenic acute regulatory protein (STAR), 17α-hydroxylase/17,20-lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase 2 (HSD3B2) in these cells (P<0.05 for all). Both ILs augmented TNF-α- and LIF-induced STAR and CYP17A1 mRNA accumulation, and TNF-α-induced cortisol production (P<0.05 for all). Both ILs also increased the apoptotic index of the cells (P<0.05), which was efficiently neutralized by their specific antibodies. The IL-induced changes in the STAR, HSD3B2, and CYP17A1 protein levels were not as evident as those in the respective mRNA levels. In conclusion, the combined effect of inflammatory cytokines at the adrenal level in acute or chronic inflammatory states could significantly stimulate glucocorticoid production, and thus explain the observed discrepancy between the cortisol and ACTH concentrations sometimes seen in sepsis and chronic inflammatory states.     

Infusion of ACTH stimulates expression of adrenal ACTH receptor and steroidogenic acute regulatory protein mRNA in fetal sheep

The late-gestation plasma cortisol surge in the sheep fetus is critical for stimulating organ development and parturition. Increased adrenal responsiveness is one of the key reasons for the surge; however, the underlying mechanisms are not fully understood. Our recent studies suggest that ACTH-mediated increased expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) may play a role in enhancing responsiveness. Hence, we examined effects of ACTH infusion in fetal sheep on mRNA expression of these two mediators of adrenal responsiveness and assessed the functional consequences of this treatment in vitro. Fetuses of approximately 118 and 138 days of gestational age (dGA) were infused with ACTH-(1-24) for 24 h. Controls received saline infusion. Arterial blood was sampled throughout the infusion. Adrenals were isolated and analyzed for ACTH-R and StAR mRNA, or cells were cultured for 48 h. Cells were stimulated with ACTH, and medium was collected for cortisol measurement. Fetal plasma ACTH and cortisol concentrations increased over the infusion period in both groups. ACTH-R mRNA levels were significantly higher in ACTH-infused fetuses in both the 118 and 138 dGA groups. StAR mRNA increased significantly in both the 118 and 138 dGA groups. Adrenal cells from ACTH-infused fetuses were significantly more responsive to ACTH stimulation in terms of cortisol secretion than those from saline-infused controls. These findings demonstrate that increases in circulating ACTH levels promote increased expression of ACTH-R and StAR mRNA and are coupled to heightened adrenal responsiveness.