Fetal ACTH Research Articles

Differential effects of betamethasone and dexamethasone fetal administration of parturition in sheep

We tested the hypothesis that betamethasone is more potent than dexamethasone in inducing the essential mechanisms of parturition in sheep. Twenty-one sheep were instrumented under general anesthesia with maternal and fetal arterial and venous catheters and myometrial electromyogram electrodes at 117 days' gestation (dGA). At 125 dGA at 12:00 PM, after 2 days of baseline recording, either saline (n = 7, control group), betamethasone (n = 7), or dexamethasone (n = 7) was administered into the fetal jugular vein at a rate of 10 micrograms/hour. A total dose of 0.48 mg was given over the next 48 hours. The animals underwent autopsy 3 days after the end of the infusion period (130 dGA), or earlier if labor resulted from the glucocorticoid administration. Daily maternal and fetal arterial blood samples (4 mL) for hormone measurement were taken at 10:00 AM throughout the study period. Additional arterial blood samples were taken if the animal developed labor. Maternal plasma progesterone and fetal ACTH and cortisol concentrations were measured by radioimmunoassay, and corticosteroid-binding globulin (CBG) binding capacity was determined by saturation analysis. Myometrial activity was monitored continuously throughout the experimental protocol. All seven betamethasone-treated animals developed labor after the glucocorticoid infusion regimen. In contrast, only two of seven dexamethasone-treated animals developed labor. Fetal treatment with betamethasone produced a greater and earlier fall in maternal plasma progesterone than fetal treatment with dexamethasone. Both betamethasone and dexamethasone treatments elevated fetal plasma CBG to similar binding capacities. Elevated fetal plasma ACTH and cortisol concentrations at the end of the infusion period in both betamethasone-and dexamethasone-treated animals were not related to the development of labor-type contractions. These data support the hypothesis that betamethasone is more potent than dexamethasone in inducing the essential mechanisms of parturition in sheep.

Functional maturation of the primate fetal adrenal in vivo. II. Ontogeny of corticosteroid synthesis is dependent upon specific zonal expression of 3 beta-hydroxysteroid dehydrogenase/isomerase.

Cortisol, produced by the primate fetal adrenal, regulates the maturation of organ systems necessary for extrauterine life. During most of primate pregnancy, however, the fetal adrenal lacks the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), which is essential for cortisol synthesis. Therefore, we used immunohistochemistry and in situ hybridization techniques to investigate the developmental expression of 3 beta HSD in the fetal rhesus monkey adrenal from 109 days' gestation until term (165 +/- 5 days) and assessed the role of ACTH in the induction of its expression and localization. We also examined whether ACTH regulates the expression of two other steroidogenic enzymes, cytochrome P450 cholesterol side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase, 17/20-lyase (P450c17), in the fetal rhesus monkey adrenal. To stimulate ACTH secretion from the fetal pituitary in vivo, we administered metyrapone to late gestation fetal rhesus monkeys for 3-7 days. Adrenals were collected from untreated fetuses at 109-125 days (n = 5), 130-148 days (n = 7), 155-172 days (n = 4), and after metyrapone treatment at 135-137 days (n = 4). The cortical width and total amount of 3 beta HSD staining were measured using an image analysis system. 3 beta HSD was localized primarily in the definitive zone cells of the adrenal from fetuses between 109-148 days, whereas at term (155-172 days), 3 beta HSD was localized in both definitive and transitional zone cells. The cortical width and total amount of 3 beta HSD staining in the adrenal increased significantly (P < 0.05) between 148 days (137 +/- 14 microns and 3,689 +/- 522 grains) and 155 days (315 +/- 61 microns and 7,321 +/- 2,008 grains). Interestingly, in metyrapone-treated fetuses at 135-137 days, 3 beta HSD messenger RNA (mRNA) and protein were localized extensively in both the definitive and transitional zones, a pattern seen only in term fetal adrenals in untreated animals. In addition, metyrapone treatment significantly (P < 0.05) increased cortical width (386 +/- 95 microns) and total 3 beta HSD immunostaining (29,063 +/- 13,692 grains) compared with age-matched controls. In contrast to 3 beta HSD, P450scc mRNA was detected in the definitive, transitional, and fetal zones, and its expression was not altered after metyrapone treatment. P450c17 mRNA was detected in the transitional and fetal zones, and the relative abundance was greater in the transitional zone. The relative abundance of P450c17 mRNA was increased in the fetal zone after metyrapone treatment. In summary, at term or after metyrapone treatment, expression of 3 beta HSD is induced in the transitional zone of the fetal rhesus monkey adrenal gland, an indication of functional maturation of the primate adrenal cortex. These data suggest that the ontogenetic increase in fetal pituitary ACTH secretion plays an important role in the induction of 3 beta HSD expression in the transitional zone.

Functional maturation of the primate fetal adrenal in vivo: I. Role of insulin-like growth factors (IGFs), IGF-I receptor, and IGF binding proteins in growth regulation.

The rapid growth of the primate fetal adrenal from midgestation until term is regulated by ACTH secreted by the fetal pituitary. Previous studies suggest that the trophic actions of ACTH are mediated by insulin-like growth factor II (IGF-II) synthesized by fetal adrenal cortical cells. To characterize further the role of IGF-II in the regulation of fetal adrenal growth, we investigated the expression of the messenger RNAs (mRNAs) encoding IGF-I, IGF-II, IGF-I receptor (IGF-IR) and IGF binding protein (IGFBP) 1-6 in the fetal rhesus monkey adrenal in vivo from 109 days of gestation until term (165 +/- 5 days) using in situ hybridization. To assess the role of ACTH in the regulation of expression of the IGF system in vivo, we administered metyrapone (3-7 days) to late gestation fetal rhesus monkeys (n = 4) in utero to increase fetal pituitary ACTH secretion. IGF-II mRNA was abundant in the definitive, transitional and fetal zones of the adrenal cortex from 109 days until term. IGF-IR mRNA was expressed in the definitive, transitional and fetal zones and decreased to nondetectable levels at term. IGFBP-2 and IGFBP-6 mRNAs were expressed in the definitive, transitional, and fetal zones, whereas IGFBP-1, -3, -4, and -5 were not detected in adrenal cells. The effects of increasing ACTH secretion on the growth of the specific zones of the adrenal were determined using morphometric techniques. Metyrapone treatment approximately doubled adrenal weight, which was due to an increase in the area of the definitive, transitional, and fetal zones with decreased cell density of the definitive, transitional, and fetal zones compared with controls and not due to a change in total cell number. Therefore, the increase in adrenal weight after metyrapone treatment was due to hypertrophy of the three cortical zones; there was no effect on adrenal medullary growth. The relative abundance of the mRNAs encoding IGF-II and the IGF-IR was increased after metyrapone treatment, whereas the localization and relative abundance of IGFBP 1-6 mRNAs were not altered by metyrapone treatment. We conclude that the ontogenetic increase in adrenal growth may be regulated, at least in part, by locally synthesized IGF-II, and the cessation of adrenal growth that occurs at term may be mediated by the decrease in the IGF-IR. The adrenal cortical expression of IGFBP-2 and IGFBP-6 suggests that these IGFBPs may modulate the IGF-IGF-IR interaction. Metyrapone treatment, which likely increased fetal pituitary ACTH secretion, causes a coordinated increase in expression of IGF-II and IGF-IR in fetal adrenal cortical cells, which may be an important mechanism of regulation of fetal adrenal cortical growth.

Hypothalamic Corticotropin-Releasing Hormone Expression in the Baboon Fetus at Mid- and Late Gestation1

We have proposed that estrogen, via regulation of the placental 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) enzyme(s) catalyzing the oxidation of cortisol to its inactive metabolite cortisone, regulates the baboon fetal pituitary-adrenocortical axis and the onset of de novo production of cortisol by the fetus near term. In support of this hypothesis we have demonstrated that the increase in expression of the mRNA for the ACTH precursor proopiomelanocortin (POMC) in the fetal pituitary and in the specific activity of steroidogenic enzymes in the fetal adrenal normally observed at term were enhanced at midgestation by maternal estrogen administration. However, it is not known whether activation of the fetal pituitary reflects a concomitant increase in corticotropin-releasing hormone (CRH) mRNA expression and/or peptide production by the fetal hypothalamus. Therefore, an aim of the present study was to determine whether the increase in POMC mRNA in fetal baboons delivered at term, and at midgestation to mothers treated with estradiol, reflected an increase in hypothalamic CRH. Fetal hypothalami were obtained on Day 100 (n = 6) and Day 165 of gestation (term = Day 184) from untreated baboons (n = 5) and on Day 100 from baboons (n = 4) whose mother had been treated daily with 1.0 mg estradiol on Days 70 to 100. Hypothalamic CRH peptide concentrations were determined by RIA, and CRH mRNA expression was quantified by in situ hybridization in sections of the fetal hypothalamus through the paraventricular nucleus (PVN) using a 48-base synthetic oligodeoxynucleotide probe 3' end-labeled with [35S]dATP. The mean (+/- SE) maternal serum estradiol concentration in baboons treated with estradiol at midgestation (2.4 +/- 0.4 ng/ml) was greater (p < 0.05) than that in untreated baboons on Day 100 (1.0 +/- 0.2), but similar to that in late gestation (2.0 +/- 0.2). The mean steady-state concentration of CRH in the baboon fetal hypothalamus at midgestation (15.8 +/- 6.0 ng/g tissue) was not altered in fetuses whose mothers had been treated with estradiol (17.6 +/- 0.9 ng/g). Hypothalamic CRH concentrations in fetal baboons of late gestation (20.7 ng/g; n = 2) were also similar to mean CRH values measured at midgestation but, owing to the marked increase in weight of the fetal hypothalamus with advancing pregnancy, the content of hypothalamic CRH in late gestation (28.8 ng/structure) exceeded (p < 0.01) that at midgestation. Mean levels of CRH mRNA at midgestation when expressed per cell (17.4 +/- 1.3 grains per cell) or per unit area of PVN (375 +/- 20 grains per area) were similar to respective values in late gestation (18.3 +/- 1.1 grains per cell; 350 +/- 55 grains per area; n = 3 per group). These findings support the suggestion that the increase in fetal pituitary POMC mRNA expression and ACTH peptide previously reported to occur normally between midgestation and term are not associated with a concomitant increase in hypothalamic CRH peptide or CRH mRNA concentrations. Moreover, it would appear that by midgestation, hypothalamic CRH is available in adequate concentrations to "drive" the fetal pituitary and that it is the levels of maternal cortisol arriving within the fetal circulation, as dictated by estrogen-regulated placental 11 beta-HSD-oxidase activity, that establish the extent to which the fetal pituitary responds to CRH.

Dopaminergic regulation of pituitary function in the late-gestation fetal sheep.

Development of the fetal ovine pituitary is essential for normal maturation and initiation of the parturition process, as well as for orchestrating endocrine responses to stress in utero. Increases in the biosynthesis of ACTH and prolactin (PRL) occur in the late-gestation fetal sheep pituitary. In the anterior lobe (AL) of the pituitary, pro-opiomelanocortin (POMC) biosynthesis and processing are primarily regulated by corticotrophin-releasing hormone and vasopressin. However, POMC in the intermediate lobe (IL) and PRL in the AL are known to be primarily regulated by dopamine, via the D2 receptor, in adult sheep. Because of the importance of ACTH and PRL during gestation we have investigated a potential role of dopamine in the control of both IL melanotrophs and AL lactotrophs and corticotrophs, in late gestation. Catheters were implanted into a maternal femoral artery and vein, fetal carotid artery and jugular vein as well as into the amniotic cavity. At day 130 of gestation, fetuses were infused intravenously with either the specific D2 receptor agonist bromocriptine (n = 5) or vehicle (n = 5), for 5 days. Blood samples were taken throughout the experiment and pituitaries were removed at the end of the treatment period. Bromocriptine caused a significant decrease (> 50%) in POMC mRNA levels in the IL. In contrast, bromocriptine had no significant effect on POMC mRNA levels or distribution in the AL. Fetal arterial ACTH and cortisol concentrations were unaffected by the bromocriptine infusion, compared with vehicle-infused controls. There was a dramatic decrease (> 80%) in plasma PRL concentrations, compared with the control fetuses. However, PRL mRNA levels in the AL were not significantly affected by bromocriptine. In conclusion, we have found that bromocriptine inhibits aspects of both melanotroph and lactotroph function in late-gestation fetal sheep. The data indicate that the fetal pituitary possesses functional D2 receptors in late gestation.

Differential regulation of proopiomelanocortin messenger ribonucleic acid in the pars distalis and pars intermedia of the pituitary gland after prolonged hypoxemia in fetal sheep.

POMC messenger RNA (mRNA) is synthesized and translated to form ACTH in the pars distalis and ACTH-related peptides in the pars intermedia of the pituitary in fetuses of species such as sheep. In the fetal sheep, maturation of hypothalamo-pituitary-adrenal (HPA) function occurs during late pregnancy accompanied by increased levels of POMC mRNA in the pars distalis, and contributes to the stimulus for birth. Activation of fetal HPA function also occurs with short term hypoxemia. However, the effects of more prolonged fetal hypoxemia, which may predispose to premature parturition, on POMC mRNA levels in the pars distalis and pars intermedia are not known. We studied the effects of sustained hypoxemia (48 h) in the absence of acidosis on fetal HPA responses at two times in late gestation, days 126-130 and days 134-136 (term = approximately 145 days). Hypoxemia was induced by lowering the fraction of oxygen in the maternal inspired gas mixture. Fetal arterial oxygen tension fell by 6-8 mm Hg, without a change in fetal arterial pH or carbon dioxide tension. Pituitary POMC mRNA was localized and quantified by in situ hybridization. At both gestational ages, hypoxemia caused a transient elevation in fetal plasma ACTH, with a maximum response at +2 h, and a sustained elevation in circulating cortisol. Cortisol responses were greater in the older fetuses. Changes in plasma cortisol were not associated with alterations in the plasma corticosteroid-binding capacity or in levels of hepatic corticosteroid-binding globulin mRNA. Pituitary POMC mRNA showed a regional distribution in the pars distalis, and mean levels increased with gestational age. After 48 h of hypoxemia, POMC mRNA levels had increased in the pars distalis. The regional distribution was unaffected by the hypoxemic insult. In contrast, POMC mRNA levels in the pars intermedia decreased after hypoxemia, and the reduction was greater in the older fetuses. We conclude that in response to prolonged hypoxemia, there is differential regulation of POMC mRNA levels in the pars distalis and pars intermedia of the pituitary in fetal sheep. The magnitude of these responses also changes with gestational age. The increase in POMC mRNA levels in the pars distalis at 48 h of hypoxemia, despite a rise in plasma cortisol and without a change in corticosteroid-binding globulin, suggests altered regulation of HPA function at this time.

Continuous intrafetal infusion of prostaglandin E2 prematurely activates the hypothalamo-pituitary-adrenal axis and induces parturition in sheep.

This study has investigated the effect of continuous intrafetal infusion of PGE2 or saline on hormone concentrations and the length of gestation in sheep. Fetal and maternal vascular catheters were surgically implanted at 112.3 +/- 1.3 days (n = 10), and the infusions were started at 121 +/- 1.2 days of gestation (term = 147). Fetuses were infused with either PGE2 (n = 5; 2 micrograms/min for 48 h and then increased to 4 micrograms/min for the remainder of the experiment) or the vehicle solution (n = 5; sterile isotonic saline) via the fetal carotid artery. In the PGE2-infused group, fetal and maternal plasma PGE2 concentrations increased (P < 0.001) after the change to the higher dose rate (4 micrograms/min) and remained elevated, fetal plasma immunoreactive ACTH (ir-ACTH) concentrations dramatically increased after the start of the infusion being maximal at 11 h before decreasing to match concentrations exhibited by the saline-infused group. Fetal plasma cortisol concentrations increased after the start of the PGE2 infusion (P = 0.05) and increased further after the change to the higher dose rate of 4 micrograms/min (P < 0.001). Concentrations of PGE2, ir-ACTH, and cortisol in the saline-infused group did not change until labor. Plasma concentrations of PGE2 (P < 0.001) and ir-ACTH (P < 0.005) increased on the day of labor in both treatment groups, and fetal cortisol concentrations increased (P < 0.001) in both groups in the last few days before labor. The proportion of low molecular weight ir-ACTH in the plasma of PGE2-infused fetuses was significantly higher than that of saline-infused fetuses (P < 0.001) during the first 15 days of infusion. In the saline-infused group, the proportion of low molecular weight ir-ACTH increased in the last few days before labour (P = 0.001), whereas no change was seen in PGE2-infused fetuses at this time. Maternal plasma progesterone concentrations decreased in both groups in the last few days before labor (P < 0.001). Fetuses infused with PGE2 delivered at 138.4 +/- 2.1 days, whereas control fetuses infused with saline delivered at 148.2 +/- 0.5 days (P < 0.001). The spontaneous increase in PGE2 preceding normal labor may thus play an important role in activation and maturation of the hypothalamic-pituitary-adrenal axis in fetal sheep.

Continuous intrafetal infusion of prostaglandin E2 prematurely activates the hypothalamo-pituitary-adrenal axis and induces parturition in sheep

This study has investigated the effect of continuous intrafetal infusion of PGE2 or saline on hormone concentrations and the length of gestation in sheep. Fetal and maternal vascular catheters were surgically implanted at 112.3 +/- 1.3 days (n = 10), and the infusions were started at 121 +/- 1.2 days of gestation (term = 147). Fetuses were infused with either PGE2 (n = 5; 2 micrograms/min for 48 h and then increased to 4 micrograms/min for the remainder of the experiment) or the vehicle solution (n = 5; sterile isotonic saline) via the fetal carotid artery. In the PGE2-infused group, fetal and maternal plasma PGE2 concentrations increased (P < 0.001) after the change to the higher dose rate (4 micrograms/min) and remained elevated, fetal plasma immunoreactive ACTH (ir-ACTH) concentrations dramatically increased after the start of the infusion being maximal at 11 h before decreasing to match concentrations exhibited by the saline-infused group. Fetal plasma cortisol concentrations increased after the start of the PGE2 infusion (P = 0.05) and increased further after the change to the higher dose rate of 4 micrograms/min (P < 0.001). Concentrations of PGE2, ir-ACTH, and cortisol in the saline-infused group did not change until labor. Plasma concentrations of PGE2 (P < 0.001) and ir-ACTH (P < 0.005) increased on the day of labor in both treatment groups, and fetal cortisol concentrations increased (P < 0.001) in both groups in the last few days before labor. The proportion of low molecular weight ir-ACTH in the plasma of PGE2-infused fetuses was significantly higher than that of saline-infused fetuses (P < 0.001) during the first 15 days of infusion. In the saline-infused group, the proportion of low molecular weight ir-ACTH increased in the last few days before labour (P = 0.001), whereas no change was seen in PGE2-infused fetuses at this time. Maternal plasma progesterone concentrations decreased in both groups in the last few days before labor (P < 0.001). Fetuses infused with PGE2 delivered at 138.4 +/- 2.1 days, whereas control fetuses infused with saline delivered at 148.2 +/- 0.5 days (P < 0.001). The spontaneous increase in PGE2 preceding normal labor may thus play an important role in activation and maturation of the hypothalamic-pituitary-adrenal axis in fetal sheep.

Biphasic developmental expression of adrenocorticotropin receptor messenger ribonucleic acid levels in the baboon fetal adrenal gland.

We have previously shown an estrogen-dependent developmental regulation of placental oxidation of cortisol to cortisone that results in enhanced fetal pituitary ACTH production and the induction of steroidogenic enzymes in and de novo cortisol production by the fetal adrenal in the second half of baboon pregnancy. However, it is not known whether the receptor for ACTH is simultaneously generated at this time in development to provide a mechanism for mediating the tropic action of ACTH on steroidogenesis in the primate fetal adrenal gland. Therefore, in the present study we determined the levels of ACTH receptor messenger RNA (mRNA) and correlated ACTH receptor expression with appearance of the mRNA for delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD), the enzyme protein that signals functional maturation of the definitive cortical zone in the baboon fetal adrenal. A baboon ACTH receptor complementary DNA was cloned and hybridized with polyadenylated RNA isolated from baboon (Papio anubis) fetal adrenals obtained in early (days 58-64; RNA from seven baboon fetuses pooled to yield three samples), mid-(days 99-103; RNA from five baboons pooled to yield four samples), and late (days 165-168; RNA of four individual baboon fetuses) gestation (term = 184 days). Expression of the primary 3.4-kilobase ACTH receptor mRNA transcript, determined by Northern blot and expressed as a ratio of beta-actin mRNA, was minimal early in gestation (mean +/- SE, 0.11 +/- 0.05 arbitrary densitometric units). However, fetal adrenal ACTH receptor mRNA levels increased (P < 0.001, by ANOVA) approximately 13-fold to 1.41 +/- 0.16 at midgestation, then declined by 70% (P < 0.001) to 0.41 +/- 0.10 in late gestation. To determine whether the decrease in ACTH receptor expression by the fetal adrenal in the second half of pregnancy reflected programmed cell death, the integrity of genomic DNA was assessed by 32P-labeled DNA gel electrophoresis and in situ DNA end labeling. Because DNA oligonucleosomes and apoptotic DNA strand breaks characteristic of apoptosis were absent in the adrenal glands of fetal baboons, the decline in ACTH receptor mRNA levels in the fetal adrenal did not seem to reflect programmed cell death. Expression of the single 2.0-kilobase mRNA transcript for 3betaHSD, an enzyme localized specifically in the definitive zone of the fetal adrenal, was minimal in early (0.01 +/- 0.00 arbitrary units) and mid- (0.10 +/- 0.01) gestation. However, 3betaHSD mRNA levels were markedly increased late in gestation to a value (1.38 =/- 0.34) approximately 13-fold greater (P < 0.001) than that in midgestation. These findings indicate that there was a biphasic monomodal developmental expression of the ACTH receptor in the baboon fetal adrenal, which contrasted with the progressive increase in adrenal weight, 3betaHSD expression, and de novo cortisol production previously determined. Because the fetal adrenal is comprised mainly of the fetal cortical zone throughout gestation, the decrease in ACTH receptor expression between mid- and late gestation seems to occur primarily in the latter zone and may signal a selective decline in tropic responsivity of and delta5-C19-steroid, e.g. dehydroepiandrosterone, biosynthesis within the baboon fetal adrenal gland.

Corticotropin-releasing hormone and related pituitary-adrenal axis hormones in fetal and maternal blood during the second half of pregnancy

There is little information available concerning the ontologic development of the human hypothalamic-pituitary-adrenal (HPA) axis nor of the potential interactions among fetal, maternal and placental-derived HPA axis hormones. This study evaluated levels of these hormones in matched maternal and fetal pairs during the second half of uncomplicated pregnancies. Immunoassays were used to measure serum concentrations of corticotropin-releasing hormone (CRH), adrenocorticotropin (ACTH) and cortisol in 104 matched fetal and maternal blood samples. Fetal specimens were obtained by percutaneous umbilical blood sampling (PUBS) between 18 and 40 weeks in patients whose pregnancies resulted in healthy, term infants. Correlations among these hormones, and the effect of gestational age were assessed. Maternal CRH concentrations [median (range)] [1.10 ng/ml (0.15 to 23.69)] were significantly greater than fetal values [0.35 ng/ml (0.07 to 1.0)]. Levels of maternal CRH (r = 0.73; p < 0.001) but not fetal CRH (r = 0.01; p = 0.98) correlated with gestational age. Maternal ACTH decreased (r = -0.21; p = 0.04) while fetal ACTH increased (r = 0.35; p < 0.003) with gestational age. Both maternal (r = 0.45; p < 0.001) and fetal (r = 0.57; p < 0.001) cortisol levels increased with gestational age. Maternal serum CRH values correlated best with fetal cortisol (r = 0.40; p = 0.0002) and correlated modestly with maternal cortisol (r = 0.28; p = 0.01), fetal ACTH (r = 0.24; p = 0.03) and fetal CRH (r = 0.23; p = 0.04); but not with maternal ACTH (r = -0.12; p = 0.3). Maternal CRH concentrations increase in the third trimester and correlate with rising fetal cortisol levels.

ACTH-like bioactivity and immunoactivity in fetal lamb pituitaries at 0.65 and 0.95 gestation.

We wished to determine if the concentration of bioactive ACTH-like activity increased during development and if there was heterogeneity in ovine fetal anterior pituitary ACTH activity as measured by bioassay and radioimmunoassay (RIA). We obtained anterior pituitaries from eight sheep fetuses (four at 0.65 and four at 0.95 gestation; term 145 +/- 5 days) and extracted and homogenized them in ice-cold 5N acetic acid, 0.3% phenylmethanesulfonyl fluoride (PMSF) and 0.2% BSA. Fractionation of each pituitary extract was performed by size-exclusion chromatography using Sepadex G-50. The ACTH-like immunoactivity (ALI) profile for each pituitary showed two well-defined peaks. One eluted with human ACTH1-39 and the other eluted with the high molecular weight fraction in the void volume. Four fractions from the first peak representing the high molecular weight forms of ACTH activity and four fractions from the second peak representing the low molecular weight forms of ACTH activity were pooled separately. These two pools were subjected to reverse-phase chromatography (RPC) on a C-8 column using a linear gradient of 70% acetonitrile in 0.8% trifluoroacetic acid over a 60 min period. Based upon the RIA, the high molecular weight forms of ACTH from the G-50 column were resolved into three main fractions, one eluting similar to the standard ACTH1-39 and the remaining two eluting after that. The low molecular weight forms of ACTH from the G-50 column were resolved into three peaks, before, with, and after the standard. We used collagenase-dispersed rat adrenal cells to test the ACTH-like bioactivity (ALB) of the crude extracts and of the different fractions obtained from the RPC of the high and low molecular weight material. The concentration of ACTH-like bioactivity in the crude extracts was similar at the two stages of gestation. However, there was a trend for the low molecular weight peak to have more peptide eluting with human ACTH1-39 and higher ratios of ALB/ALI than did the high molecular weight peak. These results suggest that multiple ACTH molecular forms with different ALB/ALI ratios are present in the ovine fetal pituitary and that there is no selective increase in ACTH1-39 concentration in the fetal pituitary in late gestation.

Effect of pulsatile oxytocin administration to the pregnant ewe in the last third of gestation on fetal ACTH and cortisol response to acute hypoxemia.

The aim of this study was to examine the effect of increased myometrial contractility throughout the last third of pregnancy on the ovine fetal response to short-term hypoxemia. Oxytocin (600 microU/kg/minute, n = 5) or saline (n = 7) was infused for 5 minutes every 20 minutes into the maternal jugular vein starting at 95-99 days of gestation and continuing throughout the last third of gestation. Fetuses were subjected to a hypoxemic challenge (1 hour) at 131 days of gestation while fetal plasma ACTH and cortisol levels and nuchal muscle electromyogram activity were monitored. The fetal plasma ACTH concentration before and during the hypoxemic challenge was similar in the control and oxytocin groups. The fetal plasma cortisol concentration in the oxytocin group was significantly lower before and during the hypoxemia than in the controls. During hypoxemia, fetal nuchal muscle activity was significantly reduced only in the control group. Increased myometrial contracture frequency throughout the last third of pregnancy alters both the neuroendocrine and behavioral responses of fetal sheep to short-term hypoxemia.

Levels of pro-opiomelanocortin and prolactin mRNA in the fetal sheep pituitary following hypoxaemia and glucocorticoid treatment in late gestation.

It is well established that corticotrophin-releasing hormone and vasopressin can induce both synthesis and release of ACTH from the ovine pituitary gland, and that glucocorticoids can inhibit these responses. Changes in the abundance, localization and distribution of pro-opiomelanocortin (POMC) mRNA and prolactin (PRL) mRNA in the ovine fetal pituitary were examined by in situ hybridization following hypoxaemia applied in the presence or absence of concomitant cortisol in late gestation (day 135). Fetuses were distributed amongst four groups; saline-infused/normoxaemic, cortisol-infused/normoxaemic (0.3 mg/h), saline-infused/hypoxaemic and cortisol-infused/hypoxaemic. Hypoxaemia (6 h) was induced by reducing the maternal PaO2, resulting in a 6-8 mmHg decrease in fetal arterial PO2. Fetal infusions were commenced 5 h prior to and maintained throughout the treatment period. Hypoxaemia, which elevated fetal plasma ACTH and cortisol, caused a significant (P < 0.05) increase in POMC mRNA in the pars distalis (PD), but was without effect on POMC mRNA in the pars intermedia (PI). Cortisol infusion attenuated the hypoxaemia-induced increase in POMC mRNA in the PD, but was without effect on non-stimulated steady-state POMC mRNA levels in either the PD or PI. PRL mRNA was only present in the PD and significantly (P < 0.05) increased after cortisol infusion and hypoxaemia. In conclusion (i) POMC and PRL mRNA in the PD are increased following moderate hypoxaemia, (ii) cortisol attenuates changes in POMC mRNA but not PRL mRNA in the PD following hypoxaemia and (iii) cortisol increases PRL mRNA levels in the PD.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Pulsatile Oxytocin Administration to the Pregnant Ewe in the Last Third of Gestation on Fetal ACTH and Cortisol Response to Acute Hypoxemia

Effect of Pulsatile Oxytocin Administration to the Pregnant Ewe in the Last Third of Gestation on Fetal ACTH and Cortisol Response to Acute Hypoxemia

Type 2 11 beta-hydroxysteroid dehydrogenase messenger ribonucleic acid and activity in human placenta and fetal membranes: its relationship to birth weight and putative role in fetal adrenal steroidogenesis.

Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) have been described which catalyze the interconversion of cortisol (F) to cortisone (E). 11 beta HSD activity has previously been reported in placenta and fetal membranes, where its role may be to protect the developing fetus from glucocorticoid excess. Furthermore, in the rat, an association between placental 11 beta HSD activity and the subsequent development of hypertension in the offspring has been reported. We have characterized the isoforms of 11 beta HSD in human fetal membranes and dissected placental tissue at term and investigated the relationship between placental 11 beta HSD activity and fetal and placental weights. 11 beta HSD activity studies in the presence of 0.1 mumol/L F and NAD (indicative of type 2 isoform activity) revealed high levels of activity in trophoblast dissected free of vessels (561 +/- 87 pmol E/h.mg protein; n = 4) > undissected placenta > cotyledenous vessels dissected away from trophoblast > placental and reflected amnion. In contrast, in the presence of 2.5 mumol/L F and NADP (indicative of type 1 isoform activity), only decidua and chorion demonstrated significant levels of 11 beta HSD activity. Type 1 11 beta HSD activity in chorion was probably due to decidual contamination, in that it was absent in decidua-free fused chorion obtained from a twin pregnancy. In keeping with these data, type 1 11 beta HSD messenger ribonucleic acid (1.5 kilobases) was detected in decidua, but in no other tissue, and high levels of type 2 11 beta HSD messenger ribonucleic acid (1.9 kilobases) were found in undissected placenta and trophoblast. In 27 term placentas, 11 beta HSD activity varied from 194-448 pmol E/h.mg protein. There was a weak, but significant, positive correlation between term placental 11 beta HSD activity and fetal weight (r = 0.408; P = 0.034), but no correlation with placental weight. Thus, in man, the reported association of a small fetus and a large placenta predisposing to adult hypertension cannot be explained on the basis of defective 11 beta HSD activity. However, the placenta offers an immense reservoir for F clearance (1.73-7.95 mumol/min.placenta) and may be a principal factor driving fetal ACTH secretion and, hence, fetal adrenal steroidogenesis.

Effect of pulsatile intravenous oxytocin administration to pregnant sheep over the last third of gestation on fetal ACTH and cortisol responses to hypotension

Effect of pulsatile intravenous oxytocin administration to pregnant sheep over the last third of gestation on fetal ACTH and cortisol responses to hypotension

Effect of Pulsatile Intravenous Oxytocin Administration to Pregnant Sheep Over the Last Third of Gestation on Fetal ACTH and Cortisol Responses to Hypotension

To examine the effect of increasing myometrial contractility in the last third of gestation on ovine fetal response to hypotension. Oxytocin (600 microU/kg/minute) or saline was infused via the maternal jugular vein as 5-minute pulses every 20 minutes, starting at 97 +/- 1 days of gestational age (mean +/- standard error of the mean) until labor. Fetal hypotension (10 minutes) was induced by intravenous nitroprusside infusion at 133 +/- 1 days' gestation. Ewes from both groups went into labor at the same gestational age. Total fetal body and adrenal gland weights were higher in controls than in the oxytocin-treated group. Maternal arterial pH and blood gas values were normal throughout the study. At 132-136 days' gestation, fetal arterial oxygen pressure was lower in the oxytocin group than in controls (P < .05). Basal fetal ACTH concentrations did not change between 130 and 136 days in both groups. The mean fetal plasma ACTH concentration was not different between the control (40.6 +/- 4.1 pg/mL) and oxytocin groups (32.6 +/- 4.9 pg/mL). Pre-hypotension fetal plasma ACTH was similar in both groups, whereas cortisol was lower in the oxytocin group. Hypotension significantly increased fetal plasma ACTH and cortisol concentrations; however, both ACTH and cortisol responses were smaller in the oxytocin group (P < .05). Increased myometrial contractility throughout the last third of gestation modifies the normal ACTH and cortisol relation at the critical time of prepartum increase in adrenocortical activity. In addition, fetal ACTH and cortisol responses to hypotension are diminished in fetuses exposed to such a prolonged increase in myometrial contractility. These observations support the hypothesis that myometrial contractility influences fetal neuroendocrine development.

Prepartum adrenocortical maturation in the fetal foal: responses to ACTH1–24

The present study was carried out on 19 chronically catheterized mares and fetuses in late gestation (term > 320 days). In six animals which were monitored up to the time of delivery of a live foal, plasma and amniotic fluid cortisol concentrations remained low until 4-5 days before parturition when there was a rapid, significant rise (P < 0.05) which was not accompanied by any corresponding changes in maternal plasma cortisol. Circulating fetal ACTH concentrations became more variable close to delivery and ANOVA revealed no significant increases during this critical period, although a negative correlation between plasma ACTH and time to delivery was observed (P < 0.05). Tests on fetal adrenal responsiveness to exogenous ACTH1-24 were carried out on ten animals. Before 295 days of gestation no significant increases in fetal plasma cortisol above its basal level of 20-30 nmol/l could be elicited by ACTH, administered as a single i.v. injection (1-2 micrograms/kg). By 304 +/- 3 days (mean +/- S.E.M.) small but significant (P < 0.05) increments in plasma cortisol were detected after ACTH, while in the oldest group (313 +/- 2 days) significant (P < 0.01) 50-60% increments were seen throughout the test period (2 h). Only one fetus was tested within 3 days of delivery and here a fourfold rise in plasma cortisol was evoked by ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Salmonella endotoxin administered to the pregnant sheep at 133-142 days gestation on fetal oxygenation, maternal and fetal adrenocorticotropic hormone and cortisol, and maternal plasma tumor necrosis factor alpha concentrations.

We studied the effects of maternal exposure to endotoxin on pregnancy outcome, stimulation of the hypothalamo-hypophyseal-adrenal axes (HHAA) of ewes and their fetuses, and maternal production of tumor necrosis factor alpha (TNF alpha), a central mediator in the pathogenesis of bacterial endotoxemia. Either endotoxin (lipopolysaccharide, LPS) from Salmonella typhimurium or saline was administered to pregnant sheep (n = 10) at 137.6 +/- 3.5 days gestational age by slow i.v. infusion at 1 microgram.kg-1 total dose over a 3-h period. Fetuses of ewes treated with LPS became hypoxemic by 2.5 h after initiation of LPS infusion. Maternal ACTH increased and peaked during LPS administration to levels approximately 10-fold greater in treated than in control ewes. In contrast, fetal ACTH levels were maximum at 6-12 h. Maternal and fetal cortisol levels were significantly different from control levels by 1 and 12 h, respectively. Maternal plasma TNF alpha peaked (6-7-fold over baseline) at 1-2 h after initiation of LPS administration and steadily declined over the following 48 h. Four of 5 treated ewes either delivered or their fetuses died within 28 h. In summary, infusion of LPS caused fetal hypoxemia and stimulated both fetal and maternal HHAA followed by preterm labor by 28 h after infusion in 4 of 5 ewes. Three of the fetuses died in utero. Maternal plasma TNF alpha rose rapidly, but the specific role it may play in initiation of preterm labor remains to be elucidated.

Parallel regulation of parentally imprinted H19 and insulin-like growth factor-II genes in cultured human fetal adrenal cells.

Adjacent, parentally imprinted, insulin-like growth factor-II (IGF-II) and H19 genes are highly expressed during embryogenesis and are important for fetal growth. Human fetal adrenals express abundantly both IGF-II and H19 genes. To clarify the significance and regulation of the H19 gene, we studied its expression in fetal adrenals. In situ hybridization experiments showed H19 RNA expression throughout the fetal adrenal cortex, with slightly higher expression in the outer definitive (adult) than in the inner fetal zone. In primary cultures of fetal adrenal cells, ACTH and other activators of the protein kinase-A signal transduction pathway increased both H19 and IGF-II RNA accumulation 1.7- to 10-fold. Staurosporine, a protein kinase-C inhibitor, increased H19 and IGF-II RNA to the same extent as did ACTH. The protein kinase-C activator 12-O-tetradecanoyl phorbol-13-acetate and cytokines, tumor necrosis factor-alpha and interferon-gamma, inhibited H19 and IGF-II RNA accumulation. Transforming growth factor-beta 1 caused a decrease in levels of H19 and IGF-II RNA, whereas the IGFs caused a slight increase. Our data show parallel multifactorial regulation of H19 and IGF-II RNAs in human fetal adrenal cells. This suggests common regulatory mechanisms for these adjacent genes.