The role of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins essential for adrenal steroidogenesis in the late-gestation ovine fetus (original) (raw)
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1. We have investigated the role of the fetal hypothalamo-pituitary axis in the control of adrenocortical growth and steroidogenesis in the sheep fetus during late gestation. Plasma concentrations of ACTH(1-39) increased between 120-125 and 136-142 days (P < 0.05), but did not change after surgical disconnection of the fetal hypothalamus and pituitary (HPD) at 106-120 days gestation. There was no effect of either gestational age or HPD on the circulating concentrations of the ACTH-containing precursors pro-opiomelanocortin (POMC) and pro-ACTH (the 22 kDa N-terminal portion of POMC). 2. In the fetal sheep adrenal, the relative abundance of the mRNAs of the steroidogenic enzymes CYPIIA1 and CYP21A1 increased between 130-135 and 136-140 days gestation (P < 0.05) and remained high after 141 days, whereas that of CYP17 mRNA increased after 141 days gestation (P < 0.05). The abundance of adrenal 3 beta-HSD mRNA did not change between 130 and 145 days. 3. Hypothalamo-pituitary disconnection significantly reduced the abundance of of CYPIIA1 mRNA, 3 beta-HSD mRNA and CYP17 mRNA by 3.4, 3.1 and 3.7 times, respectively, at 140-142 days gestation (P < 0.05). 4. In the intact group of fetal sheep, adrenal weight increased between 130-135 and 141-145 days (P < 0.05), but there was no change in the abundance of adrenal insulin-like growth factor II (IGF-II) mRNA across this gestational age range. Hypothalamo-pituitary disconnection significantly reduced fetal adrenal weight to 66% that of intact sheep (P < 0.01), but did not alter the abundance of IGF-II mRNA in the fetal adrenal at 140-142 days. 5. Our results suggest that the prepartum changes in adrenal growth and steroidogenesis are under the control of an intact hypothalamo-pituitary axis in late gestation and are dependent on an increase in circulating ACTH(1-39), rather than on ACTH precursors. We have found no evidence, however, for a direct-relationship between fetal adrenal growth or steroidogenesis and adrenal IGF-II mRNA between 130 and 145 days gestation.
We have investigated the effects of restriction of placental growth on foetal adrenal growth and adrenal expression of mRNAs for Insulin-like Growth Factor II (IGF-II), the IGF binding protein IGFBP-2, Steroidogenic Factor 1 (SF-1) and adrenocorticotrophic hormone (ACTH) receptor (ACTH-R) and the steroidogenic cytochrome P-450 enzymes: cholesterol side chain cleavage (CYP11A1), 17alpha-hydroxylase (CYP17) and 21-hydroxylase (CYP21A1); and 3beta-hydroxysteroid dehydrogenase/Delta5Delta4 isomerase (3betaHSD). Endometrial caruncles were removed from non-pregnant ewes before mating (placental restriction group; PR). The total adrenal: foetal weight ratio was higher in PR (n=6 foetuses) than in control foetuses (n=6 foetuses). There was no difference in plasma ACTH concentrations between the PR and control foetuses between 130 and 140 days gestation. Adrenal IGF-II mRNA levels were lower (P<0.05) in the PR group, however, adrenal IGFBP-2 mRNA levels were not different between the PR and control groups. Adrenal ACTH-R mRNA levels were also lower whilst CYP11A1 mRNA levels were increased (P<0.005) in the PR group. We conclude that foetal adrenal growth and steroidogenesis are stimulated as a consequence of foetal growth restriction and that factors other than ACTH are important in foetal adrenal activation during chronic, sustained hypoxaemia.
Journal of Endocrinology, 1998
A dramatic late-gestation increase in fetal plasma cortisol concentrations is critical for the timing of parturition in the sheep. This increase appears to depend upon an intact hypothalamo-pituitary unit and is characterised by increasing responsiveness of the fetal adrenal gland to ACTH. ACTH has been postulated as the critical determinant of the late-gestation cortisol increase; however, recent evidence has suggested that other factors, including the ACTH precursor, pro-opiomelanocortin, may also be involved. To further define the role of ACTH in determining the timing of parturition and the responsiveness of the fetal adrenal gland, intact (INT/ACTH) and hypophysectomised (HX/ACTH) fetuses received a continuous infusion of ACTH (1-24) from the time of surgery (115 days gestational age (GA)) at a rate we have previously shown to generate normal fetal cortisol concentrations and term parturition in HX fetuses. A third group of saline-infused intact fetuses (INT/SAL) served as the control group. Adrenal responsiveness was assessed by cortisol responses to ACTH (1-24) challenges at 120, 130 and 140 days GA.
Biology of Reproduction, 2002
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 Ͻ 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 Ͻ 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]/g 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/g 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.
Journal of Biological Chemistry
The developmental expression of adrenocortical steroid hydroxylases was studied in bovine fetuses from 40 to 280 days gestational age. The expression of P-45017, is first detected at a gestational age of 50 days and reaches a maximum at 60-70 days. The expression of P-45017, then declines and is nondetectable at a gestational age of 100 days. P-45017, is not expressed again until about 240 days, Le. shortly before birth (-280 days). P-450,, P-450c21, P-450116 and adrenodoxin were present in fetal adrenals throughout gestation. This "on-off-on" pattern of P-45017, expression during fetal development was associated with a corresponding episodic production of cortisol. Immunoreactive corticotropin (ACTH) levels in fetal plasma were elevated in small fetuses (corresponding to 5100 days) and in near-term fetuses (corresponding to >250 days) compared with those in mid-gestation fetuses. In primary culture, adrenal cells from mid-gestation fetuses contained no detectable P-45017, but rapidly responded to ACTH with an increase in P-45017, protein and mRNA. The tissue specificity of the developmental patterns is emphasized by the fact that both P-45017, and P-460, were detectable throughout the development of the fetal testes, whereas only P-450,, was detectable in fetal bovine ovary prior to 200 days. Thus, in fetal bovine adrenal it appears that ACTH is the major regulatory factor effecting the intermittent presence of P-45017,, whereas the presence of the other steroid hydroxylases is either regulated by additional factors or shows a much different sensitivity to ACTH.
Molecular and Cellular Endocrinology, 2002
We have previously demonstrated that maternal undernutrition during either the 'periconceptional' (i.e. from 60 days (d) before until 7 d after mating) or 'gestational' periods (i.e. from 8 d after mating until the end of pregnancy) have differential effects on the subsequent development of the hypothalamo-pituitary-adrenocortical (HPA) axis and on adrenal growth and steroidogenesis in the sheep fetus during late gestation (term0/1479/3 d gestation). The specific mechanisms by which periconceptional or gestational undernutrition result in activation of the fetal HPA axis in late gestation are unclear. We have therefore investigated the impact of maternal nutrient restriction imposed either during the periconceptional period, or between 8 and 147 d gestation on the expression of specific genes in the fetal pituitary and adrenal which regulate adrenal steroidogenesis in late gestation. Ewes were maintained on either a Control (C) or Restricted (R, 70% of C) diet from 60 d before until 7 d after mating (periconceptional period) and then maintained on either a Control or Restricted diet from 8 d after mating for the remainder of pregnancy (gestational period). Four nutritional treatment groups were therefore generated (C Á/C, C Á/R, R Á/R and R Á/C). Whilst periconceptional undernutrition (R Á/R and R Á/C groups) resulted in higher fetal plasma adrenocorticotrophic hormone (ACTH) at 135 Á/146 d gestation, there was no change in the relative level of expression of the ACTH receptor (MC2R), steroidogenic acute regulatory protein (StAR) or steroidogenic enzyme mRNAs in the fetal adrenal in late gestation. Exposure to gestational undernutrition (R Á/R and C Á/R groups), however, resulted in a stimulation in the relative level of expression of MC2R mRNA (P0/0.001) and StAR mRNA (P0/ 0.007) in the fetal adrenal during late gestation. This study provides new insights into the potential mechanisms by which alterations of the nutrient environment of the fetus at different stages of gestation may result in differential activation of the fetal HPA axis. #
We have investigated the effects of a 5 day infusion of cortisol into fetal sheep, in which the hypothalamus and pituitary were surgically disconnected (HPD), on fetal pituitary-adrenal function. Fetal HPD and vascular catheterization were carried out at between 104 and 124 days gestation. Cortisol was administered (3.5 mg 24 h−1) for 120 h between 134 and 140 days (HPD+F group; n=5) and saline was administered during the same gestational age range to HPD (HPD group; n=12) and intact fetal sheep (Intact group; n=6). Cortisol infusion into the HPD fetal sheep did not suppress the mRNA levels for Proopiomelanocortin (POMC) in the fetal anterior pituitary at 139/140 days gestation (POMC mRNA: 18S rRNA: Intact 0.40±0.05; HPD 0.56±0.07; HPD+F 0.49±0.07). Similarly, there was no significant effect of either HPD or cortisol infusion on the plasma concentrations of immunoreactive (ir) ACTH or ACTH(1–39). The adrenal: fetal body weight ratio was significantly higher, however, in the HPD+F (88.4±8.7 mg kg−1) and Intact groups (84.1±5.6 mg kg−1) when compared with the HPD fetal sheep (63.7±5.4 mg kg−1). The ratio of total IGF-II mRNA: 18S rRNA was similar in the adrenals of the Intact (0.48±0.09), HPD (0.78±0.09) and HPD+F (0.71±0.11) groups. The ratios of CYPIIA1, 3b-HSD and CYP21A1 mRNA: 18S rRNA were significantly lower in adrenals from the HPD group when compared to those in the Intact group and were not restored to normal by cortisol infusion. We have therefore demonstrated that cor tisol does not act directly at the fetal pituitary to suppress POMC synthesis or ACTH secretion in late gestation. Cortisol does, however, stimulate fetal adrenal growth after HPD in the absence of any effects on adrenal IGF-II or steroidogenic enzyme mRNA levels. The data provide evidence that an intact hypothalamic-pituitar y axis and cor tisol each play an important role in the stimulation of adrenal growth and steroidogenesis which occurs during the last 10–15 days of gestation in the sheep. Parturition in the sheep is dependent on the prepartum increase cortisol acts in the slow time domain at the fetal pituitary, we have infused cortisol for 5 days from around 135 days gestation in the fetal plasma concentrations of immunoreactive (ir) ACTH and cortisol (term=147±3 days (d) gestation) (1). The concomit-in HPD fetal sheep and have measured POMC mRNA levels in the fetal anterior pituitary and circulating irACTH during the ant rise in fetal plasma ACTH and cortisol before delivery is intriguing as several studies have clearly demonstrated that short infusion period. We have also previously demonstrated that the late gestational and long-term infusions of glucocorticoids can inhibit basal and stimulus induced increases in fetal plasma ACTH concentrations increase in plasma ACTH(1–39) concentrations, adrenal growth and adrenal steroidogenic enzyme expression in the sheep fetus (2–8). There have been no studies, however, on the neuroendo-crine site of action of cortisol acting in the slow time domain are dependent on an intact and functional hypothalamo-pituitary axis (11). It remains unclear, however, how adrenal growth and (i.e.>8 h) on the synthesis of the ACTH precursor, Proopiomelanocortin (POMC) in the fetal pituitary and on steroidogenesis are regulated during the last 15 days of gestation. Chromatographic studies have shown that irACTH is present in ACTH secretion. We have previously described a method of surgical disconnection of the fetal hypothalamus and pituitary the fetal circulation in a range of molecular weight forms including the bioactive ACTH(1–39) and the high molecular weight ACTH (hypothalamo-pituitary disconnection; HPD) at around 110 days gestation which results in minimal infarction of the fetal anterior precursors (POMC and Pro-ACTH) (12). One possibility therefore is that in late gestation, the coordinate regulation of fetal pituitary and does not diminish the fetal ACTH response to an exogenous bolus of Corticotrophin Releasing Hormone (CRH) adrenal growth, steroidogenesis and cortisol output is dependent on changes in the post translational processing of POMC in the (7, 9). Whilst circulating irACTH concentrations are maintained or are higher in fetal sheep after HPD, there is no prepartum fetal pituitary which in turn requires the action of a hypothalamic secretagogue. A second possibility, however, is that the functional cortisol increase after 135 d gestation and gestation is therefore prolonged in the HPD fetus (10). In order to determine whether changes in the pituitary-adrenal axis in late gestation are, in part,
Biology of Reproduction, 1995
We have previously demonstrated that increased expression of fetal pituitary proopiomelanocortin mRNA and the induction of enzymes catalyzing fetal adrenal cortisol formation at term are regulated by estrogen-induced changes in placental oxidation of maternal cortisol to cortisone. To test the hypothesis that induction of fetal pituitary adrenocorticotropic hormone (ACTH) production by estrogen-induced changes in placental cortisol oxidation results in increased responsivity of the fetal adrenal gland to ACTH, in the present study we compared fetal adrenal sensitivity to ACTH in vitro at midgestation in untreated controls and in animals treated at this time in gestation with estrogen. Fetal adrenals were obtained on Day 100 (n = 7) and Day 165 (n = 5; term = Day 184) from untreated baboons and on Day 100 following maternal treatment with estradiol (s.c.; Days 70-100; n = 10) or androgen precursor (n = 3). Adrenal slices (15-25 mg) were perifused (100 Il/min; 37'C) with Medium 199 (no phenol red); media were collected at 10-min intervals and assayed for cortisol and dehydroepiandrosterone. Secretion of cortisol and dehydroepiandrosterone reached equilibrium after 140 min of perifusion; therefore, basal release was calculated as the mean steroid concentrations during 190-240 min. Adrenal slices were then perifused for 20 min with saline or ACTH at 240 (0.001 nmol), 370 (0.01 nmol), and 490 (0.1 nmol) min, and an overall average cortisol/dehydroepiandrosterone secretion rate (pg/min/mg) between 240-600 min was calculated. Basal cortisol production at Day 165 (85 ± 11) exceeded (p < 0.05) that at Day 100 in adrenals of control (9 3) and estrogen-treated (13 + 3) animals. In contrast, dehydroepiandrosterone production by control adrenals of Day 165 (13 4) was lower than that at Day 100 in control (30 ± 6) and estrogen-treated (20 3) animals. Thus, the ratio of dehydroepiandrosterone:cortisol secretion was significantly (p < 0.05) higher at midgestation (4.93 ± 2.15) than at term (0.16 ± 0.05) and was reduced (p < 0.05) in adrenals of estrogen-treated baboons (1.87 0.38). In all groups, basal secretion of cortisol and dehydroepiandrosterone was not altered after infusion of saline. In contrast, ACTH increased (p < 0.05) cortisol secretion by 2> 50% in adrenals of Day 165 (116 ± 20) and in those of estrogen-treated (18 + 3) but not untreated (10 4) baboons of Day 100. ACTH increased (p < 0.05) androgen production by estrogen-treated adrenals of Day 100 to a greater (p < 0.05) extent (515% + 161%) than that of controls at Day 100 (154% ± 81%). These data indicate that estrogen treatment in vivo at midgestation induced a ratio of dehydroepiandrosterone:cortisol secretion in vitro in fetal adrenals that mimicked the ratio near term and was associated with increased steroid hormone production in response to ACTH. Therefore, we suggest that activation of the fetal hypothalamic-pituitary-adrenal axis at term and at midgestation following maternal estrogen administration is associated with increased responsivity of the fetal adrenal to ACTH.