Jacob T. Ross

The peptide ACTH(1-39), adrenal growth and steroidogenesis in the sheep fetus after disconnection of the hypothalamus and pituitary.

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.

Chronic stress--the key to parturition?

It is clear that the timing of parturition is dependent on a cascade of endocrine signals from an intact fetal hypothalamo-pituitary-adrenal axis. What is not known, however is the nature or source of the central neural stimulation which results in the stimulation of adrenocorticotrophic hormone (ACTH) synthesis and secretion in late gestation. The changes which occur in the synthesis and posttranslational processing of the ACTH precursor, proopiomelanocortin (POMC), in the fetal anterior pituitary before birth and the consequence of these changes for expression of the corticosteroidogenic enzymes in the fetal adrenal are described in this review. Evidence for the functional heterogeneity of corticotrophic cell types in the fetal sheep pituitary and the proposal that there is a maturational change in the populations of corticotrophic cells in late gestation are discussed. Finally, the development of cortisol negative feedback in the late gestation fetal hypothalamo-pituitary axis and the relevance of chronic stress to the timing of parturition are also discussed.

Role of Pituitary POMC-Peptides and Insulin-Like Growth Factor II in the Developmental Biology of the Adrenal Gland

During fetal life, it is critical that there is coordinate regulation of the growth, zonation and differentiation of the fetal adrenal cortex to ensure that cells in key tissues and organs are exposed in a programmed temporal sequence to the actions of glucocorticoids. Glucocorticoids are essential for maturation of key target organs before birth, including the lung, brain, liver, gut, kidney and adrenal, and the prepartum increase in glucocorticoid synthesis and secretion by the fetal adrenal gland is critical for the successful transition to postnatal life. It is also evident that premature or abnormal exposure of embryonic or fetal tissues to glucocorticoids during critical windows of development can irreversibly alter the programmed development of organ systems. Premature or abnormal exposure of the fetus to excess glucocorticoids may occur either as a consequence of endogenous stimulation of the fetal hypothalamo-pituitary-adrenal axis (HPAA) or as a consequence of exposure to exogenous glucocorticoids in a therapeutic context. Administration of synthetic glucocorticoids to women at risk of preterm labour, for example, is a routine clinical practice designed to improve respiratory function and neonatal outcome. It is clearly important to understand what endogenous factors regulate the growth and functional maturation of the adrenal cortex during development and the consequent likelihood of exposure of developing tissues to excess corticosteroids. To date, investigations have centred on the role of ACTH 1–39 in the stimulation of adrenal growth and steroidogenesis in long gestation species, such as the primate and sheep, where maturation and differentiation of organ systems occurs predominantly before birth. In this review, we will focus on the evidence that in addition to ACTH 1–39, other pro-opio-melanocortin (POMC) derived peptides, which are synthesized, processed and secreted by the fetal pituitary, play a role in the coordinate regulation of the specific phases of growth and functional development of the fetal adrenal gland in vivo. We will discuss our recent findings on the direct in vivo actions of N-POMC 1–77 and separately, insulin like growth factor II (IGF-II), as adrenal growth factors. These studies provide an understanding of the separate regulatory mechanisms which control activation of adrenal growth and stimulation of adrenal steroidogenesis in the late gestation fetus.

Infusion of N-Proopiomelanocortin-(1–77) Increases Adrenal Weight and Messenger Ribonucleic Acid Levels of Cytochrome P450 17α-Hydroxylase in the Sheep Fetus during Late Gestation1

In the sheep there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10–15 days gestation (term = 147 ± 3 days gestation). In the rat, peptides derived from the N-terminal region of POMC play a role in compensatory adrenal growth and in potentiation of ACTH-induced steroidogenesis. We therefore investigated the effects of infusion of bovine N-POMC-(1–77) and its biosynthetic derivative, N-POMC-(1–49) on adrenal growth and on the expression of adrenal steroidogenic enzymes in the late gestation sheep fetus. Twenty-seven pregnant ewes were used in this study. Fetal vascular catheters were inserted between 116–125 days gestation, and purified bovine N-POMC-(1–77) (2 μg/ml·h), N-POMC-(1–49) (2μ g/ml·h) and saline were each infused for 48 h between 136 and 138 days gestation. Intrafetal infusion of N-POMC-(1–77) resulted in an increased adrenal/fetal body weight ratio (94.6 ± 5.7 mg/kg) compared with that in saline-infused (75.6 ± 1.8 mg/kg), but not N-POMC-(1–49)-infused (82.7 ± ...

Glucocorticoids Decrease Phenylethanolamine N-Methyltransferase mRNA Expression in the Immature Foetal Sheep Adrenal

This study examined the impact of a chronic physiological elevation of plasma cortisol levels on adrenal catecholamine synthetic enzyme and proenkephalin A mRNA expression in foetal sheep. Cortisol (2.5–3.0 mg.5 ml−1.24 h−1, n=9) or saline (0.9% saline, n=6) was infused into foetal sheep for 7 days between 109 days and 116 days gestation. Foetal plasma cortisol concentrations were higher (P<0.0005) in the cortisol infused foetuses when compared with the saline infused group (43.07±4.13 nmol.l−1 vs 1.67±0.10 nmol.l−1). There were no differences, however, in the plasma ACTH levels between the two groups. Using Northern blot analysis, adrenal phenylethanolamine N‐methyltransferase (PNMT) mRNA expression was found to be reduced (P<0.005) fivefold in the cortisol infused foetuses when compared with the controls, as was the relative area of the adrenal medulla which stained positively with anti‐PNMT (28.1±2.5%vs 44.8±4.8%, P<0.007). No effect of cortisol infusion was observed on adrenal tyrosine hydroxylase mRNA and protein expression or proenkephalin A mRNA expression. We conclude that before birth, adrenaline synthesis may be suppressed by a novel direct, or indirect, inhibitory effect of glucocorticoids on PNMT mRNA expression.

Cortisol differentially regulates pituitary-adrenal function in the sheep fetus after disconnection of the hypothalamus and pituitary.

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, 3&bgr;‐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.

A premature increase in circulating cortisol suppresses expression of 11β hydroxysteroid dehydrogenase type 2 messenger ribonucleic acid in the adrenal of the fetal sheep.

Abstract We have investigated the effect of intrafetal cortisol administration, before the normal prepartum cortisol surge, on the expression of 11β hydroxysteroid dehydrogenase (11βHSD) type 2 mRNA in the fetal adrenal. We also determined whether increased fetal cortisol concentrations can stimulate growth of the fetal adrenal gland or increase expression of adrenal steroidogenic enzymes. Cortisol (hydrocortisone succinate: 2.0–3.0 mg in 4.4 ml/24 h) was infused into fetal sheep between 109 and 116 days of gestation (cortisol infused; n = 12), and saline was administered to control fetuses (saline infused; n = 13) at the same age. There was no effect of cortisol infusion on the fetal adrenal:body weight ratio (cortisol: 101.7 ± 5.3 mg/kg; saline: 108.2 ± 4.3 mg/kg). The ratio of adrenal 11βHSD-2 mRNA to 18S rRNA expression was significantly lower, however, in the cortisol-infused group (0.75 ± 0.02) compared with the group receiving saline (1.65 ± 0.14). There was no significant effect of intrafetal cortisol on the relative abundance of adrenal CYP11A1, CYP17, CYP21A1, and 3βHSD mRNA. A premature elevation in fetal cortisol therefore resulted in a suppression of adrenal 11βHSD-2. Increased intra-adrenal exposure to cortisol at this stage of gestation is, however, not sufficient to promote adrenal growth or steroidogenic enzyme gene expression.

N-Proopiomelanocortin (1–77) Suppresses Expression of Steroidogenic Acute Regulatory Protein (Star) mRNA in the Adrenal Gland of the Fetal Sheep

In the sheep, there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10–15 days gestation. Recently, we have shown that infusion of POMC (1–77) increases fetal adrenal growth and expression of CYP17 mRNA but does not significantly alter fetal plasma cortisol concentrations [1]. We therefore investigated the effects of infusion of bovine POMC (1–77) and its biosynthetic derivative POMC (1–49) on adrenal StAR mRNA expression. At 136d gestation, POMC (1–77) (n=5 fetuses; 2μg/ml/h), POMC (1–49) (n=5 fetuses, 2μg/ml/h) or Saline (n=5 fetuses, 1 ml/h) was infused for 48h. At 138d, fetal adrenal glands were collected and frozen in liquid N2 until RNA was extracted. Northern blot analyses demonstrated a major transcript for StAR mRNA at 3.0kb in fetal adrenal glands from all treatments. The membrane was stripped and re-probed with a 32P-labelled rat 18S rRNA oligo-probe to verify equal RNA loading. Infusion of POMC (1–77), but not POMC (1–49), resulted in a suppression of fetal adrenal StAR mRNA:18S rRNA when compared to adrenal StAR mRNA:18S rRNA from saline-infused controls. Our data suggest POMC (1–77) may act via separate mechanisms to increase fetal adrenal growth and to limit adrenal steroidogenesis through suppression of StAR mRNA.

Cortisol infusion decreases renin, but not PGHS-2, EP2, or EP4 mRNA expression in the kidney of the fetal sheep at days 109-116.

Renal prostaglandins (PG), renin, and cortisol are necessary for normal kidney development and function during fetal life. We examined the effects of cortisol infusion before completion of nephrogenesis (d 109–116 gestation; 2.0–3.0 mg hydrocortisone succinate/24 h) on the renal mRNA expression of PGHS-2, the PGE2 receptors, EP2 and EP4, and renin in fetal sheep. Cortisol infusion raised plasma cortisol levels to 42.8 ± 6.0 nmol/L compared with saline infusion levels of 1.5 ± 0.5 nmol/L (p < 0.001), but had no effect on fetal body weight, proportional kidney mass, or blood gases. Cortisol decreased significantly the relative expression of renin mRNA (saline: 0.93 ± 0.06 units; cortisol: 0.32 ± 0.03 units, p < 0.05), however it had no effect upon the expression of PGHS-2, EP2, or EP4 mRNA in fetal sheep kidney. Although there is substantial evidence that PGE2 acting through either the EP2 or EP4 receptor stimulates renin synthesis in the adult kidney, our results have demonstrated that before the completion of nephrogenesis, cortisol down-regulation of renin mRNA expression is independent of any change in the expression of PGHS-2, EP2, or EP4 mRNA expression. During nephrogenesis, the insensitivity of PGHS-2, EP2, and EP4 expression to down-regulation by cortisol may permit continued PG regulation of renal development and urine formation.

EFFECT OF N-PROOPIOMELANOCORTIN (1-77) AND (1-49) INFUSIONS ON ADRENAL EXPRESSION OF CYCLIN D1 IN THE FETAL SHEEP

In the sheep, there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10–15 days gestation. Recently, we have shown that infusion of POMC 1–77 increases fetal adrenal growth but does not significantly alter fetal plasma cortisol concentrations. Phosphorylation and inactivation of the pRB protein, which is required for progression into the DNA synthetic phase of the cell-cycle is conducted by a holoenzyme, for which cyclin D1 gene encodes the rate-limiting regulatory subunit. To further elucidate the mechanisms by which POMC 1–77 regulates adrenal growth, we therefore examined adrenal expression of the rate-limiting cell cycle protein, cyclin D1, from fetuses infused for 48 hr with POMC 1–77 (n = 6), POMC 1–49 or Saline (n = 6). There was no significant difference in the adrenal expression of cyclin D1 mRNA levels between POMC 1–77, 1–49 and saline infused fetuses. There was no significant correlation between cyclin D1 (4.0 Kb) and adrenal weight. In summary, these data do not demonstrate that the rate-limiting cell cycle protein, cyclin D1, is activated to stimulate adrenal growth following infusion of POMC 1–77 in the fetal sheep in late gestation.