I. D. Phillips

Placental Restriction Alters the Functional Development of the Pituitary-Adrenal Axis in the Sheep Fetus during Late Gestation

We have experimentally restricted placental growth in the sheep to investigate the impact of reduced substrate delivery on fetal pituitary proopiomelanocortin (POMC) mRNA levels and on circulating ACTH 1-39, immunoreactive ACTH, and cortisol concentrations during late gestation. Endometrial caruncles were removed in nine ewes before mating to reduce the number of placentomes formed [placental restriction group (PR)]. Fetal arterial Po2 and O2 saturation were reduced in the PR group (2.0± 0.1 kPa and 42.8 ± 1.1%, n = 9) when compared with control fetuses (3.1 ± 0.1 kPa and 66.4 ± 0.9%, n = 10). The ratio of anterior pituitary POMC mRNA:18 S ribosomal RNA was also lower (p < 0.05) in the PR group (0.49 ± 0.05) when compared with the control group (0.80 ± 0.12) after 140 d of gestation. In contrast, plasma concentrations of ACTH 1-39 and immunoreactive ACTH were similar in the PR and control groups throughout late gestation. Plasma ACTH 1-39 concentrations increased (p < 0.006) between 128 and 134 d of gestation, in both the PR (122-128 d: 2.70 ± 0.34 pmol/L: 134-141 d; 7.07 ± 1.57 pmol/L) and control (122-128 d; 3.36 ± 0.56 pmol/L: 134-141 d; 10.78 ± 2.88 pmol/L) groups. Combined adrenal weight was higher (p < 0.005) in the PR group (130 ± 10 mg/kg) compared with controls (80 ± 1 mg/kg) at 140 d of gestation, and plasma cortisol concentrations were also higher (p < 0.02) in PR than control fetuses between 127 and 141 d of gestation. These changes imply that the fetal hypothalamopituitary-adrenal axis is operating at a new central set point in the growth-restricted fetus.

CONSERVATION OF SURFACTANT PROTEIN A : EVIDENCE FOR A SINGLE ORIGIN FOR VERTEBRATE PULMONARY SURFACTANT

Abstract. Surface tension is reduced at the air–liquid interface in the lung by a mixture of lipids and proteins termed pulmonary surfactant. This study is the first to provide evidence for the presence of a surfactant-specific protein (Surfactant Protein A—SP-A) in the gas-holding structures of representatives of all the major vertebrate groups. Western blot analysis demonstrated cross-reactivity between an antihuman SP-A antibody and material lavaged from lungs or swimbladders of members from all vertebrate groups. Immunocytochemistry localized this SP-A–like protein to the air spaces of lungs from the actinopterygiian fish and lungfish. Northern blot analysis indicated that regions of the mouse SP-A cDNA sequence are complementary to lung mRNA from all species examined. The presence of an SP-A–like protein and SP-A mRNA in members of all the major vertebrate groups implies that the surfactant system had a single evolutionary origin in the vertebrates. Moreover, the evolution of the surfactant system must have been a prerequisite for the evolution of airbreathing. The presence of SP-A in the goldfish swimbladder demonstrates a role for the surfactant system in an organ that is no longer used for airbreathing.

Prolactin Receptor Gene Expression and Foetal Adipose Tissue

We have investigated the effects of increasing gestational age, maternal undernutrition or restricted placental growth on prolactin receptor (PRLR) gene expression in perirenal adipose tissue collected from foetal sheep during late gestation (term=147 d±3 d of gestation). Foetal nutrient supply was reduced by either restriction of placental growth following removal of endometrial caruncles before mating or by reducing maternal feed intake by 50% from 115 d of gestation. Total RNA was extracted from adipose tissue taken from foetal sheep between 90 and 145 d of gestation, and only at 141–145 d in placentally restricted, nutrient restricted and control foetuses. Messenger RNAs encoding the long (PRLR1) and short (PRLR2) forms of the PRLR and glyceraldehyde‐phosphate‐dehydrogenase (GAPDH) were detected and quantified in a ribonuclease protection assay using an antisense RNA probe complementary to ovine PRLR2 and GAPDH. There was a 7.5‐fold increase in the amount of perirenal adipose tissue between 90 and 125 d of gestation, compared with a 1.3‐fold increase between 125 and 145 d of gestation. The abundance of mRNA encoding PRLR1 and PRLR2 in perirenal adipose tissue increased 10‐ and sixfold, respectively, between 90 and 125 d of gestation, and then declined by 145 d of gestation. Both placental restriction and maternal undernutrition significantly reduced foetal adipose tissue deposition. The abundance of PRLR1 but not PRLR2 mRNA was reduced in adipose tissue from the placentally restricted group, where as GAPDH mRNA was three times higher than in controls. In contrast, maternal undernutrition from 115 d of gestation did not affect PRLR1, PRLR2 or GAPDH mRNA expression in foetal adipose tissue. It is concluded that during the period of rapid deposition of perirenal adipose tissue, there is a concomitant increase in PRLR gene expression. This indicates that prolactin may play an important role in the growth and maturation of foetal adipose tissue which occurs before birth.

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.

Differential Effects of Increasing Gestational Age and Placental Restriction on Tyrosine Hydroxylase, Phenylethanolamine N-Methyltransferase, and Proenkephalin A mRNA Levels in the Fetal Sheep Adrenal

Abstract: We have demonstrated that there are differential changes in the levels of tyrosine hydroxylase (TH), phenylethanolamine N‐methyltransferase (PNMT), and proenkephalin A (Pro Enk A) mRNA in the fetal sheep adrenal during late gestation. Adrenal TH mRNA:18S rRNA ratios increased between gestational days 100 (0.98 ± 0.13; n = 6) and 125 (1.40 ± 0.15; n = 6) and then decreased, whereas adrenal PNMT mRNA:18S rRNA ratios increased regularly between gestational days 100 (0.08 ± 0.01) and 146 (0.17 ± 0.03). The ratio of adrenal Pro Enk A mRNA to 18S rRNA was higher at gestational day 125 (0.085 ± 0.005) than at either 80–100 days (0.038 ± 0.007) or 140–146 days of gestation (0.055 ± 0.013). In 12 ewes, the growth and development of the placenta were restricted (placental restriction group) from conception. The ratio of adrenal PNMT mRNA to 18S rRNA was significantly reduced in the placental restriction group of fetal sheep (0.003 ± 0.002) compared with controls (0.011 ± 0.002), and there was a significant correlation between the ratio of adrenal PNMT mRNA to 18S rRNA and the mean arterial Po2 (r = 0.88, p < 0.0005). In contrast, TH mRNA and Pro Enk mRNA were unaffected by placental restriction. Adrenaline and nonadrenaline syntheses are therefore differentially regulated in the adrenal during late gestation and in response to chronic intrauterine hypoxemia.

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.

The Relative Roles of the Hypothalamus and Cortisol in the Control of Prolactin Gene Expression in the Anterior Pituitary of the Sheep Fetus

The neuroendocrine control of prolactin synthesis and secretion before birth is not well understood. We have measured the changes in the level of prolactin mRNA in the anterior pituitary of the fetal sheep throughout the last 15 days of pregnancy (term = 147 ± 3 days gestation). We have also investigated the effects of surgical disconnection of the fetal hypothalamus and pituitary (HPD) with or without long term Cortisol infusion on pituitary prolactin mRNA levels and plasma prolactin concentrations in the late gestation sheep fetus. Prolactin mRNA levels were measured in anterior pituitaries collected from a series of fetal sheep (130–134 days, n = 6; 135–140 days, n = 6; 141–145 days, n = 6) in late gestation. HPD was carried out in ten fetal sheep at 105–115 days gestation and five intact fetal sheep were used as controls. In the HPD group, either saline (HPD + saline group, n = 5) or Cortisol was infused (3.5mg/24 h) for 5 days from 134–136 days gestation (HPD + Cortisol group, n = 5). There was an increase in the ratio of prolactin mRNA:18S rRNA in the fetal pituitary between 130–134 days (0.46 + 0.08, n = 6) and 135–140 days (1.27 ± 0.17 n = 6) which was maintained after 141 days gestation, (1.27 ± 0.11, n = 6). The mean prolactin mRNA: 18 S rRNA ratio was significantly higher (P<0.05) in intact fetal sheep (1.41 ± 0.16, n = 4) than in the HPD fetal sheep after either saline (0.54 ± 0.14, n = 4) or Cortisol (0.74 ± 0.24, n = 5) administration. The mean plasma concentration of prolactin was also higher in the intact group (28.3 ± 3.9 ng/ml) when compared with the HPD + saline group (8.0±3.3 ng/ml) or the HPD+cortisol group (5.6 ± 1.9 ng/ml). We have demonstrated that there is a strong hypothalamic drive to prolactin synthesis and secretion in the fetus and that Cortisol does not act directly at the fetal pituitary to stimulate prolactin synthesis and secretion in late gestation.

Restriction of Fetal Growth has a Differential Impact on Fetal Prolactin and Prolactin Receptor mRNA Expression: Fetal growth restriction and prolactin

Prolactin is present in the fetal circulation and prolactin receptors are expressed in a wide range of fetal tissues. The factors which regulate the synthesis and secretion of prolactin, and the expression of its receptors before birth, are poorly understood. We have investigated whether experimental restriction of placental growth in the sheep has an impact on the prolactin axis in the growth restricted fetus. The majority of uterine endometrial caruncles were removed before pregnancy in 10 ewes (placental restriction; PR group). Placental, fetal liver and kidney weights were reduced in the PR compared to the control group (n = 10). The ratio of fetal prolactin mRNA : 18S rRNA was significantly lower (P < 0.01) in the PR group (1.83 ± 0.45, n = 6) than in the control group (4.11 ± 0.54, n = 6). The ratio of prolactin mRNA : 18S rRNA in the fetal pituitary was positively correlated with fetal and with placental weight. Using stepwise linear regression, it was determined that the level of fetal prolactin mRNA : 18S rRNA expression was best described (as judged by the maximum adjusted R2) by prolactin mRNA: 18 S rRNA = − 3.0378 + 0.17 PO2 + 2.772 glucose (adjusted R2 = 0.765, F = 17.53, P < 0.001). Fetal plasma prolactin concentrations were significantly reduced (P < 0.05) in the PR group compared to control animals between 109 and 141 days gestation. Fetal prolactin receptor (PRLR) mRNA transcripts encoding long (PRLR1) and short forms (PRLR2) of PRLR were present in the liver and kidney of animals in the PR and control groups at 140–141 days gestation. PR did not alter the levels of PRLR1 or PRLR2 mRNA in the fetal liver or kidney. The suppression of the synthesis and secretion of prolactin in the growth restricted fetus may limit the action of prolactin on the growth and metabolism of key fetal organs during suboptimal intrauterine conditions