Giuseppe Simonetta

Restriction of placental and fetal growth in sheep alters fetal blood pressure responses to angiotensin II and captopril

1 We have measured arterial blood pressure between 115 and 145 days gestation in normally grown fetal sheep (control group; n= 16) and in fetal sheep in which growth was restricted by experimental restriction of placental growth and development (PR group; n= 13). There was no significant difference in the mean gestational arterial blood pressure between the PR (42.7 ± 2.6 mmHg) and control groups (37.7 ± 2.3 mmHg). Mean arterial blood pressure and arterial P  O 2 were significantly correlated in control animals (r= 0.53, P < 0.05, n= 16), but not in the PR group. 2 There were no changes in mean arterial blood pressure in either the PR or control groups in response to captopril (7.5 μg captopril min−1; PR group n= 7, control group n= 6) between 115 and 125 days gestation. After 135 days gestation, there was a significant decrease (P < 0.05) in the fetal arterial blood pressure in the PR group but not in the control group during the captopril infusion (15 μg captopril min−1; PR group n= 7, control group n= 6). 3 There was a significant effect (F= 14.75; P < 0.001) of increasing doses of angiotensin II on fetal diastolic blood pressure in the PR and control groups. The effects of angiotensin II were different (F= 8.67; P < 0.05) in the PR and control groups at both gestational age ranges. 4 These data indicate that arterial blood pressure may be maintained by different mechanisms in growth restricted fetuses and normally grown counterparts and suggests a role for the fetal renin‐angiotensin system in the maintenance of blood pressure in growth restricted fetuses.

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.

Effect of maternal feed restriction on blood pressure in the adult guinea pig

Small size at birth has been associated with increased blood pressure in adult men and women. In rats, isocaloric protein restriction reduces fetal growth and increases systolic blood pressure in adult offspring. Balanced maternal undernutrition in the rat also increases adult blood pressure, but not consistently. The aim of this study was to determine the effect of moderate balanced maternal undernutrition (85% of ad libitum intake from 4 weeks before, and throughout pregnancy) on blood pressure of adult offspring in the guinea pig, a species that is relatively mature at birth. Blood pressure was measured in chronically catheterised offspring of ad libitum fed or feed‐restricted mothers, at 3 months of age (young adult). Maternal feed restriction reduced birth weight (‐17%) and increased systolic blood pressure (+9%, P < 0.03) in young adult male offspring. In offspring of ad libitum fed and feed‐restricted mothers, combined data showed that systolic blood pressure and mean arterial pressure correlated negatively with head width at birth (P = 0.02 and P = 0.04, respectively, n = 28). Systolic blood pressure also correlated negatively with birth weight and the ratio birth weight/birth length, but only in offspring of ad libitum fed mothers (P = 0.04 and P = 0.03, respectively, n = 22). The effect of maternal feed restriction on systolic blood pressure in male offspring was not significant when adjusted for these measures of size at birth. Thus, moderate balanced undernutrition in the guinea pig increases systolic blood pressure in young adult male offspring; however, these effects may be mediated, at least in part, through effects on fetal growth.

Impact of placental restriction on the development of the sympathoadrenal system.

We have investigated the impact of chronic restriction of placental function on circulating catecholamine concentrations and responses to the indirectly acting, sympathomimetic amine, tyramine, in the fetal sheep in late gestation. In 10 ewes, endometrial caruncles or placental placentation sites were removed before conception (placental restriction (PR) group). Fetal sheep in the PR group were hypoxemic throughout late gestation and growth-restricted(3.02 ± 0.35 kg) when compared with control fetal sheep (4.30 ± 0.29 kg; n = 8) at 140 d of gestation. Fetal plasma concentrations of noradrenaline and adrenaline were higher (p < 0.05) in the PR(7.06 ± 3.17 pmol/mL and 2.89 ± 2.01 pmol/mL, respectively) than in the control group (3.55 ± 0.54 pmol/mL and 1.30 ± 0.48 pmol/mL, respectively) throughout late gestation. Plasma noradrenaline, but not adrenaline concentrations, increased significantly between 110 and 140 d of gestation in both the PR and control group, and there was a significant inverse relationship between plasma noradrenaline and arterial Po2 in the PR and control groups (plasma noradrenaline = 12.34 - 0.40 Po2). In the PR group, plasma noradrenaline increased (p < 0.05) after tyramine infusion from 4.51 ± 1.28 pmol/mL to a peak of 19.40 ± 3.56 pmol/mL. In the control group, noradrenaline increased from 2.08 ± 0.30 pmol/mL to a peak of 12.23 ± 1.67 pmol/mL after tyramine infusion. There was no difference, however, in the maximal proportional changes in plasma noradrenaline concentrations in the PR (319 ± 55%) and control(449 ± 100%) groups after tyramine. We conclude that the most likely source of the increased plasma catecholamines in the PR group is enhanced catecholamine synthesis and secretion from developing sympathetic neurons.

Effects of placental insufficiency on the ovine fetal renin-angiotensin system

We postulated that chronic placental insufficiency would be associated with reduced expression of renal renin and angiotensinogen genes in the fetal sheep. Placental development was restricted in ewes by removing the majority of caruncles prior to mating (placentally restricted (PR) group). The weights of PR fetuses were significantly reduced (P < 0.05, 2.98 ± 0.33 kg) compared to control fetuses (4.20 ± 0.30 kg). Kidney weights were also significantly reduced in the PR fetuses (P < 0.05, 8.4 ± 0.9 g) compared with control fetuses (12.2 ± 1.3 g). The ratios of renal renin β‐actin mRNA levels were significantly reduced in PR fetuses (P < 0.001, 0.35 ± 0.02) when compared to control animals (0.98 ± 0.13). The renal angiotensinogen mRNA/18S rRNA ratio was significantly lower (P < 0.05, 0.28 ± 0.13) in PR fetuses compared with control fetuses (0.72 ± 0.10), while hepatic angiotensinogen was unaffected. There was a positive correlation between renal renin mRNA and renal angiotensinogen mRNA levels (r= 0.65, P < 0.05, n= 12). It is unlikely that these changes in renal angiotensinogen and renin mRNA were due to the small increment in plasma cortisol levels (< 5 nmol l‐1). There was, however, a positive correlation between arterial PO2 and renal renin mRNA (r2= 0.77, P < 0.01). Plasma renin levels were not different between the two groups. Thus, restriction of nutrient and oxygen supply throughout fetal life was associated with suppression of renal renin and renal angiotensinogen gene expression, with no effect on hepatic angiotensinogen mRNA levels. This specific suppression of fetal renal renin and angiotensinogen expression could alter the activity of the intrarenal RAS and so affect growth and development of the kidney.

The non-neurogenic catecholamine response of the fetal adrenal to hypoxia is dependent on activation of voltage sensitive Ca2+ channels.

We have investigated the cellular mechanisms underlying the catecholamine response of the fetal sheep adrenal to hypoxia before and after the development of adrenal innervation. Adrenals were collected before (80-100 days gestation: n = 7) and after (135-146 days gestation: n = 10) development of innervation and retrogradely perfused with oxygenated Krebs bicarbonate buffer in vitro via the renal vein. Adrenal hypoxia was induced by perfusion with hypoxic Krebs buffer (pO2 = 46.7 +/- 2.4 mm Hg) for 30 min periods in the presence and absence of hexamethonium (500 microM), Ca2+ (2.5 mM), nifedipine (1 microM) and KCl (10 mM). Hypoxia stimulated an increase (P < 0.001) in the output of noradrenaline at 80-100 days (3 min pre hypoxia, 0.18 +/- 0.07 nmol/3 min; 20 min hypoxia, 0.74 +/- 0.22 nmol/3 min) and at 135-146 days (3 min pre hypoxia, 0.53 +/- 0.20 nmol/3 min; 20 min hypoxia, 1.71 +/- 0.85 nmol/3 min). Adrenaline output was also higher (P < 0.001) than basal values (80-100 days, 0.11 +/- 0.06 nmol/3 min; 135-146 days, 0.53 +/- 0.15 nmol/3 min) after 20 min hypoxia (0.41 +/- 0.20 nmol/3 min and 1.35 +/- 0.56 nmol/3 min respectively). The catecholamine responses to hypoxia were abolished by removal of Ca2+ from the adrenal perfusate. There was a reduction (P < 0.05) in the catecholamine secretory response to hypoxia in the presence of nifedipine. Noradrenaline output decreased from 4.33 +/- 0.84 nmol/30 min to 0.16 +/- 0.49 nmol/30 min and adrenaline output decreased from 3.16 +/- 1.66 nmol/30 min to -0.01 +/- 0.24 nmol/30 min in the presence of nifedipine. The fetal adrenal secretes catecholamines by a direct or non-neurogenic mechanism in response to hypoxia. This secretory response is dependent on the activation of voltage sensitive Ca2+ channels in the chromaffin cell membrane.

Effect of feeding on the diurnal rhythm of plasma cortisol and adrenocorticotrophic hormone concentrations in the pregnant ewe and sheep fetus

The effects of two different feeding regimes on the 24 h profiles of maternal and fetal plasma cortisol and adrenocorticotrophic hormone (ACTH) concentrations were studied in eight pregnant ewes between 123 and 144 days of gestation. Once daily‐fed ewes (n = 4) received 1 kg of lucerne‐chaff at 11.00 h, and multi‐fed ewes (n = 4) received 100‐200 g of lucerne‐chaff at 09.00, 11.00 and 13.00 h and then 150 g until 09.00 h the following day. There were significant differences between the two feeding groups in the 24 h profile of maternal plasma osmolality; once daily feeding at 11.00 h was associated with a peak in maternal plasma osmolality at 15.00 h whereas maternal plasma osmolality reached plateau levels at around 17.00 h in the multi‐fed group. There were also differences between the two feeding groups in the 24 h profiles of maternal and fetal plasma glucose. Maternal and fetal plasma glucose reached peak concentrations at 19.00 h in the once daily‐fed ewes in contrast to the multi‐fed group, where a plateau in maternal and fetal plasma glucose was reached between 19.00 h and 09.00 h the following day. A significant diurnal variation in the plasma concentrations of cortisol was present in the once daily‐fed ewes from 123 days gestation and in their fetuses after, but not before, 135 days gestation. Plasma cortisol peaked at 11.00 h in the ewes and at 13.00 h in the fetuses of this group. In the once daily‐fed group there was also a significant diurnal variation in maternal and fetal plasma ACTH; plasma ACTH concentrations were highest at 11.00 h in the ewes aged between 123 and 144 days and in fetuses after 135 days gestation. In the multi‐fed group, whilst ACTH was highest at 09.00 h in the ewes and at 13.00 h in the fetuses, there was no significant diurnal variation in the plasma concentrations of cortisol in the ewes or fetuses of this group at any stage between 123 and 144 days gestation.

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.

Changes in renal renin gene expression in fetal sheep.

1. Renin gene expression was investigated in kidneys from 13 foetal and four adult sheep.

Plasma catecholamine and Met-Enkephalin-Arg6-Phe7 responses to hypoxaemia after adrenalectomy in the fetal sheep

We have investigated whether removal of the fetal adrenal glands alters the effect of acute hypoxaemia on the circulating concentrations of noradrenaline, adrenaline, Met-Enkephalin and Met-Enkephalin-Arg6-Phe7 (MERF) in the late gestation sheep fetus. Ewes of 6 adrenalectomized and 9 intact fetuses were subjected to a 30 min period of hypoxaemia and a 30 min period of normoxaemia between 135 and 141 days gestation. Removal of the fetal adrenals abolished the fetal adrenaline response and significantly reduced the fetal noradrenaline response to hypoxaemia. There was no significant increase in circulating Met-Enkephalin during either hypoxaemia or normoxaemia in either the intact and adrenalectomized groups. During basal conditions, plasma concentrations of MERF were significantly greater in the adrenalectomized group (1.64 +/- 0.07 ng/ml) than in the intact fetal sheep (1.04 +/- 0.05 ng/ml). There were significantly greater changes (p < 0.05) in plasma MERF concentrations during hypoxaemia than during normoxaemia in both the intact and adrenalectomized groups. Plasma concentrations of MERF were inversely correlated with arterial PO2 (r = -0.44, p < 0.01) in the adrenalectomized but not the intact group of fetal sheep during hypoxaemia. We have demonstrated therefore that the fetal adrenal is the major source of circulating catecholamines, but not Met-Enkephalin or MERF during hypoxaemia in late gestation. The increase in circulating MERF concentrations after adrenalectomy may reflect compensatory changes in the synthesis and/or secretion of MERF in developing sympathetic neurones.