The effects of pancreatectomy on the plasma concentrations of insulin-like growth factors 1 and 2 in the sheep fetus

The effects of hypoinsulinaemia and altered metabolite concentrations on the fetal plasma concentrations of insulin-like growth factors (IGF) have been investigated in chronically catheterized fetal sheep made insulin deficient by pancreatic ablation. Fetal pancreatectomy reduced significantly the plasma IGF-1 concentration and increased plasma IGF-2 activity in comparison with the values observed in sham operated fetuses. Mean plasma IGF-1 concentrations in the sham operated and pancreatectomized fetuses were 18.6 +/- 3.1 ng/ml (n = 7) and 13.4 +/- 1.4 ng/ml (n = 13) respectively. When all the data were combined, there was a significant positive correlation between the plasma concentrations of IGF-1 and insulin in utero. The mean IGF-2 activity was 2349 +/- 83 ng/ml (n = 7) in the sham operated fetuses and 3800 +/- 532 ng/ml in the pancreatectomized animals (n = 13). Plasma IGF-2 activity was correlated positively with plasma glucose, fructose and alpha-amino nitrogen levels and inversely related to the plasma insulin concentration in utero. These observations demonstrate that the fetal pancreas is essential for normal IGF production in the fetus and suggest that insulin, substrate availability and the IGFs may interact in the regulation of fetal growth.     

Thyroid hormone modulates renin and ANG II receptor expression in fetal sheep

Fetal renin-angiotensin system (RAS) activity is developmentally regulated, increasing in late gestation toward term. At the same time, fetal hemodynamic parameters change, with blood pressure increasing and heart rate decreasing. During this period, fetal plasma thyroid hormone concentrations also increase significantly. In this study we utilized the technique of thyroidectomy (TX), which removes thyroid hormone from the circulation, to investigate the importance of thyroid hormone on the developmental changes in the RAS (in plasma, kidney, heart, and lung) and hemodynamic regulation in fetal sheep. TX was performed at 120 days of gestational age (dGA), and control fetuses were sham operated. Immediately before necropsy ( approximately 137 dGA), fetuses were infused with isoproterenol and the hemodynamic responses were noted. TX significantly decreased plasma thyroid hormone concentrations and renal renin mRNA and renal active renin levels but did not change fetal plasma active renin levels. TX decreased both angiotensin II receptor subtype 1 (AT1) mRNA and protein levels in kidney and lung but not in the left ventricle. TX also was associated with increased ANG II receptor subtype 2 (AT2) mRNA and protein at the 44-kDa band in kidney, whereas AT2 protein was decreased at the 78-kDa level in kidney and lung tissue only. TX fetuses had significantly lower basal mean arterial blood pressures (MAP) and heart rates than controls. Isoproterenol infusion decreased MAP in TX fetuses. These findings support the hypothesis that thyroid hormone is important in modulating maturation of RAS and cardiovascular function in the late-gestation fetal sheep.     

Continuous measurement of biparietal distance in the intact and hypophysectomized fetal sheep using ultrasound

Piezoelectric transducers were implanted into the parietal bones of intact (n = 4) and hypophysectomized (n = 8) fetal sheep of approximately 110-120 days gestational age (term 145-150 days). Intertransducer distance was determined by measuring the time taken for an ultrasonic pulse, generated by one transducer, to elicit a piezoelectric response in an opposing transducer. The limit of sensitivity of the timer was +/- 0.033 microsec. The ultrasonic velocity through fetal sheep brain tissue was 1549.6 +/- 2.2 m.s-1 (SEM; n = 33). This velocity remained constant throughout the entire period studied in both intact and hypophysectomized fetuses. At this velocity, the sensitivity of the measuring device was +/- 0.05mm. The ultrasonic transit time was measured daily between 0900 and 1100h until term in all fetuses. Three hypophysectomized fetuses were allowed to remain in utero until day 163 of gestation. The mean biparietal distance growth rate prior to day 135 for the intact and hypophysectomized fetuses was 0.25 +/- 0.03 and 0.27 +/- 0.025 mm/day respectively. These values were not significantly different (P greater than 0.05). A significant decrease (P less than 0.05) in growth rate was detected in both experimental groups between days 135 and 147 and was more pronounced in the sham (0.05 +/- 0.04 mm/day) than in the hypophysectomized (0.14 +/- 0.03 mm/day) group. However, the growth rate of the sham animals after day 135 was not significantly different from that of the hypophysectomized animals. In the three hypophysectomized fetuses killed at day 163 the biparietal distance growth was maintained at 0.12 +/- 0.005 mm/day. We conclude that fetal biparietal distance growth is pituitary independent from day 110 of gestation and that this technique for measuring distance is a valid and extremely accurate method for the continuous measurement of this parameter of fetal growth and may have further applications in other areas of growth research.     

Ultrastructural detection of the onset of pituitary thyrotroph sensitivity to lowered thyroid hormone concentrations in the fetal sheep

The goitrogen methylthiouracil was administered orally to pregnant ewes of known gestational ages to induce hypothyroidism in both mother and fetus. Developing pituitary thyrotrophic cells were studied using electron microscopy to detect the earliest gestational age at which morphological changes occurred in response to lowered plasma thyroid hormone concentrations. At 50 days of gestation, the pituitaries of fetuses exposed to the goitrogen were indistinguishable from untreated control glands. However, at 58 days and subsequent ages, "thyroidectomy' cells were observed in pituitaries of all hypothyroid fetuses. These findings indicate that fetal sheep pituitary thyrotrophs are sensitive to lowered thyroid hormone concentrations by 58 days of gestation, suggesting that thyroid-thyrotroph interaction exists at this early stage of development.     

Ontogeny of luteinizing hormone releasing hormone (LHRH) and somatostatin (SRIF) in the hypothalamus of the sheep

Using the immunoperoxidase method, luteinizing hormone releasing hormone (LHRH) and somatostatin (SRIF) were demonstrated in the hypothalamus of fetal sheep. Both hormones were found in the perikarya at about day 60 of fetal life, i.e., at the end of the first half of pregnancy. Immunoreactive LHRH (irLHRH) perikarya were situated in the vicinity of the organum vasculosum of the lamina terminalis (OVLT), i.e., in the medial preoptic nucleus and in the nucleus of the diagonal band of Broca. They were scattered and generally sparse in these areas. In the earliest stages of fetal life (60, 75, 90 days of gestation) irSRIF perikarya grouped in the ventromedial nucleus and in the lateral preoptic nucleus, were very numerous. In the oldest fetuses (120 and 135 days of gestation) they had disappeared from these nuclei but could be found in some extrahypothalamic regions--the amygdala, septo-olfactory area and sometimes in the anterior periventricular zone of the hypothalamus. Neither irLHRH nor irSRIF material were stored in the nerve terminals of the external layer of the median eminence (ME) before day 75 of gestation. In all developmental stages examined, irLHRH material in the ME was very scarce whereas irSRIF material very aboundant.     

Effects of leptin on fetal plasma adrenocorticotropic hormone and cortisol concentrations and the timing of parturition in the sheep

We investigated whether leptin can suppress the prepartum activation of the fetal hypothalamus-pituitary-adrenal (HPA) axis and delay the timing of parturition in the sheep. First, we investigated the effects of a 4-day intravascular infusion of recombinant ovine leptin (n = 7) or saline (n = 6) on fetal plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations, starting from 136 days gestation (i.e., at the onset of the prepartum activation of the fetal HPA axis. The effects of a continuous intrafetal infusion of leptin (n = 7) or saline (n = 5) from 144 days gestation on fetal plasma ACTH and cortisol concentrations and the timing of delivery were also determined in a separate study. There was an increase in fetal plasma ACTH (P < 0.01) and cortisol (P < 0.001) concentrations when saline was infused between 136-137 and 140-141 days gestation. Plasma ACTH and cortisol concentrations did not rise, however, when leptin was infused during this period of gestation. When leptin was infused after 144 days gestation, there was no effect of a 4- to 5-fold increase in circulating leptin on fetal ACTH concentrations. In contrast, leptin infusion from 144 days gestation suppressed (P < 0.05) fetal plasma cortisol concentrations by around 40% between 90 and 42 h before delivery. There was no difference, however, in the length of gestation between the saline- and leptin-infused groups (saline infused, 150.2 +/- 0.5 days; leptin infused, 149.8 +/- 1.0 days). In saline-infused fetuses, there was a significant negative relationship between the plasma concentrations of cortisol (y) and leptin (x) between 138 and 146 days gestation (y = 81.4 - 7.7x, r = 0.38, P < 0.005). This study provides evidence for an endocrine negative feedback loop between leptin and the HPA axis in fetal life.     

Changing responsiveness to growth hormone releasing factor in the perinatal sheep

Synthetic human pancreatic growth hormone releasing factor 1-44-amide was administered (8 micrograms/kg iv bolus) to chronically catheterised fetal sheep between 77 and 135 days of gestation and to infant sheep. At all ages human pancreatic growth hormone releasing factor induced a significant growth hormone response. In fetuses less than 120 days the integrated growth hormone response to human pancreatic growth hormone releasing factor (n = 5) was 250 +/- (SE) 50 ng X hr X ml-1 compared (p less than 0.001) to -22.8 +/- 8.6 ng X hr X ml-1 in saline treated controls (n = 7). In fetuses older than 120 days (n = 5), the response to human pancreatic growth hormone releasing factor was 110.8 +/- 15.6 ng X hr X ml-1 compared to -12.0 +/- 17.6 ng X hr X ml-1 in saline treated controls (n = 4 p less than 0.001). In 4 infant lambs (4-12 days) the response to human pancreatic growth hormone releasing factor (56.5 +/- 14.5 ng X hr X ml-1) was greater than in 6 control injected lambs (0.95 +/- 1.5 ng X hr X ml-1). The magnitude of the response to growth releasing factor decreased progressively with increasing postconceptual age (r = -0.80, p less than 0.001). These observations demonstrate that the fetal somatotrope can respond to exogenous growth releasing factor from at least 77 days of gestation. The progressive decrease in responsiveness may reflect the gradual development of somatostatin mediated inhibitory control or altered responsiveness of the somatotrope.     

Effect of tracheostomy on lung development in fetal lambs

The effects of fetal tracheostomy on lung development in lambs have been investigated. Seven ewes, all with twin fetuses, were given a general anesthetic. One fetus in each set of twins was tracheostomized and the other fetus was sham operated (117-122 days gestation). The fetuses were delivered by cesarean section between 137 and 140 days gestation. Fetal tracheostomy decreased lung weight and volume, altered DNA concentration and the structural development of the acinus, and decreased lung distensibility. However, tissue and airway saturated phosphatidylcholine and lung stability during deflation were not significantly affected by fetal tracheostomy. It seems that tracheostomy in fetal lambs alters lung growth but does not affect the formation or release of surfactant. These data support the hypothesis that lung volume is actively maintained and lung growth promoted by the secretion of lung fluid against the resistance of the upper airway in fetal lambs.     

Ingestion of excessive preformed vitamin A by mothers amplifies storage of retinyl esters in early fetal livers of captive Old World monkeys

Excessive preformed vitamin A (VA) intake is contraindicated during pregnancy because of teratogenic concerns. Recent studies have provided biochemical and histologic evidence of chronic hypervitaminosis A in captive Old World monkeys consuming laboratory diets containing high concentrations of retinyl acetate. To investigate the effects of maternal chronic overconsumption of preformed VA on VA storage in early fetal liver, we analyzed monkey fetal livers ranging from 35 to 93 d gestational age (comparable with mid-first to late second trimester in humans) for VA (n = 19) and retinoic acid (n = 9). Retinyl esters were identified in all fetal livers, and retinol, on a percentage basis, was more abundant in younger fetuses. Liver VA concentration increased with gestational age, ranging from 0.0011 to 0.26 micromol/g in the youngest (35 d) and oldest fetuses (93 d), respectively. Liver VA concentrations (mean +/- 1 standard deviation) were 0.023 +/- 0.008 micromol/g in early gestation and 0.19 +/- 0.06 micromol/g in midgestation fetuses. All-trans retinoic acid concentrations were higher in early gestation (99.2 +/- 57.0 pmol/g, n = 6) than in midgestation (18.2 +/- 6.1 pmol/g, n = 3) but were variable. Liver VA concentrations from midgestation fetuses were higher than those in fetal human and monkey livers from later stages of development, when growth and VA accumulation rates are assumed to be highest. Therefore, excessive intake of preformed VA by the mothers results in amplified early fetal liver retinyl ester storage.     

Main pulmonary artery ligation reduces lung fluid production in fetal sheep.

Pulmonary hypoplasia is increasing as a cause of neonatal death. To understand the pathophysiology of pulmonary hypoplasia, the physiology of fetal lung growth must first be understood. Lung fluid production and fetal breathing are primary factors regulating lung growth. Interruption of pulmonary arterial flow also decreases fetal lung growth. To define the relationship of pulmonary arterial flow to other factors known to be important for fetal lung growth, breathing and lung fluid production were measured after postductal main pulmonary artery (MPA) ligation in fetal sheep. Surgical preparation at 107-116 d gestation included placement of vascular catheters and a tracheal catheter connected to an intrauterine collection bag for lung fluid. Five fetuses served as monitored controls (catheters only), 3 as sham operated controls (catheters and thoracotomy), and 7 had MPA ligation. MPA ligation significantly decreased lung weights at 131-140 d; mean dry weight (g): MPA ligation--6.7, sham--23.4, monitored--22.3. Mean rates of lung fluid production (mL/h) were also decreased (d gestation): 116-122 d: MPA ligation--2.2, sham--9.1, monitored--6.8; 123-129 d: MPA ligation--2.1, sham--9.1, monitored--6.2; 130-136 d: MPA ligation--1.5, sham--12.4, monitored--7.7. There were no differences between MPA ligated, sham, and monitored fetuses in the incidence or intensity of fetal breathing movements. Decreased lung fluid production after main pulmonary artery ligation is most likely due to decreased secretion of lung fluid. Pulmonary arterial flow in other models of pulmonary hypoplasia which decrease lung fluid production (i.e., oligohydramnios) should also be examined.     

The effect of hypothalamo-pituitary disconnection on the renin-angiotensin system in the late-gestation fetal sheep

The activity of the renin-angiotensin system (RAS) increases significantly in the late-gestation fetal sheep. Fetal cortisol is also increased during this time, and it is thought that the increase in cortisol may modulate the RAS changes. Previous studies have examined the effects of cortisol infusion on RAS activity, but the effects of blocking the peripartum increase in cortisol concentrations on the developmental changes in the RAS are not known. Therefore, we utilized the technique of hypothalamic-pituitary disconnection (HPD), which prevents the cortisol surge from occurring, to investigate the importance of the late-gestation increase in cortisol on the ontogenic changes in RAS activity. HPD of fetal sheep was performed at 120 days of gestational age (dGA), and fetuses were delivered between 135 and 139 dGA. Control fetuses were sham operated. HPD blocked the late-gestation cortisol increase but did not alter renal renin mRNA, renal renin or prorenin protein content, nor plasma renin levels compared with sham operated. However, HPD fetuses had increased ANG II receptor subtype 1 (AT1) mRNA and protein expression in the kidney and lungs. ANG II receptor subtype 2 (AT2) expression was not altered in these tissues at either mRNA or protein level. HPD did not change AT1 or AT2 mRNA in the left ventricle but did result in decreased protein levels for both receptors. These studies demonstrate that blockade of the naturally occurring increase in fetal cortisol concentration in late gestation is associated with tissue-specific alterations in expression of AT1 and AT2 receptors. These changes may impact on fetal tissue maturation and hence have consequences in postnatal life.     

The role of hypothalamic input on corticotroph maturation in fetal sheep

Corticotropin-releasing hormone receptor type 1 (CRH-R1) expression and vasopressin type 1b (V1b) receptor protein decrease in late-gestation fetal sheep. Because hypothalamo-pituitary disconnection (HPD) has been demonstrated to prevent the morphological maturation of corticotrophs, we hypothesized that hypothalamic input is necessary for the maturational changes in CRH-R1 and V1b receptor levels. We measured CRH-R1 and V1b receptor expression in the anterior pituitaries of fetuses at 140 days gestational age (dGA) that underwent HPD or sham surgery at 120 dGA. CRH-R1 mRNA decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. However, CRH-R1 protein levels were elevated in HPD fetuses compared with sham and were not different from 120 dGA values. V1b protein levels decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. We conclude that hypothalamic input to the pituitary is necessary for the decrease in CRH-R1 receptor protein levels in late-gestation fetal sheep. However, hypothalamic input is not necessary for the decrease in V1b receptor expression seen in late gestation.     

Studies on the growth of the fetal guinea pig. Effects of reduction in uterine blood flow on the plasma sulphation-promoting activity and on the concentration of insulin-like growth factors-I and -II

The effects of prenatal growth restriction caused by uterine artery ligation at midgestation has been studied in pregnant guinea pigs. Ligation of a uterine artery at day 30 of pregnancy commonly caused a reduction in fetal growth of greater than 45% by days 40-65 of gestation. This was associated with substantial delays in the development of a number of fetal tissues and in particular that of the skeleton which remained cartilagenous for longer than normal. Hence normally by day 50 of pregnancy clear evidence of epiphyseal ossification in the long bones of the fore- and hindlimbs was present, but in growth retarded fetuses of less than 50% of normal size such evidence was sparce. Delayed skeletal development and the slowing of fetal growth rate correlated well with marked depression of plasma sulphation-promoting activity. Indeed plasma from fetuses that were less than 40% of normal size inhibited sulphate incorporation into pig costal cartilage. This indicated the presence of inhibitory factors in the plasma of such fetuses, an interpretation that was re-inforced by the observation that plasma IGF-II concentrations were 2-4 times above normal. In contrast plasma IGF-I concentration was depressed upto 50% by growth retardation in line with the fall in fetal plasma insulin concentration. The changes in plasma sulphation-promoting activity and of IGF-I are consistent with slowing of DNA, RNA and protein synthesis and of gene expression in tissues of the growth-retarded fetus. The elevated fetal plasma IGF-II concentration provided further evidence that in the fetal guinea pig this hormone has a potentially glyconeogenic action and maintains essential glycogen stores.     

Chronic umbilical cord compression results in accelerated maturation of lung and brown adipose tissue in the sheep fetus during late gestation

Umbilical cord compression (UCC) sufficient to reduce umbilical blood flow by 30% for 3 days, results in increased fetal plasma cortisol and catecholamines that are likely to promote maturation of the fetal lung and brown adipose tissue (BAT). We determined the effect of UCC on the abundance of uncoupling protein (UCP)1 (BAT only) and -2, glucocorticoid receptor (GR), and 11beta-hydroxysteroid dehydrogenase (11beta-HSD)1 and -2 mRNA, and mitochondrial protein voltage-dependent anion channel (VDAC) and cytochrome c in these tissues. At 118 +/- 2 days of gestation (dGA; term approximately 145 days), 14 fetuses were chronically instrumented. Eight fetuses were then subjected to 3 days of UCC from 125 dGA, and the remaining fetuses were sham operated. All fetuses were then exposed to two 1-h episodes of hypoxemia at 130 +/- 1 and 134 +/- 1 dGA before tissue sampling at 137 +/- 2 dGA. In both tissues, UCC upregulated UCP2 and GR mRNA, plus VDAC and cytochrome c mitochondrial proteins. In lung, UCC increased 11beta-HSD1 mRNA but decreased 11beta-HSD2 mRNA abundance, a pattern reversed for BAT. UCC increased UCP1 mRNA and its translated protein in BAT. UCP2, GR, 11beta-HSD1 and -2 mRNA, plus VDAC and cytochrome c protein abundance were all significantly correlated with fetal plasma cortisol and catecholamine levels, but not thyroid hormone concentrations, in the lung and BAT of UCC fetuses. In conclusion, chronic UCC results in precocious maturation of the fetal lung and BAT mitochondria, an adaptation largely mediated by the surge in fetal plasma cortisol and catecholamines that accompanies UCC.     

Effect of pulsatile growth hormone administration to the growth-restricted fetal sheep on somatotrophic axis gene expression in fetal and placental tissues

We have previously reported (Bauer MK, Breier BH, Bloomfield FH, Jensen EC, Gluckman PD, and Harding JE. J Endocrinol 177: 83-92, 2003) that a chronic pulsatile infusion of growth hormone (GH) to intrauterine growth-restricted (IUGR) ovine fetuses increased fetal circulating IGF-I levels without increasing fetal growth. We hypothesized a cortisol-induced upregulation of fetal hepatic GH receptor (GH-R) mRNA levels, secondary increases in IGF-I mRNA levels, and circulating IGF-I levels, but a downregulation of the type I IGF receptor (IGF-IR) as an explanation. We, therefore, measured mRNA levels of genes of the somatotrophic axis by real-time RT-PCR in fetal and placental tissues of fetuses with IUGR (induced by uteroplacental embolization from 110- to 116-days gestation) that received either a pulsatile infusion of GH (total dose 3.5 mg/day) or vehicle from 117-126 days and in control fetuses (n = 5 per group). Tissues were collected at 127 days (term, 145 days). Fetal cortisol concentrations were significantly increased in IUGR fetuses. However, in liver, GH-R, but not IGF-I or IGF-IR, mRNA levels were decreased in both IUGR groups. In contrast, in placenta, GH-R, IGF-I, and IGF-IR expression were increased in IUGR vehicle-infused fetuses. GH infusion further increased placental GH-R and IGF-IR, but abolished the increase in IGF-I mRNA levels. GH infusion reduced IGF-I expression in muscle and increased GH-R but decreased IGF-IR expression in kidney. IUGR increased hepatic IGF-binding protein (IGFBP)-1 and placental IGFBP-2 and -3 mRNA levels with no further effect of GH infusion. In conclusion, the modest increases in circulating cortisol concentrations in IUGR fetuses did not increase hepatic GH-R mRNA expression and, therefore, do not explain the increased circulating IGF-I levels that we found with GH infusion, which are likely due to reduced clearance rather than increased production. We demonstrate tissue-specific regulation of the somatotrophic axis in IUGR fetuses and a discontinuity between GH-R and IGF-I gene expression in GH-infused fetuses that is not explained by alterations in phosphorylated STAT5b.     

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth

The fetal pituitary-adrenal axis plays a key role in the fetal response to intrauterine stress and in the timing of parturition. The fetal sheep adrenal gland is relatively refractory to stimulation in midgestation (90-120 days) before the prepartum activation, which occurs around 135 days gestation (term=147+/-3 days). The mechanisms underlying the switch from adrenal quiescence to activation are unclear. Therefore, we have investigated the expression of suppressor of cytokine signaling-3 (SOCS-3), a putative inhibitor of tissue growth in the fetal sheep adrenal between 50 and 145 days gestation and in the adrenal of the growth-restricted fetal sheep in late gestation. SOCS-3 is activated by a range of cytokines, including prolactin (PRL), and we have, therefore, determined whether PRL administered in vivo or in vitro stimulates SOCS-3 mRNA expression in the fetal adrenal in late gestation. There was a decrease (P<0.005) in SOCS-3 expression in the fetal adrenal between 54 and 133 days and between 141 and 144 days gestation. Infusion of the dopaminergic agonist, bromocriptine, which suppressed fetal PRL concentrations but did not decrease adrenal SOCS-3 mRNA expression. PRL administration, however, significantly increased adrenal SOCS-3 mRNA expression (P<0.05). Similarly, there was an increase (P<0.05) in SOCS-3 mRNA expression in adrenocortical cells in vitro after exposure to PRL (50 ng/ml). Placental and fetal growth restriction had no effect on SOCS-3 expression in the adrenal during late gestation. In summary, the decrease in the expression of the inhibitor SOCS-3 after 133 days gestation may be permissive for a subsequent increase in fetal adrenal growth before birth. We conclude that factors other than PRL act to maintain adrenal SOCS-3 mRNA expression before 133 days gestation but that acute elevations of PRL can act to upregulate adrenal SOCS-3 expression in the sheep fetus during late gestation.     

Increased placental growth and raised plasma glucocorticoid concentrations in fetal rabbits injected with insulin in utero

Individual rabbit fetuses in utero were injected intramuscularly with either 2.0 Units protamine zinc insulin or saline at 23 days gestation after surgical reduction of litter size to 4 fetuses at 9 days gestation. Insulin-treated fetuses were still hyperinsulinaemic compared with littermate controls at 28 days gestation. Neither fetal body weight nor carcase composition were affected by insulin injection, but placental weight and RNA content were increased. Placental weight was highly correlated with plasma insulin concentration in control fetuses, but not in insulin-injected fetuses. Twenty-four hours after injection of insulin into individual fetal rabbits from normal litters at 23 or 27 days gestation insulin-injected fetuses were hyperinsulinaemic and euglycaemic, but had significantly higher plasma glucocorticoid concentrations than saline-injected littermates. Stimulation of placental growth by fetal insulin may be an important component of the fetal growth stimulatory effect of endogenous insulin. Growth stimulatory effects of exogenous insulin may be antagonised by raised plasma glucocorticoid concentrations.     

The incidence of breathing movements of fetal sheep in normoxia and hypoxia after peripheral chemodenervation and brain-stem transection

The incidence of fetal breathing movements and low voltage electrocortical activity was measured in three groups of fetal sheep, at 123-137 days gestation. The first group (transected & denervated) had the brainstem transected at the level of the colliculi and also had peripheral arterial chemodenervation. The second group (denervated) had a sham brain-stem transection and peripheral arterial chemodenervation. The third group (sham-operated) had sham brain-stem transection and sham peripheral chemodenervation. No differences were observed in the incidence of fetal breathing movements or low voltage electrocortical activity between the sham-operated and the denervated groups in normoxia, or in hypoxia when all these fetuses became apnoeic. There were however differences between these 2 groups and the transected & denervated group, in which fetal breathing movements where dissociated from electrocortical activity and which in some fetuses were continuous. During isocapnic hypoxia 3 of 8 transected & denervated fetuses made fetal breathing movements. We discuss the problems of interpreting data from brain-stem transected fetuses, but conclude that the evidence reveals no tonic influence of the peripheral arterial chemoreceptors on fetal breathing movements.     

Hypothalamic-pituitary disconnection in fetal sheep blocks the peripartum increases in adrenal responsiveness and adrenal ACTH receptor expression

Although it has been recognized for over a decade that hypothalamic-pituitary disconnection (HPD) in fetal sheep prevents the late gestation rise in plasma cortisol concentrations, the underlying mechanisms remain unclear. We hypothesized that reductions in adrenal responsiveness and ACTH receptor (ACTH-R) expression may be mediating factors. HPD or sham surgery was performed at 120 days of gestation, and catheters were placed for blood sampling. At approximately 138 days of gestation, fetuses were killed, and adrenals were removed for cell culture and analyses of ACTH-R mRNA and protein. After 48 h, adrenocortical cells were stimulated with ACTH for 2 h, and the medium was collected for cortisol measurement. The same cells were incubated overnight with medium or medium containing ACTH or forskolin (FSK), followed by ACTH stimulation (as above) and cortisol and cellular ACTH-R mRNA analyses. HPD prevented the late gestation increase in plasma cortisol and bioactive ACTH and reduced adrenal ACTH-R mRNA and protein levels by over 35%. HPD cells secreted significantly less cortisol than sham cells (3.2 +/- 1.2 vs. 47.3 +/- 11.1 ng.ml(-1).2 h(-1)) after the initial ACTH stimulation. Overnight incubation of HPD cells with ACTH or FSK restored cortisol responses to acute stimulation to levels seen in sham cells initially. ACTH-R mRNA levels in cells isolated from HPD fetuses were decreased by over 60%, whereas overnight incubation with ACTH or FSK increased levels by approximately twofold. Our findings indicate that the absence of the cortisol surge in HPD fetuses is a consequence, at least in part, of decreased ACTH-R expression and adrenal responsiveness.