Periconceptional undernutrition in sheep accelerates maturation of the fetal hypothalamic-pituitary-adrenal axis in late gestation

We investigated the effects of moderate maternal periconceptional undernutrition from 60 d before to 30 d after mating on fetal hypothalamic-pituitary-adrenal axis function in late gestation. Ewes were sampled regularly during the period of undernutrition for circulating hormone levels. Vascular catheters were inserted into ewes and their singleton fetuses at 112 d gestation (term, 145 d), and fetal ACTH(1-24) and metyrapone challenge tests were performed at 127 and 128 d. Postmortems were performed at 132 d. Fetuses of undernourished ewes (UN, n = 12) had elevated baseline cortisol concentrations (P < 0.05), compared with fetuses of ad libitum-fed ewes (n = 10). There were no differences between groups in fetal responses to ACTH challenge, but only UN fetuses demonstrated ACTH and 11-deoxycortisol responses to metyrapone (P < 0.05). UN fetuses had increased mRNA levels for proopiomelanocortin and prohormone convertase-1, but not -2, in the pars intermedia of the pituitary gland (P < 0.05). Glucocorticoid receptor mRNA levels were not different between groups in pituitary or hypothalamus. Maternal cortisol and ACTH levels during undernutrition were profoundly suppressed (P < 0.001), rather than elevated, in UN ewes. Furthermore, the normal pregnancy rise in maternal serum progesterone concentrations was delayed in undernourished mothers. These data demonstrate that events around the time of conception have profound effects on fetal hypothalamic-pituitary-adrenal development in late gestation and that factors other than fetal exposure to excess glucocorticoids may be important.     

Exogenous opioid regulation of the hypothalamic-pituitary-adrenal axis in fetal sheep

To test the hypothesis that endogenous opioids participate in the regulation of the ontogenic development of the hypothalamic-pituitary-adrenal axis in fetal sheep, we measured changes in immunoreactive (ir) ACTH and cortisol concentrations in response to bolus injections of either the [Met]-enkephalin analogue, [D-Ala2,N-Phe4,Met(0)ol5]-enkephalin (FK 33-824; 25 micrograms), the opioid antagonist naloxone (1 mg), a combination of both, or saline vehicle, administered to chronically catheterized fetal sheep through late gestation. There were no effects of either FK 33-824, naloxone or saline on the release of ir-ACTH and cortisol at the earliest stage of gestation studied (days 110-115). By days 125-130, FK 33-824 caused a rapid but short-lived (30 min) increase in plasma ir-ACTH (P less than 0.05) which was accompanied by a smaller but nonsignificant increase in cortisol. Naloxone given concurrently with FK 33-824 abolished this response, thus providing evidence for a specific effect through opioid receptors. Naloxone given alone was without effect. At days 135-140, FK 33-824 caused a significant increase in ir-ACTH which was of similar duration and magnitude to that which occurred at days 125-130. There was a larger basal variation in plasma concentrations of cortisol than at days, 125-130, and a greater increase in cortisol after FK 33-824, although this did not reach statistical significance. Naloxone again reversed the effects of FK 33-824 but was without effect when given alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of maternal undernutrition on the hypothalamic-pituitary-adrenal axis responsiveness in sheep at different ages postnatal

Epidemiological and experimental data support the hypothesis of 'fetal programming', which proposes that alterations in fetal nutrition and endocrine status lead to permanent adaptations in fetal homeostatic mechanisms, producing long-term changes in physiology and determine susceptibility to later disease. Altered hypothalamic-pituitary-adrenal (HPA) axis function has been proposed to play an important role in programming of disease risk. The aim of the present study was to examine the effects of maternal nutrient restriction imposed during different periods of gestation on the HPA axis function in sheep, at different ages postnatal. Pregnant ewes were fed a 50% nutrient-restricted diet from days 0-30 (group R1, n = 7), or from days 31-100 of gestation (group R2, n = 7) or a control 100% diet throughout pregnancy, (Control, n = 8). Blood samples were collected at 10-day intervals from day 40 of gestation to term. Lambs were born naturally and fed to appetite throughout the study period. At 2, 5.5, and 10 months of age lambs were given an i.v. injection of corticotrophin-releasing hormone (CRH) and blood samples were collected at -15, 0, 15, 30, 60, 120, and 180 min postinjection. Maternal cortisol levels were significantly higher (P < 0.05) in group R1 compared with the other two groups, whereas maternal insulin levels were lower (P < 0.05) in group R2 compared with control. Birth weight of lambs was not affected by the maternal nutritional manipulation. The area under the curve for ACTH and cortisol response to CRH challenge was greater (P < 0.05) in lambs of group R1 at two months of age, whereas no difference was detected at the ages of 5.5 and 10 months. However, significantly higher (P < 0.01) basal cortisol levels were observed in lambs of R1 group at 5.5 months of age. There was no interaction between treatment and sex for both pituitary and adrenal responses to the challenge. A significant sex effect was evident with females responding with higher ACTH and cortisol levels at the age of 5.5 months (P < 0.01, P < 0.001 respectively) and with higher cortisol levels (P < 0.01) at 10 months of age than males. It is concluded that the HPA axis is programmable by altered nutrition in utero. The sensitivity of the axis to exogenous stimulation is enhanced during early postnatal life and attenuated with age, suggesting a role for the postnatal influences in resetting of the HPA axis and emphasizing the importance of identifying the impact of maternal undernutrition at several time points after birth.     

Effects of early gestation GH administration on placental and fetal development in sheep

Ovine GH (oGH) is synthesized in placental tissue during maximal placental growth and development. Our objectives were to localize oGH mRNA in the placenta, and study the impact of exogenous GH on twin pregnancies during the normal window (35-55 days of gestational age; dGA) of placental expression. In situ hybridization localized oGH mRNA in uterine luminal epithelium but not in tissues of fetal origin. While maternal GH and IGF-I concentrations were increased (P<0.001) approximately tenfold, uterine, uterine fluid, placental, and fetal weights were unaffected by treatment at either 55 or 135 dGA. Fetal length, liver weight, and liver weight per kg of body weight were unaffected by maternal GH treatment. However, in the cotyledon, IGF-binding protein (BP)-1 and IGFBP-4 mRNA concentrations were increased (P<0.05), while IGFBP-2 mRNA was decreased (P<0.05). The concentration of mRNA for IGFBP-3 was unaffected by treatment. Within the caruncle, IGFBP-1 mRNA was decreased (P<0.05), while IGFBP-3 and IGFBP-4 mRNA were increased (P<0.05), and IGFBP-2 mRNA was unchanged due to GH treatment. While our data indicate that elevated maternal GH and IGF-I concentrations during early and mid-gestation do not enhance placental and fetal growth in twin pregnancies, localization of GH mRNA in uterine luminal epithelium could explain GHs transitory expression from 35 to 55 dGA, since by the end of this period the majority of the uterine luminal epithelium has fused with chorionic binucleate cells forming the placental syncytium.     

Allopregnanolone in the brain and blood after disruption of the hypothalamic-pituitary-adrenal axis in fetal sheep

Neuroactive steroids may be synthesised in the brain either de novo from cholesterol or from blood-borne precursors. Concentrations of a GABAA receptor agonist, allopregnanolone, in the fetal brain exceed those in the circulation, and are markedly higher than adult brain concentrations. We used fetal hypophysectomy or adrenalectomy to elucidate the contribution of hypothalamic-pituitary factors and adrenal steroid secretion to the overall neuroactive steroid level in both the fetal brain and the fetal circulation. Hypophysectomy or adrenalectomy was performed between 108 and 112 days of gestation (term approximately 147 days) and fetal tissues were collected at 140 days of gestation. Immunoreactive (ir) ACTH and cortisol in the plasma were significantly reduced after hypophysectomy, whereas adrenalectomy led to increased irACTH but significantly decreased cortisol concentrations, as expected. Brain concentrations of allopregnanolone, progesterone and pregnenolone did not change significantly in fetuses that underwent either hypophysectomy or adrenalectomy; however, concentrations in the plasma and content in the adrenal gland were decreased. Expression of cytochrome P450 scc and 5alpha-reductase type II (5alphaRII) in the brain, measured by western immunoblotting, did not change after either hypophysectomy or adrenalectomy but, after hypophysectomy, expression of P450 scc in the adrenal gland was significantly decreased and that of 5alphaRII remained unchanged. These findings suggest that the regulation of the neuroactive steroid content in the fetal brain is independent of adrenal steroidogenesis and hypothalamic-pituitary factors. Furthermore, the absence of a change in enzyme expression in the brain suggests that the control of the expression of these enzymes is independent of hypothalamic-pituitary factors. Thus local control mechanisms within the brain may be responsible for maintaining the high neurosteroid content present during fetal life, as these mechanisms are independent of adrenal steroid production.     

Maternal nutrient restriction during early to mid gestation up-regulates cardiac insulin-like growth factor (IGF) receptors associated with enlarged ventricular size in fetal sheep

Intrauterine undernutrition is associated with a high incidence of cardiovascular diseases in adulthood. We previously showed that maternal nutrient restriction during early to mid gestation produces ventricular enlargement, although the mechanism is unknown. We examined myocardial expression of insulin-like growth factor I (IGF-1), IGF-2, IGF binding protein 3 (IGFBP-3), IGF-receptor 1 (IGF-1R) and IGF-2R in fetal sheep with maternal undernutrition. Multiparous ewes were fed with 50% (nutrient-restricted, NR) or 100% (control-fed, C) of NRC requirements from day 28 to 78 of gestation. Some of NR and C ewes were euthanized on day 78, and the rest were fed 100% NRC requirements from day 79 to 135 of gestation. At necropsy on day 78 or day 135 of gestation, gravid uteri were recovered. mRNA expression of IGF-1 and IGF-2 in ventricles were measured with RT-PCR, and protein expression of IGF-1R, IGF-2R, IGFBP-3 was quantitated with Western blot. Crown-rump length was reduced and left ventricle was enlarged in NR fetuses on day 78. At day 135 after re-alimentation, ventricular weights were similar between the two groups although ventricular wall thicknesses were greater in NR than C fetuses. No difference was found in IGF-1, IGF-2 or IGFBP-3 levels between the NR and C groups at either gestational age. Protein expression of IGF-1R and IGF-2R in the left ventricle and IGF-1R in the right ventricle was significantly elevated in the NR group on day 78 of gestation. Only IGF-1R expression remained elevated after late gestational re-alimentation in association with increases in ventricular wall thickness. Our study suggest that maternal undernutrition from early to mid gestation may change the expression of IGF-1R and IGF-2R in fetal myocardium, and play a role in cardiac ventricular enlargement in fetal sheep.     

Effect of periconceptional undernutrition and gender on hypothalamic-pituitary-adrenal axis function in young adult sheep

Glucocorticoids are proposed to act as intermediary factors that transcribe the developmental programming sequelae of maternal nutrient restriction (NR). Periconceptional under-nutrition of sheep markedly activates fetal hypothalamic-pituitary-adrenal (HPA) axis activity leading to preterm birth, while transient undernutrition during late gestation in sheep programs adult HPA axis function. To date, no study has examined resting or stimulated HPA axis function in young adult offspring following a periconceptional nutritional challenge. In the present study, 20 ewes were either periconceptionally undernourished (50% metabolisable energy requirements from days 1 to 30 gestation; NR, n = 8) or fed to control levels (100% requirement; controls, n = 12) to term (147 days gestation). Ewes were blood sampled remotely at 2 and 30 days using automated blood sampling equipment. Thereafter, offspring (controls, n = 6/6 males/females; NR, n = 4/4 males/females) were reared to 1 year of age and on separate days received either an i.v. corticotrophin-releasing hormone (CRH; 0.5 microg/kg) and vasopressin (AVP; 0.1 microg/kg) challenge or a synthetic ACTH i.v. bolus (Synacthen; 1.25 microg/kg), and blood samples were taken (manually and remotely) at appropriate intervals for measurement of plasma ACTH and cortisol accordingly. Resting plasma cortisol, assessed remotely, was similar in ewes during undernutrition (control 18.3 +/- 1.4 vs NR 23.4 +/- 1.9 nmol/l) and in offspring at 4 months of age (control male 17.6 +/- 2.9; control female 17.2 +/- 0.4, NR male 16.5 +/- 3.1, NR female 21.7 +/- 4.0 nmol/l). At 12 months of age, however, resting plasma cortisol was significantly increased in NR females (control male 28.0 +/- 1.5, control female 32.9 +/- 9, NR male 32 +/- 7, NR female 53 +/- 10 nmol/l, F 5.7, P = 0.02) despite no difference in plasma ACTH concentration. There was an interaction between nutritional group and gender for both the pituitary and adrenal responses to CRH and AVP, i.e. for controls, females exhibited increased plasma ACTH or cortisol relative to males but for NR this trend was either not present or reversed. The adrenocortical response to synthetic ACTH was gender-dependent only, being greater in female offspring. Combined CRH and AVP provoked a transient hypertension and marked bradycardia in all animals, irrespective of dietary group or gender and could be effectively reproduced by an AVP bolus alone. In conclusion, the present study has shown that periconceptional undernutrition of sheep has only a minor influence on HPA axis function in their young adult offspring when considered alongside the effect of gender per se.     

Influence of cortisol on adipose tissue development in the fetal sheep during late gestation

The present study examined the extent to which the late gestation rise in fetal plasma cortisol influenced adipose tIssue development in the fetus. The effect of cortisol on the abundance of adipose tIssue mitochondrial proteins on both the inner (i.e. uncoupling protein (UCP)1) and outer (i.e. voltage-dependent anion channel (VDAC)) mitochondrial membrane, together with the long and short forms of the prolactin receptor (PRLR) protein and leptin mRNA was determined. Perirenal adipose tIssue was sampled from ovine fetuses to which (i) cortisol (2-3 mg/day for 5 days) or saline was infused up to 127-130 days of gestation, and (ii) adrenalectomised and intact controls at between 142 and 145 days of gestation (term=148 days). UCP1 protein abundance was significantly lower in adrenalectomised fetuses compared with age-matched controls, and UCP1 was increased by cortisol infusion and with gestational age. Adrenalectomy reduced the concentration of the long form of PRLR, although this effect was only significant for the highest molecular weight isoform. In contrast, neither the short form of PRLR, VDAC protein abundance or leptin mRNA expression was significantly affected by gestational age or cortisol status. Fetal plasma triiodothyronine concentrations were increased by cortisol and with gestational age, an affect abolished by adrenalectomy. When all treatment groups were combined, both plasma cortisol and triiodothyronine concentrations were positively correlated with UCP1 protein abundance. In conclusion, an intact adrenal is necessary for the late gestation rise in UCP1 protein abundance but cortisol does not appear to have a major stimulatory role in promoting leptin expression in fetal adipose tIssue. It remains to be established whether effects on UCP1 protein are directly regulated by cortisol alone or mediated by other anabolic fetal hormones such as triiodothyronine.     

Maternal nutrient restriction during late gestation and early postnatal growth in sheep differentially reset the control of energy metabolism in the gastric mucosa

Fetal growth restriction followed by accelerated postnatal growth contributes to impaired metabolic function in adulthood. The extent to which these outcomes may be mediated centrally within the hypothalamus, as opposed to in the periphery within the digestive tract, remains unknown. In a sheep model, we achieved intrauterine growth restriction experimentally by maternal nutrient restriction (R) that involved a 40% reduction in food intake through late gestation. R offspring were then either reared singly to accelerate postnatal growth (RA) or as twins and compared with controls also reared singly. From weaning, all offspring were maintained indoors until adulthood. A reduced litter size accelerated postnatal growth for only the first month of lactation. Independently from postnatal weight gain and later fat mass, R animals developed insulin resistance as adults. However, restricted accelerated offspring compared with both the control accelerated and restricted restricted offspring ate less and had higher fasting plasma leptin as adults, an adaptation which was accompanied by changes in energy sensing and cell proliferation within the abomasum. Additionally, although fetal restriction down-regulated gene expression of mammalian target of rapamycin and carnitine palmitoyltransferase 1-dependent pathways in the abomasum, RA offspring compensated for this by exhibiting greater activity of AMP-activated kinase-dependent pathways. This study demonstrates a role for perinatal nutrition in the peripheral control of food intake and in energy sensing in the gastric mucosal and emphasizes the importance of diet in early life in regulating energy metabolism during adulthood.     

Responses of the fetal pituitary-adrenal axis to acute and chronic hypoglycemia during late gestation in the sheep

We investigated the response of the fetal pituitary-adrenal axis to acute and chronic hypoglycemia before and after the normal prepartum activation of this axis at around 135 days gestation (term = 147 +/- 3 days). Pregnant ewes were either well nourished (control group; n = 22) or undernourished (UN; 50% reduction in maternal nutrient intake; n = 23) during the last 30 days of pregnancy. Acute hypoglycemia was induced by intrafetal administration of insulin between 125 and 130 days gestation (control, n = 7; UN, n = 12) and between 138 and 141 days gestation (control, n = 6; UN = 9). Fetal plasma glucose concentrations were significantly lower (P < 0.005) in the UN compared with the control group throughout the insulin infusion period at both gestational age ranges. In the control group, there was no fetal ACTH response to insulin infusion before 135 days gestation, but there was a significant (P < 0.001) response after 136 days gestation. In the UN group, there was a significant ACTH response to insulin infusion both before and after 135 days gestation, and there was no difference in the fetal ACTH response between the two gestational age ranges. The plasma cortisol responses to insulin were greater (P < 0.001) after 136 days compared with before 135 days gestation in both the UN and control groups. In the control group there was no significant relationship between basal fetal plasma ACTH and glucose concentrations between 115-135 days gestation or between 136-145 days gestation. In the UN group, fetal glucose ranged from 0.5-2.0 mM, and plasma ACTH and glucose concentrations were inversely related at 115-135 days gestation [log ACTH = -0.31 (glucose) + 2.21; r = -0.37; P < 0.001] and at 136-145 days gestation [log ACTH = -0.40 (glucose) + 2.50; r = -0.54; P < 0.001]. When the UN and control groups were combined, fetal plasma ACTH concentrations were significantly greater (F = 13.5; P < 0.05) when plasma glucose concentrations were less than 1.0 mM at either 115-135 days or 136-147 days gestation. Similarly, fetal plasma cortisol concentrations were also significantly greater (F = 18.7; P < 0.05) when plasma glucose concentrations were less than 1.0 mM at each gestational age range. Therefore, there is an increased sensitivity of the fetal hypothalamo-pituitary axis to acute falls in glucose concentrations below 1.2 mM after 135 days compared with earlier in gestation. The fetal hypothalamo-pituitary axis can respond, however, when plasma glucose concentrations fall below 1.0 mM, before and after 135 days gestation, independently of whether the low glucose concentrations are a consequence of insulin-induced hypoglycemia or maternal nutrient restriction.     

The effect of hypothalamo-pituitary disconnection on the functional and morphologic development of the pituitary-adrenal axis in the fetal sheep in the last third of gestation.

We have investigated the effect of hypothalamo-pituitary disconnection (HPD) on the maturation of basal ir-ACTH and cortisol concentrations in fetal sheep plasma, and on the development of the anterior pituitary corticotroph population in the last third of gestation. After HPD, fetal plasma ir-ACTH concentrations were significantly elevated, and continued to rise with increasing gestational age. However, despite elevated ir-ACTH concentrations, there was no increase in fetal plasma cortisol concentrations, and parturition was delayed for at least 8 days beyond normal term. Furthermore, HPD resulted in a significant disruption of the maturation of the pars distalis corticotrophs. We also examined the change in fetal plasma concentrations of ir-ACTH and cortisol to exogenous CRF after HPD. There was a significant increase in plasma ir-ACTH in response to CRF administration in the HPD fetuses, which was qualitatively similar to that observed in sham-operated fetuses. In contrast, the plasma cortisol response was less in HPD fetuses when compared to that in sham-operated fetuses. The results of this study demonstrate that ir-ACTH secretion is not maintained by the fetal hypothalamus in the last third of gestation, and that ir-ACTH secretion is tonically inhibited by the hypothalamus during this time. The disconnection of the pituitary from the hypothalamus disrupts the maturation of the pituitary-adrenal axis, thus demonstrating the fundamental importance of the hypothalamo-pituitary axis in the normal maturational cascade which culminates in birth in this species.     

Effect of indomethacin on the hypothalamic-pituitary-adrenal axis in man.

The effect of the cyclo-oxygenase inhibitor, indomethacin, on hypothalamic-pituitary-adrenal function was investigated in five normal males. Baseline 0800 and 1600 h plasma ACTH and cortisol, plasma ACTH and 11-deoxycortisol responses to metyrapone, and plasma cortisol responses to dexamethasone and exogenous ACTH were not affected by indomethacin. However, the area under the curve for plasma ACTH after iv injection of regular insulin (0.1 U/kg) was significantly decreased during indomethacin administration (control, 88.0 +/- 32.6 cm2, mean +/- sd; indomethacin, 47.6 +/- 23.1, P less than 0.01). Plasma cortisol levels were decreased only at 30 min. Our results support the hypothesis that prostaglandins or any of their precursors play a role in the hypothalamic control of ACTH secretion and indicate that evaluation of hypophyseal ACTH secretory capacity by means of insulin-induced hypoglycemia may yield abnormal results in patients receiving indomethacin.     

Brief undernutrition in late-gestation sheep programs the hypothalamic-pituitary-adrenal axis in adult offspring

Reduced size at birth in humans has been associated with altered function of the hypothalamic-pituitary-adrenal (HPA) axis in childhood and adult life. Experimentally, maternal undernutrition has also been associated with altered fetal HPA function. However, the relationship between birth size, fetal nutrition, and adult pathophysiology is not clear. We recently have reported that glucose tolerance, blood pressure, and IGF-I levels in adult sheep were more closely associated with birth weight than with nutritional insult in late gestation or with current weight. Here, we report adult HPA function in the same group of animals. Pregnant ewes were severely undernourished for 10 d (UN10) or 20 d (UN20) from 105 d gestation (term, 146 d), or were ad libitum-fed controls. At 30 months, female offspring were subjected to an insulin tolerance test and a CRH plus arginine vasopressin (AVP) challenge. UN20 lambs were lighter at birth, but there were no significant differences in weight at 30 months. Adult UN10 ewes had an increased ACTH response to both CRH+AVP challenge and insulin tolerance test, but no differences in cortisol response. UN10 ewes also demonstrated elevated 11-deoxycortisol concentrations, but lower progesterone concentrations, in response to CRH+AVP challenge. In contrast, the responses of UN20 ewes to these challenges were not different from ad libitum controls. Protein levels of P450(c17) and P450(11beta1) were not significantly different among groups. We conclude that brief maternal undernutrition for 10 d, but not 20 d, in late gestation alters HPA function in adult offspring. In contrast to our previous findings, these HPA effects are independent of birth weight and current weight, suggesting that different mechanisms may be involved in programming different physiological axes.     

A short period of maternal nutrient restriction in late gestation modifies pituitary-adrenal function in adult guinea pig offspring

Altered fetal environment can program the hypophyseal-pituitary-adrenal (HPA) axis development and thus affect endocrine function in later life. We hypothesized that 48 h of maternal nutrient restriction during the period of maximal fetal brain growth alters HPA function in adult offspring and leads to modified blood pressure regulation. Pregnant guinea pigs (n = 15) were deprived of food (water ad libitum) or fed normally (n = 13) on days 50 and 51 of gestation, after which they were all fed normally (birth = 68 days). Carotid artery and jugular vein catheters were implanted in adult guinea pig offspring (day 65). Animals were treated with corticotropin (ACTH(1-24); 0.5 microg/kg), corticotropin-releasing hormone (CRH; 0.5 microg/kg) and insulin (5 units/kg), and pituitary-adrenal responses were measured. Guinea pigs were then euthanized and pituitaries removed for analysis of pro-opiomelanocortin (POMC) and glucocorticoid receptor (GR) mRNA levels. There was no effect of prenatal treatment on body weight, blood pressure or heart rate. In male offspring, both basal ACTH (p < 0.007) and basal cortisol (p < 0.05) levels were significantly reduced in animals whose mothers had been nutrient restricted (NR). In contrast, in female offspring, basal plasma ACTH was not different between offspring from NR mothers and controls; however, basal plasma cortisol concentrations were significantly (p < 0.01) elevated at 13.00 h in females born to NR mothers. Responses to HPA challenge were different between offspring from NR mothers and control offspring, and these differences were consistent with alterations in basal adrenocortical function. There was no effect of prenatal treatment on POMC mRNA levels in the pars distalis or pars intermedia. However, GR mRNA levels were significantly (p < 0.05) reduced in adult female offspring born to NR mothers. In conclusion, 48 h of maternal nutrient restriction during pregnancy has a long-term effect on HPA function in adult offspring, and this effect is highly sex specific, but does not result in alteration of blood pressure.     

Effect of cortisol infusion on the pituitary-adrenal axis of the hypothalamo-pituitary-disconnected fetal sheep.

In order to determine whether cortisol acts directly at the level of the fetal pituitary to promote pars distalis corticotroph maturation, we have infused cortisol into the hypothalamo-pituitary-disconnected (HPD) fetal sheep from 111 to 117 days of gestation. In this study we have measured fetal plasma cortisol and immunoreactive adrenocorticotrophic hormone (ir-ACTH) concentrations between 105 and 116 days of gestation, and we have determined the proportions of adult- and fetal-type corticotrophs in the pars distalis of catheter control fetuses and in HPD fetuses infused with either saline (HPD+SAL) or cortisol (2 mg/day; HPD+F). The fetal plasma cortisol concentrations did not change significantly following HPD. The mean fetal plasma cortisol concentration between 113 and 116 days was threefold higher in the HPD+F fetuses than that measured in HPD fetuses. Following HPD, fetal plasma ir-ACTH concentrations were significantly higher than in catheter control fetuses. Despite the significant elevation in plasma cortisol concentrations in HPD+F fetuses between 113 and 116 days, plasma ir-ACTH concentrations were not different in these fetuses from HPD fetuses infused with saline. At 117 days of gestation in HPD+F fetuses, the proportion of fetal-type corticotrophs in the pars distalis was significantly less than in the HPD+SAL fetuses; however, there was no significant change in the proportion of adult-type corticotrophs in the pars distalis following cortisol infusion. We have shown that cortisol has a direct trophic effect on the maturation of the pars distalis corticotrophs; however, the full maturation of these cells requires an intact hypothalamo-pituitary axis. These findings demonstrate the importance of the fetal hypothalamus in anterior pituitary corticotroph maturation during the last third of gestation.     

Effect of maternal food restriction on fetal rat lung lipid differentiation program

Although “fetal programming” has been extensively studied in many organs, there is only limited information on pulmonary effects in the offspring following intrauterine growth restriction (IUGR). We aimed to determine the effects of nutrient restriction on the lung structure and lung lipid differentiation programs in offspring using an animal mode of maternal food restriction (MFR). We utilized a rodent model of 50% MFR from day 10 of gestation to term and then using lung morphology, Western blotting, Real Time RT‐PCR and Oil Red O staining, lung structure and development of the offspring were examined at postnatal days (p) 1, p21, and 9 months (9M). At postnatal day 1, MFR pups weighed significantly less compared to control pups, but at p21 and 9M, they weighed significantly more. However, lung weight, expressed as a percentage of body weight between the two groups was not different at all time‐points examined. The MFR group had significantly decreased alveolar number and significantly increased septal thickness at p1 and 9M, indicating significantly altered lung structure in the MFR offspring. Furthermore, although at p1, compared to the control group, lung lipid accumulation was significantly decreased in the MFR group, at 9M, it was significantly increased. There were significant temporal changes in the parathyroid hormone‐related protein/peroxisome proliferator‐activated receptor gamma signaling pathway and surfactant synthesis. We conclude that MFR alters fetal lung lipid differentiation programming and lung morphometry by affecting specific epithelial–mesenchymal signaling pathways, offering the possibility for specific interventions to overcome these effects. Pediatr Pulmonol. 2009; 44:635–644. © 2009 Wiley‐Liss, Inc.     

Regulation of the hypothalamo-pituitary-adrenocortical axis in fetal sheep.

Development of the fetal hypothalamo-pituitary-adrenal (HPA) axis is required for normal fetal life and subsequent neonatal health. Activation of the fetal pituitary gland results in the synthesis and release of glucocorticoids from the adrenal cortex. Glucocorticoids promote maturation of several organ systems, are important in responses of the fetus to stress, and are involved in the initiation of parturition in several species. The expression of hypothalamic and pituitary genes associated with HPA function is apparent early in gestation in fetal sheep, although the endocrine changes associated with maturation and parturition do not occur until the last fifth of gestation. In this connection, the fetal HPA axis can be activated by treatment with hypophysiotrophic factors or moderate stress throughout gestation. This review focuses on the development of neuroendocrine mechanisms controlling HPA function during fetal life.     

Subpopulations of corticotrophs in the sheep pituitary during late gestation: effects of development and placental restriction

The prepartum surge in fetal plasma cortisol is essential for the normal timing of parturition in sheep and may result from an increase in the ratio of ACTH to proopiomelanocortin (POMC) in the fetal circulation. In fetuses subjected to experimental induction of placental restriction, the prepartum surge in fetal cortisol is exaggerated, whereas pituitary POMC mRNA levels are decreased, and in vitro, unstimulated ACTH secretion is elevated in corticotrophs nonresponsive to CRH. We therefore investigated the changes in the relative proportions of cells expressing POMC, ACTH, and the CRH type 1 receptor (CRHR(1)) shortly before birth and during chronic placental insufficiency. Placental restriction (PR) was induced by removal of the majority of placental attachment sites in five ewes before mating. Pituitaries were collected from control and PR fetal sheep at 140 d (control, n = 4; PR, n = 4) and 144 d (control, n = 6; PR, n = 4). Pituitary sections were labeled with specific antisera raised against POMC, ACTH, and CRHR(1). Three major subpopulations of corticotrophs were identified that expressed POMC + ACTH + CRHR(1), ACTH + CRHR(1), or POMC only. The proportion of pituitary corticotrophs expressing POMC + ACTH + CRHR(1) decreased (P < 0.05) between 140 (control, 60 +/- 1%; PR, 66 +/- 4%) and 144 (control, 45 +/- 2%; PR, 56 +/- 6%) d. A significantly higher (P < 0.05) proportion of corticotrophs expressed POMC + ACTH + CRHR(1) in the pituitary of the PR group compared with controls. This study is the first to demonstrate subpopulations of corticotrophs in the fetal sheep pituitary that differentially express POMC, ACTH, and CRHR(1) and the separate effects of gestational age and placental restriction on these subpopulations of corticotrophs.     

Evidence that the central noradrenergic and adrenergic pathways activate the hypothalamic-pituitary-adrenal axis in the sheep.

These studies were undertaken to test the hypothesis that stimulation of the central noradrenergic and adrenergic pathways activates the hypothalamic-pituitary-adrenal axis in vivo in the conscious sheep. Blood samples were taken at 10-min intervals over 4 h to establish the baseline state, and then each animal received an intracerebroventricular (icv) injection of NaCl (control animals) or catecholamine [norepinephrine (NE) or epinephrine (EPI)]. A more frequent rate of venous sampling was used for the 30-min period after the icv injection, after which time the 10-min rate of blood sampling was continued for another 3.5 h. NaCl (n = 4) caused no change in pituitary-adrenal secretion. In contrast, 10 micrograms NE (n = 4) caused acute 1.9- and 3.2-fold increases in mean plasma ACTH and cortisol levels over the 1 h period post injection, and 1.6- and 2.3-fold increments in their concentrations over the 4 h postinjection period. Although 10 micrograms EPI (n = 4) did not elevate mean plasma ACTH, it produced significant 1.7- and 1.5-fold increases in plasma cortisol during the 1- and 4-h periods post injection. However, when 100 micrograms EPI was injected (n = 4), acute 9.5- and 5.5-fold increases in plasma ACTH and cortisol were seen over the 1 h period post injection, and 6.1- and 4.2-fold increments in their plasma concentration were noted during the entire post-injection period. To determine the predominant site of action of the catecholamines, we also examined the ability of NE and EPI to release ACTH from cultured ovine anterior pituitary cells. NE and EPI (10(-9)-10(-6) M) stimulated the release of ACTH in a dose-dependent manner, but with maximal increments only 1.5-fold greater than the basal secretion. NE and EPI also increased the maximal ACTH response to CRF, but did not alter the maximal ACTH release induced by arginine vasopressin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of morphine and naloxone on plasma prolactin concentrations in the fetal sheep and pregnant ewe during late gestation

This study investigated the effects of intrafetal morphine or naloxone administration on fetal and maternal plasma prolactin concentrations in the sheep during late pregnancy (117-143 days gestation). After intravenous morphine (3 mg/kg) there was a significant increase (p less than 0.05) in fetal plasma prolactin concentrations which was sustained for 180 min post-injection. There was no significant effect of either gestational age (125-133 days compared to 134-143 days gestation) or repeated administration (up to 3 treatments) of morphine on the fetal prolactin response to morphine. Intrafetal administration of naloxone (3.8 mg/kg bolus + 9.9 mg/kg/60 min), blocked the fetal prolactin response to morphine. Maternal plasma concentrations of prolactin were significantly increased (p less than 0.05) at 180 min after the intrafetal morphine bolus. When naloxone alone was infused, there was no change in fetal plasma prolactin concentrations, but there was a significant fall (p less than 0.05) in maternal plasma prolactin from 25 min after the start of the naloxone infusion. Thus, acute administration of morphine is associated with fetal and maternal hyperprolactinaemia. Although the endogenous opioids do not appear to mediate basal prolactin secretion in the fetus, they may have a role in the control of prolactin release in the pregnant ewe during late gestation.