Journal of Developmental Origins of Health and Disease (2012), 3(1), 52–58. & Cambridge University Press and the International Society for Developmental Origins of Health and Disease 2011 doi:10.1017/S2040174411000626
ORIGINAL ARTICLE
Periconceptional undernutrition suppresses cortisol response to arginine vasopressin and corticotropinreleasing hormone challenge in adult sheep offspring M. H. Oliver*, F. H. Bloomfield, A. L. Jaquiery, S. E. Todd, E. B. Thorstensen and J. E. Harding Liggins Institute, University of Auckland, Auckland, New Zealand
Poor maternal nutrition during pregnancy can result in increased disease risk in adult offspring. Many of these effects are proposed to be mediated via altered hypothalamo–pituitary–adrenal axis (HPAA) function, and are sex and age specific. Maternal undernutrition around the time of conception alters HPAA function in foetal and early postnatal life, but there are limited conflicting data about later effects. The aim of this study was to investigate the effect of moderate periconceptional undernutrition on HPAA function of offspring of both sexes longitudinally, from juvenile to adult life. Ewes were undernourished from 61 days before until 30 days after conception or fed ad libitum. HPAA function in offspring was assessed by arginine vasopressin plus corticotropin-releasing hormone challenge at 4, 10 and 18 months. Plasma cortisol response was lower in males than in females, and was not different between singles and twins. Periconceptional undernutrition suppressed offspring plasma cortisol but not adrenocorticotropic hormone responses. In males, this suppression was apparent by 4 months, and was more profound by 10 months, with no further change by 18 months. In females, suppression was first observed at 10 months and became more profound by 18 months. Maternal undernutrition limited to the periconceptional period has a prolonged, sex-dependent effect on adrenal function in the offspring. Received 5 July 2011; Revised 12 September 2011; Accepted 23 September 2011; First published online 20 October 2011 Key words: ACTH, cortisol, maternal nutrition, periconceptional, sheep
Introduction There is increasing evidence that poor maternal nutrition extending from before conception into early pregnancy can have important effects on the postnatal health of offspring. Offspring born to mothers exposed to the Dutch Famine around the time of conception had increased risk of cardiovascular and metabolic disease in adult life.1 In rats, maternal protein restriction for as little as 4.5 days in the period before implantation leads to hypertension in the offspring.2 Periconceptional undernutrition in sheep also resulted in impaired glucose tolerance of adult offspring.3 Altered hypothalamo–pituitary–adrenal axis (HPAA) function may play an important role in mediating the effects of an adverse intrauterine environment on the development of disease in postnatal life. In rats, protein malnutrition of pregnant dams leads to hypertension in the offspring, an effect dependent on altered HPAA function in the offspring4 and mediated by in utero exposure to excess maternal glucocorticoids.5 Early pregnancy exposure of ewes to synthetic glucocorticoids also results in hypertension in the adult offspring.6 Subsequent studies in sheep showed perturbed glucose metabolism in the offspring after maternal exposure to synthetic glucocorticoids.7,8 *Address for correspondence: Dr M. H. Oliver, Ngapouri Research Farm, Liggins Institute, University of Auckland, 2739 State Highway 5, RD2, Reporoa 3083, South Waikato, New Zealand. (Email [email protected])
However, in this case, although offspring HPAA function was enhanced in foetal and early postnatal life, it was profoundly suppressed in adult life.9,10 We have shown in sheep that maternal undernutrition before and for the first 30 days of pregnancy leads to precocious activation of the foetal HPAA11 and an increased incidence of preterm birth.12 Regulation of early postnatal growth in offspring from periconceptionally undernourished (PCUN) mothers also appears to be perturbed, with dissociation of growth velocity from circulating concentrations of key endocrine regulators of growth.13 There was also advanced pancreatic maturation in the foetus,14 followed by impaired glucose tolerance in early adulthood, and this effect was sex specific.3 Previous studies in sheep have also shown sex differences in the development of HPAA function.15–19 Others have reported enhanced plasma adrenocorticotropic hormone (ACTH) and cortisol responses to arginine vasopressin plus corticotropin-releasing hormone (AVP 1 CRH) in 3-monthold lambs born to ewes undernourished from 14 days before to 70 days after mating,20 but reduced ACTH and cortisol responses to AVP 1 CRH stimulation in 12-month-old female offspring following a restricted maternal diet for the first 30 days of pregnancy.15 Recent sheep studies using the same 30-day period of maternal undernutrition report enhanced adrenocorticoid output in 2.5-year-old female offspring.18 Further, behaviour (including stress responses) of offspring of ewes exposed to periconceptional undernutrition, is modified by sex and singleton/twin status.21,22
Undernutrition and cortisol response There are no longitudinal data on postnatal HPAA function following establishment of a stable plane of maternal undernutrition at conception, in both females and intact males, and distinguishing singletons and twins. We therefore aimed to investigate the changes in postnatal HPAA function over time in offspring of ewes undernourished in the periconceptional period, studying both females and intact males, as well as singletons and twins.
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catheter insertion, animals underwent AVP 1 CRH challenge. After a 3 ml baseline blood sample, equimolar doses of bovine CRH 0.5 mg/kg and AVP 0.1 mg/kg, (both Sigma Chemical Co., St Louis, MO, USA) were given intravenously. Further 3 ml samples were taken at 15, 30, 45, 60 and 120 min. Catheters were removed after the tests and the animals were returned to the pasture. Hormone analysis
Materials and methods Animals Ethical approval was obtained from the University of Auckland Animal Ethics Committee. Four- to 5-year-old Romney ewes were acclimatized for 10 days to indoor conditions, and to a pelleted concentrate diet (65% lucerne, 30% barley with limestone, molasses and trace element supplements; Camtech Nutrition Ltd, Hamilton, New Zealand). Ewes were randomly allocated to one of the two treatment groups. Controls (N) were fed a maintenance ration of concentrates at 3–4% body weight/day from 61 days before mating. PCUN ewes were undernourished from 61 days before until 30 days after mating, then fed in the same way as N for the remainder of the experiment. During undernutrition, feeds were adjusted individually to achieve and maintain a weight reduction of 10–15% body weight.3 Restricted feed intake was initially 1–2% of body weight/day, increasing to approximately 80% of N levels (4% of body weight per day) by the end of the undernutrition period. Ewes were housed indoors in a photoperiod-controlled feedlot from 71 days before mating until 2 weeks after lambing. Ewes were kept in individual pens during the undernutrition period (PCUN ewes only), and from 2 weeks before until 2 weeks after lambing. At all other times, animals were housed in group pens. Two weeks before mating with Dorset rams, oestrus was synchronized using an intravaginal progesterone-release device (Eazi-Breed CIDR; Pfizer, NZ, Ltd).23 After birth, lambs were weighed and remained with their mothers indoors for the first 2 weeks. In the cohort described in this study, length of pregnancy was not affected by nutritional group or lamb sex; however, twins were born 2 days later than singletons (147.4 6 0.3 v. 145.3 6 0.5 days, P , 0.001). Ewes and lambs were then returned to the pasture outdoors and managed as part of a single flock. Although on pasture, animals were kept acquainted with concentrate feeds and handling to allow better acclimatization on re-entry to the feedlot for challenge tests. At 4, 10 and 18 months of age the offspring were again brought indoors, housed in individual pens and fed concentrate feeds. After an acclimatization period of 2 days, animals were weighed and indwelling catheters (size 040, Critchley Electrical, Auburn, Australia) were inserted into both jugular veins under local anaesthesia. At 10 and 18 months, ewes were also fitted with CIDRs to prevent oestrus during the tests. Five days after
Blood samples were collected on ice, centrifuged at 3000 rpm for 10 min at 48C, and the plasma were divided into two aliquots and stored at 2208C until analysis. Plasma ACTH was measured using a commercial radioimmunoassay kit (DiaSorin Inc., Stillwater, MN, USA) previously validated for sheep,24 with inter- and intra-assay coefficients of variation being 13.1% and 7.6%, respectively (n 5 37). Plasma cortisol concentrations were measured by liquid chromatography tandem mass spectrometry as described previously25 with mean inter- and intra-assay coefficients of variation of 11% and 4.3%, respectively (n 5 50). Statistics Data are presented as means 6 S.E.M. Total area under the curve (AUC) for cortisol and ACTH responses were calculated from baseline until 60 min following AVP 1 CRH. Where a single data point was missing (because of practical difficulties of sampling or assay), the missing value was obtained by extrapolation for the purposes of calculating AUC. If more than one data point were missing, the data were excluded from this calculation. AUC for cortisol and ACTH at different ages were compared using analysis of variance (ANOVA) with the Tukey correction for multiple comparisons. Body weight, baseline plasma concentrations of cortisol, ACTH and AUC parameters at each age were analysed using factorial ANOVA with nutrition group, sex and single/twin status as independent variables. The time courses of the plasma cortisol and ACTH concentrations during the AVP 1 CRH challenge were analysed using repeated measures of ANOVA with group, sex and single/twin status as covariates. The independent effects of nutritional group, sex, single/twin status, birth weight and current weight were analysed using multiple regression. Results A total of 74 offspring were born to 49 ewes (Table 1); 41 offspring from 26 N ewes (11 singles, 30 twins) and 33 offspring from 23 PCUN ewes (13 singles, 20 twins). Mean birth weight was 5.4 6 1 kg, and nutrition group had no effect on weight at any age (Table 1). Males were heavier than females at all ages (P , 0.05; Table 1), whereas singletons were heavier than twins at birth (5.7 6 0.1 v. 5.3 6 0.1 kg, P , 0.05) and 4 months of age (33 6 1 v. 30 6 1 kg, P , 0.05). Baseline plasma cortisol concentrations were similar in all groups at 4 and 10 months of age (Table 2). At 18 months,
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M. H. Oliver et al. Table 1. Offspring weight (kg) at birth, 4, 10 and 18 months of age Females
Birth* 4 months* 10 months* 18 months*
Males
N
PCUN
N
PCUN
5.2 6 0.1 (23) 30 6 1 (23) 48 6 2 (22) 76 6 1 (17)
5.3 6 0.2 (19) 31 6 1 (18) 46 6 2 (18) 72 6 2 (16)
5.7 6 0.2 (18) 32 6 1 (18) 54 6 2 (17) 85 6 2 (16)
5.5 6 0.2 (14) 34 6 2 (13) 58 6 3 (12) 84 6 2 (9)
PCUN, periconceptionally undernourished; N, well nourished. Data are mean 6 S.E.M. (n). *P , 0.05 for sex effect. Table 2. Plasma cortisol and ACTH concentrations at 4, 10 and 18 months of age Females N Cortisol at baseline (ng/ml) 4 months 10 months 18 months Cortisol AUC (ng/ml/min) 4 months 10 monthsb 18 monthsb ACTH at baseline (pg/ml) 4 months 10 months 18 monthsd ACTH AUC (ng/ml/min) 4 months 10 monthsb 18 monthsd Cortisol:ACTH (AUC ratio) 4 months 10 months 18 monthsb
5.6 6 0.9 (23) 3.4 6 0.4 (22) 5.2 6 0.7 (17)
Males PCUN
4.6 6 0.8 (18) 4.0 6 0.7 (18) 3.8 6 0.7 (16)
N
4.7 6 0.6 (18) 4.2 6 0.6 (17) 1.8 6 0.4 (16)c
PCUN
4.7 6 0.7 (13) 3.1 6 0.4 (12) 2.4 6 0.8 (9)
1784 6 82 2614 6 137c 2507 6 109
1658 6 111 2023 6 91a,c 1985 6 78a
1647 6 112 1771 6 134 1426 6 112c
1414 6 106 1374 6 50a 1173 6 113
35 6 5 21 6 3c 23 6 3
28 6 3 27 6 4 35 6 5a
35 6 8 21 6 4 31 6 6
47 6 5 32 6 6 22 6 7
6.4 6 0.8 9.3 6 0.7c 6.6 6 0.5c
7.8 6 1.2 8.7 6 1.0 9.1 6 1.0a
6.6 6 0.7 7.1 6 0.6 7.9 6 1.1
7.8 6 0.9 6.9 6 0.7 7.4 6 0.7
344 6 31 311 6 22 401 6 24c
299 6 42 279 6 28 257 6 30a
299 6 49 267 6 23 221 6 31
204 6 21 234 6 42 162 6 18
ACTH, adrenocorticotropic hormone; PCUN, periconceptionally undernourished; AUC, area under the curve; N, well nourished. Data are mean 6 S.E.M. (n). a P , 0.05 for nutrition effect within sex. b P , 0.05 for sex effect. c P , 0.05 for comparison with value at previous age. d P , 0.05 nutrition group 3 sex interaction.
baseline plasma cortisol concentrations were approximately 60% lower in N males than in other animals (Table 2). Plasma cortisol AUC was similar in all groups at 4 months of age (Table 2). However, over the first 30 min of the challenge, plasma cortisol concentrations were higher in the N than in the PCUN group, with the effect more marked in males (P , 0.05; Fig. 1). Plasma cortisol AUC increased by approximately 25% from 4 to 10
months in females, remaining relatively stable until 18 months (Table 2). However, plasma cortisol AUC in males did not change from 4 to 10 months, but decreased by 20% at 18 months in the N group (Table 2). At both 10 and 18 months of age, plasma cortisol AUC was greater in females than in males, and was approximately 30% greater in N than PCUN animals, with no significant sex times nutrition group interaction (Table 2).
Undernutrition and cortisol response
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Fig. 1. Plasma cortisol (left) and adrenocorticotropic hormone (ACTH; right) responses to arginine vasopressin plus corticotropinreleasing hormone stimulation at 4 months (top), 10 months (middle) and 18 months (bottom) in control (N) females (J), periconceptionally undernourished (PCUN) females (K), N males (&), and PCUN males (’). Data are mean 6 S.E.M. aEffect of nutrition group (P , 0.05); beffect of sex (P , 0.05); dnutrition group 3 sex interaction (P , 0.05); etime 3 nutrition group interaction (P , 0.05); ftime 3 sex interaction (P , 0.05).
Baseline plasma ACTH concentrations were similar in all groups at 4 months of age, but decreased in N females at 10 months, remaining lower than in PCUN females at 18 months (Table 2). In males, baseline plasma ACTH concentrations did not change with age and were similar among nutrition groups (Table 2). Plasma ACTH AUC was similar in all groups at 4 months of age, and increased approximately 30% by 10 months in N females, but not in males (Table 2). At 18 months, plasma ACTH AUC decreased again in N, but not in PCUN females (P , 0.05 for nutrition group times sex interaction; Table 2). Cortisol:ACTH AUC was similar in all groups at 4 and 10 months of age, then increased approximately 30% by 18 months in N females (Table 2). At 18 months, the cortisol:ACTH AUC was thus greater in females than males, and greater in N than in PCUN females (Table 2). There were no associations between weight at birth or at the time of study and baseline or stimulated hormone concentrations. There was also no effect of singleton or twin status on any of the outcomes measured.
Discussion Maternal undernutrition from 61 days before to 30 days after conception resulted in suppression of plasma cortisol response to AVP 1 CRH challenge during postnatal life. This suppression of cortisol response first appeared in the male offspring as early as 4 months of age; however, was clearly evident in both sexes at 10 months, increasing in females but not males at 18 months. Following puberty (,6 months) cortisol responses were lower in males than females, but were not different between singles and twins. These effects of maternal periconceptional undernutrition on response to AVP 1 CRH stimulation and the interaction with sex, may contribute to the long-term effects of periconceptional undernutrition on postnatal metabolic and cardiovascular regulation. The literature describing the consequences of maternal undernutrition for postnatal HPAA function in sheep includes a variety of differently timed and managed nutritional insults, and a mixture of sex and single/twin outcomes. Hawkins et al.26 reported that moderate nutritional restriction
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M. H. Oliver et al.
of ewes (at 85% of theoretical requirement) for 14 days before to 70 days after conception resulted in foetuses with suppressed plasma cortisol responses to AVP 1 CRH stimulation in late gestation. However, in a cohort from the same study exposed to the same nutritional insult, cortisol response to AVP 1 CRH stimulation was enhanced at 3 months after birth.26 Similarly, Poore et al.18 reported that maternal nutritional restriction for 30 days after mating increased cortisol responses to AVP 1 CRH in female offspring at 2.5 years of age. In the Hawkins studies, the period of restriction extended into mid-gestation, and the lambs may have been subjected to weaning stress at the time of testing, whereas in the Poore study there was an acute change in the maternal nutrition introduced at the time of conception. Clearly, the hormonal and metabolic milieu of the uterine environment in those studies would be quite different from those reported here, potentially explaining the contrasting effects on postnatal HPAA function. Diminished cortisol response to AVP 1 CRH challenge in adult offspring of PCUN ewes may be because of embryonic and foetal development in an environment of altered maternal HPAA function. In rats, maternal undernutrition leads to decreased placental 11b-hydroxysteroid dehydrogenase (11bHSD-2) activity, and thus increased foetal exposure to maternal glucocorticoids.27 Exposure of ewes to 11bHSDresistant synthetic glucocorticoids in mid-pregnancy results in adult offspring with hypertension6 and altered glucose–insulin homeostasis.8 Consistent effects on glucose metabolism are also seen in adult offspring of ewes treated with repeated synthetic glucocorticoid doses in mid-to-late gestation.7 In the same experiment, HPAA responsiveness in foetal offspring is enhanced in late gestation,9 but suppressed in adulthood.10 It is possible that altered maternal HPAA function also mediated the effects of periconceptional undernutrition observed here. Periconceptional undernutrition similar to that reported in this study resulted in the suppression of circulating maternal plasma ACTH and cortisol concentrations,11 and reduced maternal responsiveness to corticotropic stimulation during the period of undernutrition and for at least 20 days after refeeding.28 However, in mid-gestation, 55 days after the end of undernutrition, placental 11bHSD-2 activity was decreased and the ratio of cortisol to cortisone in foetal blood was increased.29 Thus, the embryonic period, during which the developing foetal adrenal gland is capable of producing large amounts of cortisol relative to body weight,30 was characterized by development in a low maternal cortisol environment, whereas by mid-gestation, at a time when the developing ovine adrenal gland undergoes a period of quiescence,30 the foetuses could have been exposed to higher concentrations of maternal cortisol after, rather than during, the period of undernutrition. Either of these abnormal exposures to maternal glucocorticoid concentrations could alter the developmental trajectory of the foetal adrenal gland, resulting in accelerated foetal HPAA maturation and function in late gestation11 and preterm birth.12 In general, our findings after periconceptional undernutrition are largely consistent with
previous findings after exposure to synthetic glucocorticoids in sheep, leading to enhanced foetal but suppressed postnatal HPAA function.9 We have described similar enhanced foetal maturation, followed by impaired postnatal function, in the glucose–insulin axis in these sheep.3 The relationships between maternal periconceptional undernutrition, twinning, birth size, growth rate up to 12 weeks of postnatal age and postnatal cortisol and other endocrine factors have been reported previously.13 Our findings also suggest that the suppressed response to AVP 1 CRH stimulation in postnatal offspring is due to reduced adrenal cortisol production and release, rather than a defect at the level of the hypothalamus or pituitary, leading to reduced ACTH response to corticotropic stimulation. Cortisol response to AVP 1 CRH was suppressed at 18 months in both sexes, without any reduction in ACTH response. Indeed, in female PCUN offspring at 18 months of age both baseline ACTH concentrations and ACTH AUC in response to AVP 1 CRH challenge appear to have increased compared with N offspring, perhaps indicating a compensatory increase in ACTH response reflecting adrenal ACTH resistance. Once again, this is the opposite of our findings in the lategestation foetus, where PCUN resulted in a decreased ACTH response to AVP 1 CRH stimulation, but an accentuated 11-deoxycortisol response to metyrapone-induced suppression of cortisol synthesis, suggesting enhanced adrenal responsiveness to ACTH.25 Consistent with this, PCUN foetuses also had increased expression of placental 17a-hydroxylase (P450C17), the rate-limiting enzyme in cortisol synthesis.31 Overall, these data suggest that a diminished cortisol response to corticotropic stimulation in postnatal life follows precocious activation of cortisol synthesis during foetal life. Such a response could reflect a trade-off between continued growth v. early maturation in foetal life, representing an adaptive response to an adverse intrauterine environment that has long-term consequences in postnatal life.32 Similar findings have been reported for the glucose–insulin axis, with evidence of accelerated pancreatic beta cell maturation in foetal life after periconceptional undernutrition,14 but impaired insulin secretion in postnatal life that worsens with age.3 Mechanisms underlying these effects remain to be determined, but could include alterations in cell cycle genes or in epigenetic changes, similar to those we have recently reported in proopiomelanocortin and the glucocorticoid receptor in the arcuate nucleus of the hypothalamus in late-gestation foetuses exposed to periconceptional undernutrition.33 A consistent feature in this study was the greater cortisol response in female than in male offspring, regardless of nutritional group. There are sex differences in function at all levels of the HPAA, which may result primarily from stimulatory effects of oestrogen.17 Differences in cortisol secretion between the sexes are reported to originate mainly from a higher adrenal steroidogenic enzyme response to ACTH rather than to reception or signal transduction.17 Sex differences consistent with our findings have also been reported in other experiments involving maternal nutritional restriction in sheep.18,19 One-year-old
Undernutrition and cortisol response female offspring born to ewes restricted for the first 30 days of pregnancy showed suppressed ACTH response, but enhanced cortisol response to AVP 1 CRH, whereas males had enhanced ACTH responses, but similar cortisol responses compared with N.15 In guinea pigs34 and rats,35 sex differences in the development of postnatal HPAA function have been reported after maternal treatment with synthetic glucocorticoids during pregnancy. These findings emphasize the importance of ensuring that experiments have sufficient power to determine outcomes separately for offspring of both sexes after intrauterine nutritional manipulation. Interestingly, we did not find any differences between singletons and twins in the outcomes measured. Evidence for the effect of twinning on the risk of adult onset disease in humans is conflicting,36–43 and most experimental studies are undertaken on polytocous species. However, we25 and others44 have reported in late-gestation foetal sheep that twins have suppressed HPAA function compared with singletons. In contrast, after birth there is increased responsiveness of the central HPAA in twins compared with singletons.45 This responsiveness is associated with the within-twin coefficient for birth weight, rather than the between-twin coefficient, suggesting an effect of factors related to the growth of individual foetuses rather than to their shared maternal environment. Thus, it is perhaps not surprising that in this study we did not detect any effect of twinning per se, as the substantial effect of periconceptional undernutrition, a shared maternal environmental influence on both twins, may have obscured any subtle changes due to twinning itself. In conclusion, moderate maternal undernutrition from 61 days before to 30 days after conception results in offspring that develop impaired adrenal response to corticotropic stimulation after birth, an effect seen earlier in males than females. Previously published data show that maternal HPAA function is suppressed during periconceptional undernutrition and that the embryo develops in a low cortisol environment,11 followed by possible increased exposure to maternal cortisol by mid-gestation29 and accelerated maturation of the foetal HPAA in late gestation.11 Together, these longitudinal data suggest that early maturation of the foetal HPAA, perhaps as an adaptive response to the altered intrauterine corticosteroid environment, incurs the cost of profound postnatal HPAA suppression that worsens with increased age and affects males earlier than females. Periconceptional undernutrition has long-term effects on the endocrinology of the adult offspring that may impact on later health. Acknowledgements The authors thank the Health Research Council of New Zealand and the National Research Centre for Growth and Development for funding, and the staff of Ngapouri Research station and the Fetal and Neonatal Growth Group at the Liggins Institute for invaluable technical assistance.
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References 1. Roseboom T, de Rooij S, Painter R. The Dutch famine and its long-term consequences for adult health. Early Hum Dev. 2006; 82, 485–491. 2. Kwong WY, Wild AE, Roberts P, Willis AC, Fleming TP. Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development. 2000; 127, 4195–4202. 3. Todd SE, Oliver MH, Jaquiery AL, Bloomfield FH, Harding JE. Periconceptional undernutrition of ewes impairs glucose tolerance in their adult offspring. Pediatr Res. 2009; 65, 409–413. 4. Langley-Evans SC, Gardner DS, Jackson AA. Maternal protein restriction influences the programming of the rat hypothalamic–pituitary–adrenal axis. J Nutr. 1996; 126, 1578–1585. 5. Lesage J, Blondeau B, Grino M, Breant B, Dupouy JP. Maternal undernutrition during late gestation induces fetal overexposure to glucocorticoids and intrauterine growth retardation, and disturbs the hypothalamo–pituitary–adrenal axis in the newborn rat. Endocrinology. 2001; 142, 1692–1702. 6. Dodic M, Hantzis V, Duncan J, et al. Programming effects of short prenatal exposure to cortisol. FASEB J. 2002; 16, 1017–1026. 7. Sloboda DM, Challis JR, Moss TJ, Newnham JP. Synthetic glucocorticoids: antenatal administration and long-term implications. Curr Pharm Des. 2005; 11, 1459–1472. 8. De Blasio MJ, Gatford KL, Robinson JS, Owens JA. Placental restriction of fetal growth reduces size at birth and alters postnatal growth, feeding activity, and adiposity in the young lamb. Am J Physiol Regul Integr Comp Physiol. 2007; 292, R875–R886. 9. Sloboda DM, Moss TJ, Li S, et al. Prenatal betamethasone exposure results in pituitary–adrenal hyporesponsiveness in adult sheep. Am J Physiol Endocrinol Metab. 2007; 292, E61–E70. 10. Sloboda DM, Moss TJ, Li S, et al. Expression of glucocorticoid receptor, mineralocorticoid receptor, and 11beta-hydroxysteroid dehydrogenase 1 and 2 in the fetal and postnatal ovine hippocampus: ontogeny and effects of prenatal glucocorticoid exposure. J Endocrinol. 2008; 197, 213–220. 11. Bloomfield FH, Oliver MH, Hawkins P, et al. Periconceptional undernutrition in sheep accelerates maturation of the fetal hypothalamic–pituitary–adrenal axis in late gestation. Endocrinology. 2004; 145, 4278–4285. 12. Bloomfield FH, Oliver MH, Hawkins P, et al. A periconceptional nutritional origin for noninfectious preterm birth. Science. 2003; 300, 606. 13. Jaquiery AL, Oliver MH, Bloomfield FH, Harding JE. Periconceptional events perturb postnatal growth regulation in sheep. Pediatr Res. 2011; 70, 261–266. 14. Oliver MH, Hawkins P, Breier BH, et al. Maternal undernutrition during the periconceptual period increases plasma taurine levels and insulin response to glucose but not arginine in the late gestational fetal sheep. Endocrinology. 2001; 142, 4576–4579. 15. Gardner DS, Van Bon BW, Dandrea J, et al. Effect of periconceptional undernutrition and gender on
58
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
M. H. Oliver et al. hypothalamic–pituitary–adrenal axis function in young adult sheep. J Endocrinol. 2006; 190, 203–212. Chadio SE, Kotsampasi B, Papadomichelakis G, et al. Impact of maternal undernutrition on the hypothalamic–pituitary–adrenal axis responsiveness in sheep at different ages postnatal. J Endocrinol. 2007; 192, 495–503. Canny BJ, O’Farrell KA, Clarke IJ, Tilbrook AJ. The influence of sex and gonadectomy on the hypothalamo–pituitary–adrenal axis of the sheep. J Endocrinol. 1999; 162, 215–225. Poore KR, Boullin JP, Cleal JK, et al. Sex- and age-specific effects of nutrition in early gestation and early postnatal life on hypothalamo–pituitary–adrenal axis and sympathoadrenal function in adult sheep. J Physiol. 2010; 588, 2219–2237. Wallace JM, Milne JS, Green LR, Aitken RP. Postnatal hypothalamic–pituitary–adrenal function in sheep is influenced by age and sex, but not by prenatal growth restriction. Reprod Fertil Dev. 2011; 23, 275–284. Hawkins P, Steyn C, McGarrigle HH, et al. Cardiovascular and hypothalamic–pituitary–adrenal axis development in late gestation fetal sheep and young lambs following modest maternal nutrient restriction in early gestation. Reprod Fertil Dev. 2000; 12, 443–456. Hernandez CE, Harding JE, Oliver MH, et al. Effects of litter size, sex and periconceptional ewe nutrition on side preference and cognitive flexibility in the offspring. Behav Brain Res. 2009; 204, 82–87. Hernandez CE, Matthews LR, Oliver MH, Bloomfield FH, Harding JE. Effects of sex, litter size and periconceptional ewe nutrition on offspring behavioural and physiological response to isolation. Physiol Behav. 2010; 101, 588–594. Wheaton JE, Windels HF, Johnston LJ. Accelerated lambing using exogenous progesterone and the ram effect. J Anim Sci. 1992; 70, 2628–2635. Jeffray TM, Matthews SG, Hammond GL, Challis JR. Divergent changes in plasma ACTH and pituitary POMC mRNA after cortisol administration to late-gestation ovine fetus. Am J Physiol. 1998; 274, E417–E425. Rumball CW, Oliver MH, Thorstensen EB, et al. Effects of twinning and periconceptional undernutrition on late-gestation hypothalamic–pituitary–adrenal axis function in ovine pregnancy. Endocrinology. 2008; 149, 1163–1172. Hawkins P, Hanson MA, Matthews SG. Maternal undernutrition in early gestation alters molecular regulation of the hypothalamic–pituitary–adrenal axis in the ovine fetus. J Neuroendocrinol. 2001; 13, 855–861. Lesage J, Hahn D, Leonhardt M, et al. Maternal undernutrition during late gestation-induced intrauterine growth restriction in the rat is associated with impaired placental GLUT3 expression, but does not correlate with endogenous corticosterone levels. J Endocrinol. 2002; 174, 37–43. Jaquiery AL, Oliver MH, Bloomfield FH, et al. Fetal exposure to excess glucocorticoid is unlikely to explain the effects of periconceptional undernutrition in sheep. J Physiol. 2006; 572, 109–118. Connor KL, Challis JR, van Zijl P, et al. Do alterations in placental 11beta-hydroxysteroid dehydrogenase (11betaHSD) activities explain differences in fetal hypothalamic–pituitary–adrenal (HPA) function following
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
periconceptional undernutrition or twinning in sheep? Reprod Sci. 2009; 16, 1201–1212. Wintour EM, Brown EH, Denton DA, et al. The ontogeny and regulation of corticosteroid secretion by the ovine foetal adrenal. Acta Endocrinol (Copenh). 1975; 79, 301–316. Connor KL, Bloomfield FH, Oliver MH, Harding JE, Challis JR. Effect of periconceptional undernutrition in sheep on late gestation expression of mRNA and protein from genes involved in fetal adrenal steroidogenesis and placental prostaglandin production. Reprod Sci. 2009; 16, 573–583. Gluckman PD, Hanson MA. The consequences of being born small – an adaptive perspective. Horm Res. 2006; 65(Suppl 3), 5–14. Stevens A, Begum G, Cook A, et al. Epigenetic changes in the hypothalamic proopiomelanocortin and glucocorticoid receptor genes in the ovine fetus after periconceptional undernutrition. Endocrinology. 2010; 151, 3652–3664. Kapoor A, Petropoulos S, Matthews SG. Fetal programming of hypothalamic–pituitary–adrenal (HPA) axis function and behavior by synthetic glucocorticoids. Brain Res Rev. 2008; 57, 586–595. Liu L, Li A, Matthews SG. Maternal glucocorticoid treatment programs HPA regulation in adult offspring: sex-specific effects. Am J Physiol Endocrinol Metab. 2001; 280, E729–E739. Bo S, Cavallo-Perin P, Ciccone G, Scaglione L, Pagano G. The metabolic syndrome in twins: a consequence of low birth weight or of being a twin? Exp Clin Endocrinol Diabetes. 2001; 109, 135–140. Ijzerman RG, Stehouwer CD, de Geus EJ, et al. The association between low birth weight and high levels of cholesterol is not due to an increased cholesterol synthesis or absorption: analysis in twins. Pediatr Res. 2002; 52, 868–872. Iliadou A, Cnattingius S, Lichtenstein P. Low birthweight and Type 2 diabetes: a study on 11,162 Swedish twins. Int J Epidemiol. 2004; 33, 948–953; discussion 53-4. Levine RS, Hennekens CH, Jesse MJ. Blood pressure in prospective population based cohort of newborn and infant twins. BMJ. 1994; 308, 298–302. Loos RJ, Fagard R, Beunen G, Derom C, Vlietinck R. Birth weight and blood pressure in young adults: a prospective twin study. Circulation. 2001; 104, 1633–1638. Poulsen P, Vaag AA, Kyvik KO, Moller Jensen D, Beck-Nielsen H. Low birth weight is associated with NIDDM in discordant monozygotic and dizygotic twin pairs. Diabetologia. 1997; 40, 439–446. Williams S, Poulton R. Twins and maternal smoking: ordeals for the fetal origins hypothesis? A cohort study. BMJ. 1999; 318, 897–900. Zhang J, Brenner RA, Klebanoff MA. Differences in birth weight and blood pressure at age 7 years among twins. Am J Epidemiol. 2001; 153, 779–782. Gardner DS, Jamall E, Fletcher AJ, Fowden AL, Giussani DA. Adrenocortical responsiveness is blunted in twin relative to singleton ovine fetuses. J Physiol. 2004; 557, 1021–1032. Bloomfield FH, Oliver MH, Harding JE. Effects of twinning, birth size, and postnatal growth on glucose tolerance and hypothalamic–pituitary–adrenal function in postpubertal sheep. Am J Physiol Endocrinol Metab. 2007; 292, E231–E237.
ORIGINAL ARTICLE
Periconceptional undernutrition suppresses cortisol response to arginine vasopressin and corticotropinreleasing hormone challenge in adult sheep offspring M. H. Oliver*, F. H. Bloomfield, A. L. Jaquiery, S. E. Todd, E. B. Thorstensen and J. E. Harding Liggins Institute, University of Auckland, Auckland, New Zealand
Poor maternal nutrition during pregnancy can result in increased disease risk in adult offspring. Many of these effects are proposed to be mediated via altered hypothalamo–pituitary–adrenal axis (HPAA) function, and are sex and age specific. Maternal undernutrition around the time of conception alters HPAA function in foetal and early postnatal life, but there are limited conflicting data about later effects. The aim of this study was to investigate the effect of moderate periconceptional undernutrition on HPAA function of offspring of both sexes longitudinally, from juvenile to adult life. Ewes were undernourished from 61 days before until 30 days after conception or fed ad libitum. HPAA function in offspring was assessed by arginine vasopressin plus corticotropin-releasing hormone challenge at 4, 10 and 18 months. Plasma cortisol response was lower in males than in females, and was not different between singles and twins. Periconceptional undernutrition suppressed offspring plasma cortisol but not adrenocorticotropic hormone responses. In males, this suppression was apparent by 4 months, and was more profound by 10 months, with no further change by 18 months. In females, suppression was first observed at 10 months and became more profound by 18 months. Maternal undernutrition limited to the periconceptional period has a prolonged, sex-dependent effect on adrenal function in the offspring. Received 5 July 2011; Revised 12 September 2011; Accepted 23 September 2011; First published online 20 October 2011 Key words: ACTH, cortisol, maternal nutrition, periconceptional, sheep
Introduction There is increasing evidence that poor maternal nutrition extending from before conception into early pregnancy can have important effects on the postnatal health of offspring. Offspring born to mothers exposed to the Dutch Famine around the time of conception had increased risk of cardiovascular and metabolic disease in adult life.1 In rats, maternal protein restriction for as little as 4.5 days in the period before implantation leads to hypertension in the offspring.2 Periconceptional undernutrition in sheep also resulted in impaired glucose tolerance of adult offspring.3 Altered hypothalamo–pituitary–adrenal axis (HPAA) function may play an important role in mediating the effects of an adverse intrauterine environment on the development of disease in postnatal life. In rats, protein malnutrition of pregnant dams leads to hypertension in the offspring, an effect dependent on altered HPAA function in the offspring4 and mediated by in utero exposure to excess maternal glucocorticoids.5 Early pregnancy exposure of ewes to synthetic glucocorticoids also results in hypertension in the adult offspring.6 Subsequent studies in sheep showed perturbed glucose metabolism in the offspring after maternal exposure to synthetic glucocorticoids.7,8 *Address for correspondence: Dr M. H. Oliver, Ngapouri Research Farm, Liggins Institute, University of Auckland, 2739 State Highway 5, RD2, Reporoa 3083, South Waikato, New Zealand. (Email [email protected])
However, in this case, although offspring HPAA function was enhanced in foetal and early postnatal life, it was profoundly suppressed in adult life.9,10 We have shown in sheep that maternal undernutrition before and for the first 30 days of pregnancy leads to precocious activation of the foetal HPAA11 and an increased incidence of preterm birth.12 Regulation of early postnatal growth in offspring from periconceptionally undernourished (PCUN) mothers also appears to be perturbed, with dissociation of growth velocity from circulating concentrations of key endocrine regulators of growth.13 There was also advanced pancreatic maturation in the foetus,14 followed by impaired glucose tolerance in early adulthood, and this effect was sex specific.3 Previous studies in sheep have also shown sex differences in the development of HPAA function.15–19 Others have reported enhanced plasma adrenocorticotropic hormone (ACTH) and cortisol responses to arginine vasopressin plus corticotropin-releasing hormone (AVP 1 CRH) in 3-monthold lambs born to ewes undernourished from 14 days before to 70 days after mating,20 but reduced ACTH and cortisol responses to AVP 1 CRH stimulation in 12-month-old female offspring following a restricted maternal diet for the first 30 days of pregnancy.15 Recent sheep studies using the same 30-day period of maternal undernutrition report enhanced adrenocorticoid output in 2.5-year-old female offspring.18 Further, behaviour (including stress responses) of offspring of ewes exposed to periconceptional undernutrition, is modified by sex and singleton/twin status.21,22
Undernutrition and cortisol response There are no longitudinal data on postnatal HPAA function following establishment of a stable plane of maternal undernutrition at conception, in both females and intact males, and distinguishing singletons and twins. We therefore aimed to investigate the changes in postnatal HPAA function over time in offspring of ewes undernourished in the periconceptional period, studying both females and intact males, as well as singletons and twins.
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catheter insertion, animals underwent AVP 1 CRH challenge. After a 3 ml baseline blood sample, equimolar doses of bovine CRH 0.5 mg/kg and AVP 0.1 mg/kg, (both Sigma Chemical Co., St Louis, MO, USA) were given intravenously. Further 3 ml samples were taken at 15, 30, 45, 60 and 120 min. Catheters were removed after the tests and the animals were returned to the pasture. Hormone analysis
Materials and methods Animals Ethical approval was obtained from the University of Auckland Animal Ethics Committee. Four- to 5-year-old Romney ewes were acclimatized for 10 days to indoor conditions, and to a pelleted concentrate diet (65% lucerne, 30% barley with limestone, molasses and trace element supplements; Camtech Nutrition Ltd, Hamilton, New Zealand). Ewes were randomly allocated to one of the two treatment groups. Controls (N) were fed a maintenance ration of concentrates at 3–4% body weight/day from 61 days before mating. PCUN ewes were undernourished from 61 days before until 30 days after mating, then fed in the same way as N for the remainder of the experiment. During undernutrition, feeds were adjusted individually to achieve and maintain a weight reduction of 10–15% body weight.3 Restricted feed intake was initially 1–2% of body weight/day, increasing to approximately 80% of N levels (4% of body weight per day) by the end of the undernutrition period. Ewes were housed indoors in a photoperiod-controlled feedlot from 71 days before mating until 2 weeks after lambing. Ewes were kept in individual pens during the undernutrition period (PCUN ewes only), and from 2 weeks before until 2 weeks after lambing. At all other times, animals were housed in group pens. Two weeks before mating with Dorset rams, oestrus was synchronized using an intravaginal progesterone-release device (Eazi-Breed CIDR; Pfizer, NZ, Ltd).23 After birth, lambs were weighed and remained with their mothers indoors for the first 2 weeks. In the cohort described in this study, length of pregnancy was not affected by nutritional group or lamb sex; however, twins were born 2 days later than singletons (147.4 6 0.3 v. 145.3 6 0.5 days, P , 0.001). Ewes and lambs were then returned to the pasture outdoors and managed as part of a single flock. Although on pasture, animals were kept acquainted with concentrate feeds and handling to allow better acclimatization on re-entry to the feedlot for challenge tests. At 4, 10 and 18 months of age the offspring were again brought indoors, housed in individual pens and fed concentrate feeds. After an acclimatization period of 2 days, animals were weighed and indwelling catheters (size 040, Critchley Electrical, Auburn, Australia) were inserted into both jugular veins under local anaesthesia. At 10 and 18 months, ewes were also fitted with CIDRs to prevent oestrus during the tests. Five days after
Blood samples were collected on ice, centrifuged at 3000 rpm for 10 min at 48C, and the plasma were divided into two aliquots and stored at 2208C until analysis. Plasma ACTH was measured using a commercial radioimmunoassay kit (DiaSorin Inc., Stillwater, MN, USA) previously validated for sheep,24 with inter- and intra-assay coefficients of variation being 13.1% and 7.6%, respectively (n 5 37). Plasma cortisol concentrations were measured by liquid chromatography tandem mass spectrometry as described previously25 with mean inter- and intra-assay coefficients of variation of 11% and 4.3%, respectively (n 5 50). Statistics Data are presented as means 6 S.E.M. Total area under the curve (AUC) for cortisol and ACTH responses were calculated from baseline until 60 min following AVP 1 CRH. Where a single data point was missing (because of practical difficulties of sampling or assay), the missing value was obtained by extrapolation for the purposes of calculating AUC. If more than one data point were missing, the data were excluded from this calculation. AUC for cortisol and ACTH at different ages were compared using analysis of variance (ANOVA) with the Tukey correction for multiple comparisons. Body weight, baseline plasma concentrations of cortisol, ACTH and AUC parameters at each age were analysed using factorial ANOVA with nutrition group, sex and single/twin status as independent variables. The time courses of the plasma cortisol and ACTH concentrations during the AVP 1 CRH challenge were analysed using repeated measures of ANOVA with group, sex and single/twin status as covariates. The independent effects of nutritional group, sex, single/twin status, birth weight and current weight were analysed using multiple regression. Results A total of 74 offspring were born to 49 ewes (Table 1); 41 offspring from 26 N ewes (11 singles, 30 twins) and 33 offspring from 23 PCUN ewes (13 singles, 20 twins). Mean birth weight was 5.4 6 1 kg, and nutrition group had no effect on weight at any age (Table 1). Males were heavier than females at all ages (P , 0.05; Table 1), whereas singletons were heavier than twins at birth (5.7 6 0.1 v. 5.3 6 0.1 kg, P , 0.05) and 4 months of age (33 6 1 v. 30 6 1 kg, P , 0.05). Baseline plasma cortisol concentrations were similar in all groups at 4 and 10 months of age (Table 2). At 18 months,
54
M. H. Oliver et al. Table 1. Offspring weight (kg) at birth, 4, 10 and 18 months of age Females
Birth* 4 months* 10 months* 18 months*
Males
N
PCUN
N
PCUN
5.2 6 0.1 (23) 30 6 1 (23) 48 6 2 (22) 76 6 1 (17)
5.3 6 0.2 (19) 31 6 1 (18) 46 6 2 (18) 72 6 2 (16)
5.7 6 0.2 (18) 32 6 1 (18) 54 6 2 (17) 85 6 2 (16)
5.5 6 0.2 (14) 34 6 2 (13) 58 6 3 (12) 84 6 2 (9)
PCUN, periconceptionally undernourished; N, well nourished. Data are mean 6 S.E.M. (n). *P , 0.05 for sex effect. Table 2. Plasma cortisol and ACTH concentrations at 4, 10 and 18 months of age Females N Cortisol at baseline (ng/ml) 4 months 10 months 18 months Cortisol AUC (ng/ml/min) 4 months 10 monthsb 18 monthsb ACTH at baseline (pg/ml) 4 months 10 months 18 monthsd ACTH AUC (ng/ml/min) 4 months 10 monthsb 18 monthsd Cortisol:ACTH (AUC ratio) 4 months 10 months 18 monthsb
5.6 6 0.9 (23) 3.4 6 0.4 (22) 5.2 6 0.7 (17)
Males PCUN
4.6 6 0.8 (18) 4.0 6 0.7 (18) 3.8 6 0.7 (16)
N
4.7 6 0.6 (18) 4.2 6 0.6 (17) 1.8 6 0.4 (16)c
PCUN
4.7 6 0.7 (13) 3.1 6 0.4 (12) 2.4 6 0.8 (9)
1784 6 82 2614 6 137c 2507 6 109
1658 6 111 2023 6 91a,c 1985 6 78a
1647 6 112 1771 6 134 1426 6 112c
1414 6 106 1374 6 50a 1173 6 113
35 6 5 21 6 3c 23 6 3
28 6 3 27 6 4 35 6 5a
35 6 8 21 6 4 31 6 6
47 6 5 32 6 6 22 6 7
6.4 6 0.8 9.3 6 0.7c 6.6 6 0.5c
7.8 6 1.2 8.7 6 1.0 9.1 6 1.0a
6.6 6 0.7 7.1 6 0.6 7.9 6 1.1
7.8 6 0.9 6.9 6 0.7 7.4 6 0.7
344 6 31 311 6 22 401 6 24c
299 6 42 279 6 28 257 6 30a
299 6 49 267 6 23 221 6 31
204 6 21 234 6 42 162 6 18
ACTH, adrenocorticotropic hormone; PCUN, periconceptionally undernourished; AUC, area under the curve; N, well nourished. Data are mean 6 S.E.M. (n). a P , 0.05 for nutrition effect within sex. b P , 0.05 for sex effect. c P , 0.05 for comparison with value at previous age. d P , 0.05 nutrition group 3 sex interaction.
baseline plasma cortisol concentrations were approximately 60% lower in N males than in other animals (Table 2). Plasma cortisol AUC was similar in all groups at 4 months of age (Table 2). However, over the first 30 min of the challenge, plasma cortisol concentrations were higher in the N than in the PCUN group, with the effect more marked in males (P , 0.05; Fig. 1). Plasma cortisol AUC increased by approximately 25% from 4 to 10
months in females, remaining relatively stable until 18 months (Table 2). However, plasma cortisol AUC in males did not change from 4 to 10 months, but decreased by 20% at 18 months in the N group (Table 2). At both 10 and 18 months of age, plasma cortisol AUC was greater in females than in males, and was approximately 30% greater in N than PCUN animals, with no significant sex times nutrition group interaction (Table 2).
Undernutrition and cortisol response
55
Fig. 1. Plasma cortisol (left) and adrenocorticotropic hormone (ACTH; right) responses to arginine vasopressin plus corticotropinreleasing hormone stimulation at 4 months (top), 10 months (middle) and 18 months (bottom) in control (N) females (J), periconceptionally undernourished (PCUN) females (K), N males (&), and PCUN males (’). Data are mean 6 S.E.M. aEffect of nutrition group (P , 0.05); beffect of sex (P , 0.05); dnutrition group 3 sex interaction (P , 0.05); etime 3 nutrition group interaction (P , 0.05); ftime 3 sex interaction (P , 0.05).
Baseline plasma ACTH concentrations were similar in all groups at 4 months of age, but decreased in N females at 10 months, remaining lower than in PCUN females at 18 months (Table 2). In males, baseline plasma ACTH concentrations did not change with age and were similar among nutrition groups (Table 2). Plasma ACTH AUC was similar in all groups at 4 months of age, and increased approximately 30% by 10 months in N females, but not in males (Table 2). At 18 months, plasma ACTH AUC decreased again in N, but not in PCUN females (P , 0.05 for nutrition group times sex interaction; Table 2). Cortisol:ACTH AUC was similar in all groups at 4 and 10 months of age, then increased approximately 30% by 18 months in N females (Table 2). At 18 months, the cortisol:ACTH AUC was thus greater in females than males, and greater in N than in PCUN females (Table 2). There were no associations between weight at birth or at the time of study and baseline or stimulated hormone concentrations. There was also no effect of singleton or twin status on any of the outcomes measured.
Discussion Maternal undernutrition from 61 days before to 30 days after conception resulted in suppression of plasma cortisol response to AVP 1 CRH challenge during postnatal life. This suppression of cortisol response first appeared in the male offspring as early as 4 months of age; however, was clearly evident in both sexes at 10 months, increasing in females but not males at 18 months. Following puberty (,6 months) cortisol responses were lower in males than females, but were not different between singles and twins. These effects of maternal periconceptional undernutrition on response to AVP 1 CRH stimulation and the interaction with sex, may contribute to the long-term effects of periconceptional undernutrition on postnatal metabolic and cardiovascular regulation. The literature describing the consequences of maternal undernutrition for postnatal HPAA function in sheep includes a variety of differently timed and managed nutritional insults, and a mixture of sex and single/twin outcomes. Hawkins et al.26 reported that moderate nutritional restriction
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M. H. Oliver et al.
of ewes (at 85% of theoretical requirement) for 14 days before to 70 days after conception resulted in foetuses with suppressed plasma cortisol responses to AVP 1 CRH stimulation in late gestation. However, in a cohort from the same study exposed to the same nutritional insult, cortisol response to AVP 1 CRH stimulation was enhanced at 3 months after birth.26 Similarly, Poore et al.18 reported that maternal nutritional restriction for 30 days after mating increased cortisol responses to AVP 1 CRH in female offspring at 2.5 years of age. In the Hawkins studies, the period of restriction extended into mid-gestation, and the lambs may have been subjected to weaning stress at the time of testing, whereas in the Poore study there was an acute change in the maternal nutrition introduced at the time of conception. Clearly, the hormonal and metabolic milieu of the uterine environment in those studies would be quite different from those reported here, potentially explaining the contrasting effects on postnatal HPAA function. Diminished cortisol response to AVP 1 CRH challenge in adult offspring of PCUN ewes may be because of embryonic and foetal development in an environment of altered maternal HPAA function. In rats, maternal undernutrition leads to decreased placental 11b-hydroxysteroid dehydrogenase (11bHSD-2) activity, and thus increased foetal exposure to maternal glucocorticoids.27 Exposure of ewes to 11bHSDresistant synthetic glucocorticoids in mid-pregnancy results in adult offspring with hypertension6 and altered glucose–insulin homeostasis.8 Consistent effects on glucose metabolism are also seen in adult offspring of ewes treated with repeated synthetic glucocorticoid doses in mid-to-late gestation.7 In the same experiment, HPAA responsiveness in foetal offspring is enhanced in late gestation,9 but suppressed in adulthood.10 It is possible that altered maternal HPAA function also mediated the effects of periconceptional undernutrition observed here. Periconceptional undernutrition similar to that reported in this study resulted in the suppression of circulating maternal plasma ACTH and cortisol concentrations,11 and reduced maternal responsiveness to corticotropic stimulation during the period of undernutrition and for at least 20 days after refeeding.28 However, in mid-gestation, 55 days after the end of undernutrition, placental 11bHSD-2 activity was decreased and the ratio of cortisol to cortisone in foetal blood was increased.29 Thus, the embryonic period, during which the developing foetal adrenal gland is capable of producing large amounts of cortisol relative to body weight,30 was characterized by development in a low maternal cortisol environment, whereas by mid-gestation, at a time when the developing ovine adrenal gland undergoes a period of quiescence,30 the foetuses could have been exposed to higher concentrations of maternal cortisol after, rather than during, the period of undernutrition. Either of these abnormal exposures to maternal glucocorticoid concentrations could alter the developmental trajectory of the foetal adrenal gland, resulting in accelerated foetal HPAA maturation and function in late gestation11 and preterm birth.12 In general, our findings after periconceptional undernutrition are largely consistent with
previous findings after exposure to synthetic glucocorticoids in sheep, leading to enhanced foetal but suppressed postnatal HPAA function.9 We have described similar enhanced foetal maturation, followed by impaired postnatal function, in the glucose–insulin axis in these sheep.3 The relationships between maternal periconceptional undernutrition, twinning, birth size, growth rate up to 12 weeks of postnatal age and postnatal cortisol and other endocrine factors have been reported previously.13 Our findings also suggest that the suppressed response to AVP 1 CRH stimulation in postnatal offspring is due to reduced adrenal cortisol production and release, rather than a defect at the level of the hypothalamus or pituitary, leading to reduced ACTH response to corticotropic stimulation. Cortisol response to AVP 1 CRH was suppressed at 18 months in both sexes, without any reduction in ACTH response. Indeed, in female PCUN offspring at 18 months of age both baseline ACTH concentrations and ACTH AUC in response to AVP 1 CRH challenge appear to have increased compared with N offspring, perhaps indicating a compensatory increase in ACTH response reflecting adrenal ACTH resistance. Once again, this is the opposite of our findings in the lategestation foetus, where PCUN resulted in a decreased ACTH response to AVP 1 CRH stimulation, but an accentuated 11-deoxycortisol response to metyrapone-induced suppression of cortisol synthesis, suggesting enhanced adrenal responsiveness to ACTH.25 Consistent with this, PCUN foetuses also had increased expression of placental 17a-hydroxylase (P450C17), the rate-limiting enzyme in cortisol synthesis.31 Overall, these data suggest that a diminished cortisol response to corticotropic stimulation in postnatal life follows precocious activation of cortisol synthesis during foetal life. Such a response could reflect a trade-off between continued growth v. early maturation in foetal life, representing an adaptive response to an adverse intrauterine environment that has long-term consequences in postnatal life.32 Similar findings have been reported for the glucose–insulin axis, with evidence of accelerated pancreatic beta cell maturation in foetal life after periconceptional undernutrition,14 but impaired insulin secretion in postnatal life that worsens with age.3 Mechanisms underlying these effects remain to be determined, but could include alterations in cell cycle genes or in epigenetic changes, similar to those we have recently reported in proopiomelanocortin and the glucocorticoid receptor in the arcuate nucleus of the hypothalamus in late-gestation foetuses exposed to periconceptional undernutrition.33 A consistent feature in this study was the greater cortisol response in female than in male offspring, regardless of nutritional group. There are sex differences in function at all levels of the HPAA, which may result primarily from stimulatory effects of oestrogen.17 Differences in cortisol secretion between the sexes are reported to originate mainly from a higher adrenal steroidogenic enzyme response to ACTH rather than to reception or signal transduction.17 Sex differences consistent with our findings have also been reported in other experiments involving maternal nutritional restriction in sheep.18,19 One-year-old
Undernutrition and cortisol response female offspring born to ewes restricted for the first 30 days of pregnancy showed suppressed ACTH response, but enhanced cortisol response to AVP 1 CRH, whereas males had enhanced ACTH responses, but similar cortisol responses compared with N.15 In guinea pigs34 and rats,35 sex differences in the development of postnatal HPAA function have been reported after maternal treatment with synthetic glucocorticoids during pregnancy. These findings emphasize the importance of ensuring that experiments have sufficient power to determine outcomes separately for offspring of both sexes after intrauterine nutritional manipulation. Interestingly, we did not find any differences between singletons and twins in the outcomes measured. Evidence for the effect of twinning on the risk of adult onset disease in humans is conflicting,36–43 and most experimental studies are undertaken on polytocous species. However, we25 and others44 have reported in late-gestation foetal sheep that twins have suppressed HPAA function compared with singletons. In contrast, after birth there is increased responsiveness of the central HPAA in twins compared with singletons.45 This responsiveness is associated with the within-twin coefficient for birth weight, rather than the between-twin coefficient, suggesting an effect of factors related to the growth of individual foetuses rather than to their shared maternal environment. Thus, it is perhaps not surprising that in this study we did not detect any effect of twinning per se, as the substantial effect of periconceptional undernutrition, a shared maternal environmental influence on both twins, may have obscured any subtle changes due to twinning itself. In conclusion, moderate maternal undernutrition from 61 days before to 30 days after conception results in offspring that develop impaired adrenal response to corticotropic stimulation after birth, an effect seen earlier in males than females. Previously published data show that maternal HPAA function is suppressed during periconceptional undernutrition and that the embryo develops in a low cortisol environment,11 followed by possible increased exposure to maternal cortisol by mid-gestation29 and accelerated maturation of the foetal HPAA in late gestation.11 Together, these longitudinal data suggest that early maturation of the foetal HPAA, perhaps as an adaptive response to the altered intrauterine corticosteroid environment, incurs the cost of profound postnatal HPAA suppression that worsens with increased age and affects males earlier than females. Periconceptional undernutrition has long-term effects on the endocrinology of the adult offspring that may impact on later health. Acknowledgements The authors thank the Health Research Council of New Zealand and the National Research Centre for Growth and Development for funding, and the staff of Ngapouri Research station and the Fetal and Neonatal Growth Group at the Liggins Institute for invaluable technical assistance.
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References 1. Roseboom T, de Rooij S, Painter R. The Dutch famine and its long-term consequences for adult health. Early Hum Dev. 2006; 82, 485–491. 2. Kwong WY, Wild AE, Roberts P, Willis AC, Fleming TP. Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development. 2000; 127, 4195–4202. 3. Todd SE, Oliver MH, Jaquiery AL, Bloomfield FH, Harding JE. Periconceptional undernutrition of ewes impairs glucose tolerance in their adult offspring. Pediatr Res. 2009; 65, 409–413. 4. Langley-Evans SC, Gardner DS, Jackson AA. Maternal protein restriction influences the programming of the rat hypothalamic–pituitary–adrenal axis. J Nutr. 1996; 126, 1578–1585. 5. Lesage J, Blondeau B, Grino M, Breant B, Dupouy JP. Maternal undernutrition during late gestation induces fetal overexposure to glucocorticoids and intrauterine growth retardation, and disturbs the hypothalamo–pituitary–adrenal axis in the newborn rat. Endocrinology. 2001; 142, 1692–1702. 6. Dodic M, Hantzis V, Duncan J, et al. Programming effects of short prenatal exposure to cortisol. FASEB J. 2002; 16, 1017–1026. 7. Sloboda DM, Challis JR, Moss TJ, Newnham JP. Synthetic glucocorticoids: antenatal administration and long-term implications. Curr Pharm Des. 2005; 11, 1459–1472. 8. De Blasio MJ, Gatford KL, Robinson JS, Owens JA. Placental restriction of fetal growth reduces size at birth and alters postnatal growth, feeding activity, and adiposity in the young lamb. Am J Physiol Regul Integr Comp Physiol. 2007; 292, R875–R886. 9. Sloboda DM, Moss TJ, Li S, et al. Prenatal betamethasone exposure results in pituitary–adrenal hyporesponsiveness in adult sheep. Am J Physiol Endocrinol Metab. 2007; 292, E61–E70. 10. Sloboda DM, Moss TJ, Li S, et al. Expression of glucocorticoid receptor, mineralocorticoid receptor, and 11beta-hydroxysteroid dehydrogenase 1 and 2 in the fetal and postnatal ovine hippocampus: ontogeny and effects of prenatal glucocorticoid exposure. J Endocrinol. 2008; 197, 213–220. 11. Bloomfield FH, Oliver MH, Hawkins P, et al. Periconceptional undernutrition in sheep accelerates maturation of the fetal hypothalamic–pituitary–adrenal axis in late gestation. Endocrinology. 2004; 145, 4278–4285. 12. Bloomfield FH, Oliver MH, Hawkins P, et al. A periconceptional nutritional origin for noninfectious preterm birth. Science. 2003; 300, 606. 13. Jaquiery AL, Oliver MH, Bloomfield FH, Harding JE. Periconceptional events perturb postnatal growth regulation in sheep. Pediatr Res. 2011; 70, 261–266. 14. Oliver MH, Hawkins P, Breier BH, et al. Maternal undernutrition during the periconceptual period increases plasma taurine levels and insulin response to glucose but not arginine in the late gestational fetal sheep. Endocrinology. 2001; 142, 4576–4579. 15. Gardner DS, Van Bon BW, Dandrea J, et al. Effect of periconceptional undernutrition and gender on
58
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
M. H. Oliver et al. hypothalamic–pituitary–adrenal axis function in young adult sheep. J Endocrinol. 2006; 190, 203–212. Chadio SE, Kotsampasi B, Papadomichelakis G, et al. Impact of maternal undernutrition on the hypothalamic–pituitary–adrenal axis responsiveness in sheep at different ages postnatal. J Endocrinol. 2007; 192, 495–503. Canny BJ, O’Farrell KA, Clarke IJ, Tilbrook AJ. The influence of sex and gonadectomy on the hypothalamo–pituitary–adrenal axis of the sheep. J Endocrinol. 1999; 162, 215–225. Poore KR, Boullin JP, Cleal JK, et al. Sex- and age-specific effects of nutrition in early gestation and early postnatal life on hypothalamo–pituitary–adrenal axis and sympathoadrenal function in adult sheep. J Physiol. 2010; 588, 2219–2237. Wallace JM, Milne JS, Green LR, Aitken RP. Postnatal hypothalamic–pituitary–adrenal function in sheep is influenced by age and sex, but not by prenatal growth restriction. Reprod Fertil Dev. 2011; 23, 275–284. Hawkins P, Steyn C, McGarrigle HH, et al. Cardiovascular and hypothalamic–pituitary–adrenal axis development in late gestation fetal sheep and young lambs following modest maternal nutrient restriction in early gestation. Reprod Fertil Dev. 2000; 12, 443–456. Hernandez CE, Harding JE, Oliver MH, et al. Effects of litter size, sex and periconceptional ewe nutrition on side preference and cognitive flexibility in the offspring. Behav Brain Res. 2009; 204, 82–87. Hernandez CE, Matthews LR, Oliver MH, Bloomfield FH, Harding JE. Effects of sex, litter size and periconceptional ewe nutrition on offspring behavioural and physiological response to isolation. Physiol Behav. 2010; 101, 588–594. Wheaton JE, Windels HF, Johnston LJ. Accelerated lambing using exogenous progesterone and the ram effect. J Anim Sci. 1992; 70, 2628–2635. Jeffray TM, Matthews SG, Hammond GL, Challis JR. Divergent changes in plasma ACTH and pituitary POMC mRNA after cortisol administration to late-gestation ovine fetus. Am J Physiol. 1998; 274, E417–E425. Rumball CW, Oliver MH, Thorstensen EB, et al. Effects of twinning and periconceptional undernutrition on late-gestation hypothalamic–pituitary–adrenal axis function in ovine pregnancy. Endocrinology. 2008; 149, 1163–1172. Hawkins P, Hanson MA, Matthews SG. Maternal undernutrition in early gestation alters molecular regulation of the hypothalamic–pituitary–adrenal axis in the ovine fetus. J Neuroendocrinol. 2001; 13, 855–861. Lesage J, Hahn D, Leonhardt M, et al. Maternal undernutrition during late gestation-induced intrauterine growth restriction in the rat is associated with impaired placental GLUT3 expression, but does not correlate with endogenous corticosterone levels. J Endocrinol. 2002; 174, 37–43. Jaquiery AL, Oliver MH, Bloomfield FH, et al. Fetal exposure to excess glucocorticoid is unlikely to explain the effects of periconceptional undernutrition in sheep. J Physiol. 2006; 572, 109–118. Connor KL, Challis JR, van Zijl P, et al. Do alterations in placental 11beta-hydroxysteroid dehydrogenase (11betaHSD) activities explain differences in fetal hypothalamic–pituitary–adrenal (HPA) function following
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
periconceptional undernutrition or twinning in sheep? Reprod Sci. 2009; 16, 1201–1212. Wintour EM, Brown EH, Denton DA, et al. The ontogeny and regulation of corticosteroid secretion by the ovine foetal adrenal. Acta Endocrinol (Copenh). 1975; 79, 301–316. Connor KL, Bloomfield FH, Oliver MH, Harding JE, Challis JR. Effect of periconceptional undernutrition in sheep on late gestation expression of mRNA and protein from genes involved in fetal adrenal steroidogenesis and placental prostaglandin production. Reprod Sci. 2009; 16, 573–583. Gluckman PD, Hanson MA. The consequences of being born small – an adaptive perspective. Horm Res. 2006; 65(Suppl 3), 5–14. Stevens A, Begum G, Cook A, et al. Epigenetic changes in the hypothalamic proopiomelanocortin and glucocorticoid receptor genes in the ovine fetus after periconceptional undernutrition. Endocrinology. 2010; 151, 3652–3664. Kapoor A, Petropoulos S, Matthews SG. Fetal programming of hypothalamic–pituitary–adrenal (HPA) axis function and behavior by synthetic glucocorticoids. Brain Res Rev. 2008; 57, 586–595. Liu L, Li A, Matthews SG. Maternal glucocorticoid treatment programs HPA regulation in adult offspring: sex-specific effects. Am J Physiol Endocrinol Metab. 2001; 280, E729–E739. Bo S, Cavallo-Perin P, Ciccone G, Scaglione L, Pagano G. The metabolic syndrome in twins: a consequence of low birth weight or of being a twin? Exp Clin Endocrinol Diabetes. 2001; 109, 135–140. Ijzerman RG, Stehouwer CD, de Geus EJ, et al. The association between low birth weight and high levels of cholesterol is not due to an increased cholesterol synthesis or absorption: analysis in twins. Pediatr Res. 2002; 52, 868–872. Iliadou A, Cnattingius S, Lichtenstein P. Low birthweight and Type 2 diabetes: a study on 11,162 Swedish twins. Int J Epidemiol. 2004; 33, 948–953; discussion 53-4. Levine RS, Hennekens CH, Jesse MJ. Blood pressure in prospective population based cohort of newborn and infant twins. BMJ. 1994; 308, 298–302. Loos RJ, Fagard R, Beunen G, Derom C, Vlietinck R. Birth weight and blood pressure in young adults: a prospective twin study. Circulation. 2001; 104, 1633–1638. Poulsen P, Vaag AA, Kyvik KO, Moller Jensen D, Beck-Nielsen H. Low birth weight is associated with NIDDM in discordant monozygotic and dizygotic twin pairs. Diabetologia. 1997; 40, 439–446. Williams S, Poulton R. Twins and maternal smoking: ordeals for the fetal origins hypothesis? A cohort study. BMJ. 1999; 318, 897–900. Zhang J, Brenner RA, Klebanoff MA. Differences in birth weight and blood pressure at age 7 years among twins. Am J Epidemiol. 2001; 153, 779–782. Gardner DS, Jamall E, Fletcher AJ, Fowden AL, Giussani DA. Adrenocortical responsiveness is blunted in twin relative to singleton ovine fetuses. J Physiol. 2004; 557, 1021–1032. Bloomfield FH, Oliver MH, Harding JE. Effects of twinning, birth size, and postnatal growth on glucose tolerance and hypothalamic–pituitary–adrenal function in postpubertal sheep. Am J Physiol Endocrinol Metab. 2007; 292, E231–E237.
