Article

Sex Differences in the Ovine Fetal Cortisol Response to Stress

Article metrics

Abstract

This study tested the hypothesis that the sexually dimorphic adrenocortical response to stress is already established before birth. Chronically instrumented late gestation pregnant sheep carrying 16 male and 15 female age-matched singleton fetuses were subjected to an acute episode of hypoxic stress. Maternal and fetal blood gases, adrenocorticotrophic hormone (ACTH), and cortisol were measured. In addition, six male and six female fetuses received the ACTH analog, Synacthen, and plasma cortisol was measured. During hypoxic stress, the increment in plasma cortisol was 2-fold greater in male versus females fetuses (30.6 ± 3.2 versus 14.3 ± 2.0 ng/mL; p < 0.001) mediated, in part, by greater adrenocortical sensitivity to ACTH. The data support the hypothesis tested and show that sex-specific differences in the cortisol stress response are present before birth with the output of cortisol being much greater in male than in female fetuses.

Main

During threatening situations, the stress system coordinates adaptive responses that adjust homeostatic mechanisms to increase the individual's chance of survival. The hypothalamic-pituitary-adrenal (HPA) axis constitutes one of the main efferent limbs of the stress system, and measurement of circulating plasma adrenocorticotrophic hormone (ACTH) and cortisol concentrations are established measures of the level of psychological, social, and/or physiological stress (1). An adequate capacity for secretion of adrenal glucocorticoids is essential for fetal development and maintenance of an independent life (2,3); the adrenal glands are thus highly perfused with blood and richly innervated. Adrenocortical secretion of glucocorticoids is often used as an example of classical endocrinology, with the principal control provided by the release of ACTH from the distant anterior pituitary and with excess cortisol production switching off the stimulatory signal through negative feedback.

It is widely acknowledged that there are differences in the response to stress between the sexes, but the reason underlying these differences is unclear. The literature is often contradictory due, in part, to variations in the type of stress imposed, the particular species being studied, and the age of the subject. For example, in adult rats, females have greater stress responses (4,5); however, in humans, it is young men and old women who show an increased stress response relative to old men and young women (6). In addition, when compared with age-matched women, men showed consistently higher plasma cortisol responses to stress in four independent studies (7). It is also accepted that sex hormones can alter the magnitude of the stress response, with androgens being suppressive and estrogens being stimulatory to the HPA axis (8,9) and that manipulation of gonadal steroids soon after birth can have activational and organizational effects on the function of the stress axis later in life (9). Development of the fetal HPA axis is exquisitely controlled and critical for the appropriate maturation of multiple vital organs and systems before term (10). Preventing the action of glucocorticoids in the fetus by knock out of the glucocorticoid receptor is invariably embryonically lethal (>90%) (2). By contrast, fetal exposure to excess glucocorticoids, either as a result of maternal stress or maternal treatment with steroids to accelerate fetal lung maturation, has been shown to induce changes on the offspring's stress axis (11,12) in a sex-specific manner (1315). In addition, it has been reported that a sexually dimorphic response to exogenous ACTH seems to develop postnatally (16) and that this can be affected by adult lifestyle (1720). To date, what remains completely unknown is whether there is a difference between the sexes in the HPA axis response to stress that is already established before birth and, thereby, that is independent of exposure to maternal antenatal exogenous steroid therapy, changes in sex hormones throughout the postnatal life course, and/or environmental influences determined by lifestyle.

In this study, we have used the chronically instrumented sheep preparation to compare the circulating plasma concentrations of ACTH and cortisol in age-matched singleton male and female late gestation ovine fetuses during basal conditions and during an episode of acute hypoxic stress, a well-characterized stressor relevant to fetal life (2123). To mechanistically explore potential differences between the sexes in the adrenocortical sensitivity to ACTH, a separate cohort of age-matched singleton male and female sheep fetuses were challenged with a bolus of synthetic ACTH and the plasma cortisol response determined.

MATERIALS AND METHODS

Animals.

All procedures were performed under the UK Animals (Scientific Procedures) Act 1986 and were approved by the Ethical Review Committee of the University of Cambridge. Under general anesthesia, 43 (22 male and 21 female) Welsh Mountain singleton sheep fetuses and their mothers were surgically prepared for long-term recording with vascular and amniotic catheters at 125 ± 1 d of gestation (term is 145 d) as previously described (24,25). During recovery, ewes were housed in individual pens in rooms with a 12 h/12 h light/dark cycle where they had free access to hay and water and were fed concentrates twice daily. Antibiotics were administered daily to the ewe i.m. and the fetus i.v. and into the amniotic cavity. Five days after surgery, 31 of the fetuses (16 male and 15 female) were exposed to a 3 h protocol consisting of 1 h of normoxia, 1 h of hypoxic stress, and 1 h of recovery at the same time of the day. Acute hypoxia in the fetus was induced by maternal inhalational of a hypoxic mixture, using well-established protocols (24,25).

Hormone measurements.

Maternal and fetal arterial blood samples were taken at 15 and 45 min of each experimental period for measurement of blood gases, pH, and the plasma concentrations of ACTH and cortisol. In the remaining 12 fetuses (6 male and 6 female), arterial blood samples were taken for measurement of plasma ACTH and cortisol concentrations during basal conditions and after i.v. treatment with exogenous ACTH (2.5 μg; Synacthen; Ciba Pharmaceuticals, United Kingdom). The dose of Synacthen used was based on previous studies from this laboratory (26,27). Hormone measurements were performed within 4 mo from plasma collection by the same investigators using the same established procedures, which were validated for use with ovine plasma (26,27).

Statistics.

All values are expressed as mean + SEM unless otherwise indicated. The residuals for all measured variables were first assessed for equality of variance across fitted values. Data for plasma ACTH were highly positively skewed and, therefore, were analyzed using a generalized linear mixed model regression procedure incorporating a gamma distribution and logarithm-link function (Genstat v12; VSNi, United Kingdom). Sex, time, and sex × time were included as fixed effects with each fetus included as a random effect in the model. The estimated means (back transformed) are presented together with estimated error at each time point. Linear regression analysis of paired logACTH and plasma cortisol for each sex were conducted using Graphpad Prism 5.0. For all comparisons, statistical significance was accepted when p < 0.05.

RESULTS

Blood gases during hypoxia.

During baseline conditions, there were no differences in arterial blood gases or pH between ewes bearing male or female fetuses or between the male or female fetuses themselves (Table 1). Acute hypoxia reduced the maternal and fetal arterial partial pressure of oxygen (Pao2) to similar levels in pregnancies bearing either male or female fetuses (Table 1). By the end of the hypoxic challenge, female fetuses had become more acidemic [lower arterial pH (pHa)] than males (Table 1) despite maintenance of arterial partial pressure of carbon dioxide (Paco2) at similar levels.

Table 1 Maternal and fetal arterial blood gases and pH

ACTH and cortisol during hypoxia.

Basal maternal plasma ACTH and cortisol were similar in ewes carrying male or female fetuses and remained unchanged throughout the hypoxic challenge (Table 2). Fetal plasma ACTH and cortisol concentrations were also not different from each other during baseline (male ACTH, 38.5 ± 2.9 pg/mL and male cortisol, 23.4 ± 2.3 ng/mL; female ACTH, 32.3 ± 3.1 pg/mL and female cortisol, 22.2 ± 3.6 ng/mL). Fetal hypoxia resulted in a significant increase in ACTH in both groups of fetuses, with no effect of sex (Fig. 1A). In contrast, the increase in fetal cortisol triggered by fetal hypoxia was markedly greater in male compared with female fetuses (p < 0.05, Fig. 1B). Linear regression analyses of paired plasma ACTH and cortisol concentrations throughout the hypoxia protocol indicated that the slope of the linear relationship was twice as steep in male (10.77 ± 0.63; r2 = 0.98) compared with female (3.43 ± 0.18; r2 = 0.99) fetuses (p < 0.001, Fig. 1C).

Table 2 Maternal plasma ACTH and cortisol
Figure 1
figure1

Male and female fetal HPA axis function during hypoxic stress. Values are mean ± SEM for plasma ACTH (A) and cortisol (B) concentrations at 15 (N15) and 45 (N45) min of normoxia, 15 (H15) and 45 (H45) min of hypoxia (shading), and 15 (R15) and 45 (R45) min of recovery in 16 male () and 15 female (•) fetuses during the experimental protocol. (C) Relation between values for ACTH and cortisol in male and female fetuses. Significant differences (p < 0.05): *, differences by post hoc analysis indicating a significant main effect of time compared with normoxia; †, differences by post hoc analysis indicating a significant main effect of sex. Analysis of slopes and intercepts revealed a significant main effect of sex on the slopes, but not the intercept, of the ACTH and cortisol relationship.

ACTH challenge.

After an exogenous bolus of synthetic ACTH, fetal plasma ACTH increased significantly in all fetuses, but the increment was significantly greater (F = 5.0, p = 0.003) in females compared with males (peak plasma ACTH; females, 522 ± 130 versus males, 246 ± 55 pg/mL; Fig. 2A). Synthetic ACTH significantly increased fetal plasma cortisol with the increment being variable but not significantly different between male and female fetuses (Fig. 2B). When the measured fetal plasma cortisol was considered relative to the concentration of measured fetal plasma ACTH (cortisol/ACTH ratio), male fetuses compared with female fetuses showed a significantly greater increment in plasma cortisol at 30 min after the ACTH bolus administration (F = 3.27, p = 0.02; Fig. 2C).

Figure 2
figure2

Adrenal cortical response to synthetic ACTH in the male and female fetuses. Values are mean ± SEM for the change from baseline in plasma ACTH (A) and cortisol (B) concentrations before (−15 and −5 min) and at 5, 15, and 30 min after i.v. treatment with exogenous ACTH (2.5 μg bolus; Synacthen) in six male (□) and six female (▪) fetuses. (C) Values are the mean ± SEM increment from baseline in the ratio of plasma cortisol to plasma ACTH. Significant differences (p < 0.05): *, differences by post hoc analysis indicating a significant main effect of time compared with baseline; †, differences by post hoc analysis indicating a significant main effect of sex.

DISCUSSION

Fetal hypoxia is one of the major challenges that the unborn child may face during gestation (28,29). Reductions in fetal oxygenation may occur in healthy pregnancies during compressions of the umbilical cord (30), in high-altitude pregnancy (31), or during spinal or epidural anesthesia (32). In complicated pregnancies, fetal hypoxia is common and may develop as a result of maternal diabetes, preeclampsia, Rhesus sensitization, maternal infection, sickle cell anemia, chronic substance abuse, asthma, and/or smoking (33). Much of our basic scientific and clinical knowledge on the fetal physiological and endocrine responses to hypoxic stress has been obtained from seminal studies using the chronically instrumented, unanaesthetized fetal sheep preparation. The ovine fetal HPA axis responds to hypoxic stress in late gestation with increases in arterial plasma concentrations of ACTH and adrenocortical output of cortisol (3436). The data in this study show that the male fetus responds to the same magnitude and duration of hypoxia with a much greater increment in plasma cortisol, despite a similar increment in plasma ACTH during the acute episode of stress in both sexes. Indeed, this sex difference exists despite a greater fall in pHa in the female fetus during hypoxia, which itself is known to exacerbate the plasma cortisol response (37). To isolate one particular element of this response, we specifically stimulated the adrenal glands of fetuses under normoxic conditions to determine whether adrenal glands from male fetuses were more responsive to exogenous ACTH. Although the data were more variable than those collected during hypoxia, treatment of the fetus with exogenous ACTH led to an enhanced cortisol response per mole of ACTH in male versus female fetuses. Combined, these data show a sexually dimorphic fetal cortisol response to intrauterine hypoxic stress and that a part of the greater plasma cortisol response in the male compared with female fetus is mechanistically linked to sex-specific changes in adrenocortical sensitivity to ACTH.

Several factors can affect adrenocortical sensitivity. Only relatively recently has the importance of the innervation of the gland on adrenocortical secretion been determined. Thus, Edwards and Jones (38) using conscious, hypophysectomized calves showed that stimulation of the splanchnic input to the adrenal gland doubled, whereas splanchnic denervation halved (39), the output of cortisol in response to an exogenous infusion of ACTH. In the fetus, similar neural mechanisms operate in the control of stimulated adrenocortical secretion, because functional innervation of the ovine fetal adrenal gland is present by the final third of gestation (40,41), and Myers et al. (42) showed that splanchnic nerve section in the ovine fetus had no effect on basal plasma cortisol concentration but significantly attenuated the cortisol increment during acute hypotensive stress. Other mechanisms that may affect adrenocortical sensitivity in the fetus include the actions of neuropeptides such as vasoactive intestinal peptide, corticotropin releasing hormone (CRH), and the eicosanoid prostaglandin E2 (PGE2), because all have been shown to promote steroidogenesis, even in the absence of changes in circulating ACTH (43). It is also possible that the density of ACTH receptors in the fetal adrenal may differ between the sexes. However, to date, there have been no reports, even in the adult, on sex-specific differences in adrenocortical responsiveness resulting from alterations in sympathetic control, or in adrenocortical sensitivity to neuropeptides or eicosanoids, or in the expression of ACTH receptors within the adrenal gland, warranting further investigation in all of these areas of interest.

The metabolic responses to acute hypoxia in the late gestation fetus involve an increase in the circulating concentrations of glucose and lactate (44). The fetal hyperglycemia results from decreased glucose uptake and utilization by peripheral tissues (45) and an increase in hepatic glucose production (46). Fetal lactic acidemia results from anaerobic metabolism of glucose in hypoxic fetal tissues, particularly in the carcass where blood flow and oxygen delivery are markedly declined. Increased sympathetic outflow inhibits insulin release from the fetal pancreas, thereby decreasing glucose uptake and utilization by the fetal tissues (45). As hypoxia progresses, catecholamines (47) and neuropeptide Y (NPY; 48) are released into the fetal circulation and act to maintain the peripheral vasoconstrictor response. In addition, catecholamines are also known to mobilize and release glucose from glycogen stores in the fetal liver (49). The greater acidemic response to hypoxia in the female compared with the male fetus may therefore represent a greater adrenergic and/or peripheral constrictor response to the challenge, warranting focus on these outcome variables in future studies.

In adulthood, it is established that hypercortisolemia is associated with increased morbidity and mortality (50) and that cardiovascular morbidity and mortality are much higher in men than similarly aged premenopausal women (51). Ovarian hormones have long been thought to explain the higher resistance of females to cardiovascular stress because estrogen replacement therapy can reduce postmenopausal morbidity by 50% (51). Our study is the first to show that a sex-specific adrenocortical response to stress is already established during the fetal period, a time when the major source of the high circulating concentrations of estrogen in plasma of male or female fetuses is the placenta. A component of the well-described increased susceptibility to stress in men may therefore be predetermined even before birth, independent of exposure to maternal antenatal exogenous steroid therapy, changes in sex hormones throughout the postnatal life course, or environmental influences determined by lifestyle.

Abbreviations

HPA:

hypothalamic-pituitary-adrenal

pHa:

arterial pH

Paco2:

arterial partial pressure of carbon dioxide

Pao2:

arterial partial pressure of oxygen

References

  1. 1

    Chrousos GP 2009 Stress and disorders of the stress system. Nat Rev Endocrinol 5: 374–381

  2. 2

    Cole TJ, Myles K, Purton JF, Brereton PS, Solomon NM, Godfrey DI, Funder JW 2001 GRKO mice express an aberrant dexamethasone-binding glucocorticoid receptor, but are profoundly glucocorticoid resistant. Mol Cell Endocrinol 173: 193–202

  3. 3

    Dallman MF, Strack AM, Akana SF, Bradbury MJ, Hanson ES, Scribner KA, Smith M 1993 Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. Front Neuroendocrinol 14: 303–347

  4. 4

    Kitay JI 1961 Sex differences in adrenal cortical secretion in the rat. Endocrinology 68: 818–824

  5. 5

    Rivier C 1999 Gender, sex steroids, corticotropin-releasing factor, nitric oxide, and the HPA response to stress. Pharmacol Biochem Behav 64: 739–751

  6. 6

    Roca CA, Schmidt PJ, Deuster PA, Danaceau MA, Altemus M, Putnam K, Chrousos GP, Nieman LK, Rubinow DR 2005 Sex-related differences in stimulated hypothalamic-pituitary-adrenal axis during induced gonadal suppression. J Clin Endocrinol Metab 90: 4224–4231

  7. 7

    Kirschbaum C, Wust S, Hellhammer D 1992 Consistent sex differences in cortisol responses to psychological stress. Psychosom Med 54: 648–657

  8. 8

    Handa RJ, Burgess LH, Kerr JE, O'Keefe JA 1994 Gonadal steroid hormone receptors and sex differences in the hypothalamo-pituitary-adrenal axis. Horm Behav 28: 464–476

  9. 9

    McCormick CM, Furey BF, Child M, Sawyer MJ, Donohue SM 1998 Neonatal sex hormones have ‘organizational' effects on the hypothalamic-pituitary-adrenal axis of male rats. Brain Res Dev Brain Res 105: 295–307

  10. 10

    Fowden AL, Li J, Forhead AJ 1998 Glucocorticoids and the preparation for life after birth: are there long-term consequences of the life insurance?. Proc Nutr Soc 57: 113–122

  11. 11

    Dodic M, May CN, Wintour EM, Coghlan JP 1998 An early prenatal exposure to excess glucocorticoid leads to hypertensive offspring in sheep. Clin Sci (Lond) 94: 149–155

  12. 12

    Nyirenda MJ, Welberg LA, Seckl JR 2001 Programming hyperglycaemia in the rat through prenatal exposure to glucocorticoids-fetal effect or maternal influence?. J Endocrinol 170: 653–660

  13. 13

    McCormick CM, Smythe JW, Sharma S, Meaney MJ 1995 Sex-specific effects of prenatal stress on hypothalamic-pituitary-adrenal responses to stress and brain glucocorticoid receptor density in adult rats. Brain Res Dev Brain Res 84: 55–61

  14. 14

    Levine S 1994 The ontogeny of the hypothalamic-pituitary-adrenal axis. The influence of maternal factors. Ann N Y Acad Sci 746: 275–288

  15. 15

    Kapoor A, Dunn E, Kostaki A, Andrews MH, Matthews SG 2006 Fetal programming of hypothalamo-pituitary-adrenal function: prenatal stress and glucocorticoids. J Physiol 572: 31–44

  16. 16

    Turner AI, Hosking BJ, Parr RA, Tilbrook AJ 2006 A sex difference in the cortisol response to tail docking and ACTH develops between 1 and 8 weeks of age in lambs. J Endocrinol 188: 443–449

  17. 17

    Gardner DS, Van Bon BW, Dandrea J, Goddard PJ, May SF, Wilson V, Stephenson T, Symonds ME 2006 Effect of periconceptional undernutrition and gender on hypothalamic-pituitary-adrenal axis function in young adult sheep. J Endocrinol 190: 203–212

  18. 18

    Andrew R, Phillips DI, Walker BR 1998 Obesity and gender influence cortisol secretion and metabolism in man. J Clin Endocrinol Metab 83: 1806–1809

  19. 19

    Lindquist TL, Beilin LJ, Knuiman MW 1997 Influence of lifestyle, coping, and job stress on blood pressure in men and women. Hypertension 29: 1–7

  20. 20

    Fukuda S, Morimoto K 2001 Lifestyle, stress and cortisol response: review II. Environ Health Prev Med 6: 15–21

  21. 21

    Jones CT 1977 The development of some metabolic responses to hypoxia in the foetal sheep. J Physiol 265: 743–762

  22. 22

    Giussani DA, Spencer JA, Moore PJ, Bennet L, Hanson MA 1993 Afferent and efferent components of the cardiovascular reflex responses to acute hypoxia in term fetal sheep. J Physiol 461: 431–449

  23. 23

    Giussani DA, McGarrigle HH, Moore PJ, Bennet L, Spencer JA, Hanson MA 1994 Carotid sinus nerve section and the increase in plasma cortisol during acute hypoxia in fetal sheep. J Physiol 477: 75–80

  24. 24

    Gardner DS, Fowden AL, Giussani DA 2002 Adverse intrauterine conditions diminish the fetal defense against acute hypoxia by increasing nitric oxide activity. Circulation 106: 2278–2283

  25. 25

    Giussani DA, Gardner DS, Cox DT, Fletcher AJ 2001 Purinergic contribution to circulatory, metabolic, and adrenergic responses to acute hypoxemia in fetal sheep. Am J Physiol Regul Integr Comp Physiol 280: R678–R685

  26. 26

    Gardner DS, Jamall E, Fletcher AJ, Fowden AL, Giussani DA 2004 Adrenocortical responsiveness is blunted in twin relative to singleton ovine fetuses. J Physiol 557: 1021–1032

  27. 27

    Fowden AL, Mijovic J, Silver M 1993 The effects of cortisol on hepatic and renal gluconeogenic enzyme activities in the sheep fetus during late gestation. J Endocrinol 137: 213–222

  28. 28

    Dawes GS 1968 Foetal blood-gas homeostasis during development. Proc R Soc Med 61: 1227–1231

  29. 29

    Rudolph AM, Itskovitz J, Iwamoto H, Reuss ML, Heymann MA 1981 Fetal cardiovascular responses to stress. Semin Perinatol 5: 109–121

  30. 30

    Lee ST, Hon EH 1963 Fetal hemodynamic response to umbilical cord compression. Obstet Gynecol 22: 553–562

  31. 31

    Moore LG, Niermeyer S, Zamudio S 1998 Human adaptation to high altitude: regional and life-cycle perspectives. Am J Phys Anthropol Suppl 27: 25–64

  32. 32

    Ratcliffe FM, Evans JM 1993 Neonatal wellbeing after elective caesarean delivery with general, spinal, and epidural anaesthesia. Eur J Anaesthesiol 10: 175–181

  33. 33

    Giussani DA, Gardner DS 2004 Intrauterine hypoxaemia and cardiovascular development. In: Langley-Evans SC (ed) Fetal Nutrition and Adult Disease: Programming of Chronic Disease through Fetal Exposure to Undernutrition. CAB International, Wallingford, CT, pp 55–87

  34. 34

    Alexander DP, Forsling ML, Martin MJ, Nixon DA, Ratcliffe JG, Redstone D, Tunbridge D 1972 The effect of maternal hypoxia on fetal pituitary hormone release in the sheep. Biol Neonate 21: 219–228

  35. 35

    Keller-Wood M, Wood CE 1991 Does the ovine placenta secrete ACTH under normoxic or hypoxic conditions?. Am J Physiol 260: R389–R395

  36. 36

    Gardner DS, Fletcher AJ, Fowden AL, Giussani DA 2001 Plasma adrenocorticotropin and cortisol concentrations during acute hypoxemia after a reversible period of adverse intrauterine conditions in the ovine fetus during late gestation. Endocrinology 142: 589–598

  37. 37

    Gardner DS, Fletcher AJ, Bloomfield M, Fowden AL, Giussani DA 2002 The effects of prevailing hypoxaemia, acidaemia or hypoglycaemia upon the cardiovascular, endocrine and metabolic responses to acute hypoxaemia in the ovine fetus. J Physiol 540: 351–366

  38. 38

    Edwards AV, Jones CT 1987 The effect of splanchnic nerve stimulation on adrenocortical activity in conscious calves. J Physiol 382: 385–396

  39. 39

    Edwards AV, Jones CT 1987 The effect of splanchnic nerve section on the sensitivity of the adrenal cortex to adrenocorticotrophin in the calf. J Physiol 390: 23–31

  40. 40

    Comline RS, Silver M 1961 The release of adrenaline and noradrenaline from the adrenal glands of the foetal sheep. J Physiol 156: 424–444

  41. 41

    Engeland WC, Wotus C, Rose JC 1998 Ontogeny of innervation of rat and ovine fetal adrenals. Endocr Res 24: 889–898

  42. 42

    Myers DA, Robertshaw D, Nathanielsz PW 1990 Effect of bilateral splanchnic nerve section on adrenal function in the ovine fetus. Endocrinology 127: 2328–2335

  43. 43

    Bloom SR, Edwards AV, Jones CT 1987 Adrenal cortical responses to vasoactive intestinal peptide in conscious hypophysectomized calves. J Physiol 391: 441–450

  44. 44

    Jones CT, Ritchie JW 1976 Endocrine and metabolic changes associated with periods of spontaneous hypoxia in fetal sheep. Biol Neonate 29: 286–293

  45. 45

    Jones CT, Ritchie JW, Walker D 1983 The effects of hypoxia on glucose turnover in the fetal sheep. J Dev Physiol 5: 223–235

  46. 46

    Jones CT, Ashton IK 1976 The appearance, properties, and functions of gluconeogenic enzymes in the liver and kidney of the guinea pig during fetal and early neonatal development. Arch Biochem Biophys 174: 506–522

  47. 47

    Jones CT, Robinson RO 1975 Plasma catecholamines in foetal and adult sheep. J Physiol 248: 15–33

  48. 48

    Fletcher AJ, Edwards CMB, Gardner DS, Fowden AL, Giussani DA 2000 Neuropeptide Y in the sheep fetus: effects of acute hypoxemia and dexamethasone during late gestation. Endocrinology 141: 3976–3982

  49. 49

    Apatu RS, Barnes RJ 1991 Release of glucose from the liver of fetal and postnatal sheep by portal vein infusion of catecholamines or glucagon. J Physiol 436: 449–468

  50. 50

    Whitworth JA, Williamson PM, Mangos G, Kelly JJ 2005 Cardiovascular consequences of cortisol excess. Vasc Health Risk Manag 1: 291–299

  51. 51

    Stampfer MJ, Colditz GA, Willett WC, Manson JE, Rosner B, Speizer FE, Hennekens CH 1991 Postmenopausal estrogen therapy and cardiovascular disease. Ten-year follow-up from the nurses' health study. N Engl J Med 325: 756–762

Download references

Acknowledgements

We thank Professor A.L. Fowden for her continued support with our studies, Mr. Paul Hughes and Mr. Scott Gentle for their help during surgery, Mrs. Sue Nicholls and Miss Victoria Johnson for the routine care of the animals used in this study, and Mr. Malcolm Bloomfield for the radioimmunoassays.

This article is dedicated to Dr. Andrew J.W. Fletcher. Andy was a promising young doctor and an excellent scientist, who died suddenly, at the age of 33, after finishing a charity run in Norfolk in 2008.

Author information

Correspondence to Dino A Giussani.

Rights and permissions

Reprints and Permissions

About this article

Further reading