Activation of the hypothalmic-pituitary-adrenal (HPA) axis is important for many of the adaptive processes essential for life ex utero. In all species studied to date, including the horse, there is an increase in the cortisol concentration in the fetal circulation during the period immediately before birth as a result of developmental changes at all levels of the HPA axis (Fig 1) (Liggins 1994; Challis et al. 2000). This increase in fetal glucocorticoid availability induces structural and functional changes in a variety of different tissues, including the lungs, liver, kidneys and gastrointestinal tract (Silver 1990; Sangild et al. 2000). It also activates many of the physiological processes that are vital at birth but have little or no function in fetal life, such as pulmonary ventilation, glucogenesis and renal sodium conservation (Fowden et al. 1998). In many species, prepartum activation of the fetal HPA axis is also involved in the onset of labour. In man and other primates, parturition is facilitated by increased adrenal output of oestrogen precursors, whereas in ruminants, it is the cortisol surge itself that induces labour by actions on uteroplacental steroidogenesis and prostaglandin production (Wood and Cudd 1997; Challis et al. 2000). Consequently, in some species, adrenal glucocorticoids act to coordinate prepartum maturation of the fetus with the onset of uterine contractile activity, which maximises the chances of delivering viable young (Silver 1990). In horses, the signal for the onset of labour is not as clear as in other species but may involve increased adrenal secretion of steroids (Silver 1994).
The hypothalmic-pituitary-adrenal axis and cortisol production
In the horse, activation of the HPA axis occurs late in gestation compared with other species (Rossdale et al. 1973; Fowden and Silver 1995; Wood and Cudd 1997). Fetal cortisol concentrations only begin to rise about 5 days before birth and then escalate rapidly towards term in pony foals (Fig 1b). This is associated with a doubling of adrenal weight over the last 5% of gestation, primarily due to increased growth of the zona fasciculata in the adrenal cortex (Comline and Silver 1971). Similar increments in adrenal weight and cortisol concentrations are observed in fetal sheep and pigs during late gestation but these begin proportionately earlier in gestation and occur more slowly than in fetal horses (Wood and Cudd 1997). In general, plasma adrenocorticotropic hormone (ACTH) concentrations rise in parallel with the cortisol concentrations in the fetus (Silver and Fowden 1994; Cudd et al. 1995). Thus, in sheep, there is a progressive increase in plasma ACTH concentrations from about 15 days before birth, whereas, in horses, fetal plasma ACTH is not elevated until the day of delivery (Fig 1a). Fetal adreno-cortical activity, therefore, appears to rise over the last 15% of gestation in sheep and pigs but is confined to the final 1–2% of gestation in the horse (Fowden and Silver 1995). However, fetal plasma cortisol binding capacity declines over a slightly longer period before birth in the horse than in other species; thus, the period of increased prepartum cortisol exposure may be longer than appears from measurements of total cortisol concentrations alone (Cudd et al. 1995). Nevertheless, overall, there is a very narrow window just before delivery of the foal for glucocorticoids to stimulate fetal maturation in preparation for birth. This may explain the greater incidence of prematurity and maladaptation in newborn foals than seen in other precocious species (Rossdale and Silver 1982).
In contrast to other species (Silver 1990), cortisol concentrations continue to rise for 2 h after birth in full-term pony foals before falling to basal values 24 h after delivery (Fig 1b). Concentrations then remain stable for the next 10–14 days (Fig 1b). Plasma ACTH concentrations fall progressively from birth to reach basal level by 8 h after delivery (Fig 1a). Similar decreases in plasma ACTH and cortisol are observed between the first and subsequent days after birth in Thoroughbred (TB), Standardbred and Quarter Horse (QH) foals (Wong et al. 2009; Hart et al. 2009a). In pony foals delivered prematurely before 320 days, cortisol concentrations are lower than normal at birth and fail to increase significantly over the next 2 h (Silver et al. 1984). In contrast, plasma ACTH concentrations continue to rise after birth in premature foals and are 2–5-fold higher than normal during the first 2 h after birth (Silver et al. 1984). In term newborn foals showing mixed premature and full-term characteristics (Rossdale et al. 1984), cortisol concentrations are intermediate between those of premature and mature foals at birth and rise in the 2 h after birth but to a lesser extent than seen in mature neonates born spontaneously at full-term (Silver et al. 1984). If the premature foals born close to full-term survive beyond the first 48 h, their cortisol concentrations are raised compared with healthy mature foals (Panzani et al. 2009). Even when delivery is induced 24–48 h before full-term on the basis of milk parameters (Ousey et al. 1984), plasma cortisol concentrations are elevated 2–3 fold throughout the first 10 days of life in pony foals that otherwise appear normal and mature by clinical criteria (Holdstock et al. 2011). Overall, these observations show that the cortisol concentrations observed in the immediate neonatal period are determined by the maturity of the foal, and possibly, by its method of delivery.
High concentrations of cortisol and ACTH are also seen in foals aged 1–7 days hospitalised for a range of illnesses including septic and nonseptic conditions (Gold et al. 2007; Hurcombe et al. 2008; Panzani et al. 2009; Hart et al. 2009b). Nonsurvival of sick foals is associated with both higher and lower cortisol concentrations and with a high ACTH:cortisol ratio compared with healthy foals (Gold et al. 2007; Hurcombe et al. 2008; Panzani et al. 2009; Hart et al. 2009b), which suggests that some sick foals are capable of mounting a stress response including activation of adrenal cortisol secretion while others suffer from adrenal exhaustion or insufficiency. Foals born prematurely following maternal placentitis may have precocious adrenocortical maturation, despite displaying other clinical features of prematurity, and can survive with minimal intensive care (Rossdale et al. 1991; Ousey 2004). When mares with placentitis are treated with antimicrobials and immunomodulators, gestational length is close to normal and foals delivered alive have cortisol concentrations within the normal range in the majority of cases (Bailey et al. 2010). In contrast, in fescue toxicosis when fetal HPA development is suppressed by the dopaminergic action of the maternally ingested alkaloids, foals delivered after a full term have low concentrations of cortisol and ACTH at birth and usually do not survive (Brendemeuhl et al. 1995; Cross et al. 1995).
Adrenocortical sensitivity to ACTH in terms of cortisol secretion changes dramatically in the foal during the perinatal period. Before about 290 days of gestation, the fetal adrenal cortex appears to be unresponsive to exogenously administered ACTH1-24 but, thereafter, there is a small but significant rise in fetal plasma cortisol when ACTH is administered (Fig 2a) (Silver and Fowden 1994; Lyle et al. 2010). This response then increases with increasing proximity to delivery (Fig 2a). Adrenocortical sensitivity to exogenous ACTH1-24 is maximal at birth and then declines again in newborn pony foals, although the postnatal cortisol responses are greater than those observed prenatally (Fig 2a). Similar temporal changes in adrenocortical responsiveness to exogenous administration of ACTH have been observed in newborn TB and QH foals, in response to low and high doses of ACTH (Wong et al. 2009; Hart et al. 2009a). In pony foals delivered prematurely before 320 days, the cortisol response to exogenous ACTH1-24 is small at birth and resembles that of the fetus in late gestation (Rossdale et al. 1982; Silver et al. 1984; Silver and Fowden 1995). It remains suppressed for the next 2 days if the foal survives, despite high endogenous ACTH concentrations (Silver et al. 1984). When gestational length is shortened by maternal administration of dexamethasone beginning after 315 days, the foals are small at birth yet viable with normal neonatal cortisol concentrations (Ousey et al. 2011). However, there is a tendency for reduced cortisol responses to exogenous ACTH1-24 in the 15–24 h period after birth of these foals (Ousey et al. 2011). Blunted cortisol responses to exogenous ACTH are also observed in sick foals hospitalised for sepsis and other disorders, such as multiple organ dysfunction syndrome, which suggests that adrenal exhaustion can occur when stress is prolonged (Hart et al. 2009b). These observations show that the ability of the equine adrenal gland to secrete cortisol develops only late in gestation and is directly related to the viability of the foal. The cortisol response to ACTH administration, therefore, provides a useful clinical test of the maturity and health status of newborn foals (Rossdale et al. 1984; Wong et al. 2009).
Figure 2. Mean ± s.e. increment (change) in plasma cortisol concentration from baseline (0 min) in response to (a) administration of adrenocorticotropic hormone (ACTH1-24 1–2 µg/kg bwt i.v.) and (b) hypoglycaemia induced by insulin (0.5 u/kg bwt i.v.) in pony fetuses before term (<300 days of gestation, open circles) and near term (>320 days of gestation, filled circles) and in newborn pony foals at <12 h (open triangles), 3–5 days (filled triangles) and at 14–21 days after birth (grey triangles). * Significant increase from baseline (P<0.05, paired t test). Arrow indicates time of administration of ACTH1-24 or insulin. Number of animals = 4–9. Data from Silver et al. (1984, 1987); Silver and Fowden (1994, 1995); Ousey et al. (2004); Jellyman et al. (2011); A.L. Fowden and A.J. Forhead, unpublished observation).
Download figure to PowerPoint
The developmental profile of the adrenocortical response to stressful stimuli, such as hypoglycaemia and hypotension, differs from that seen in response to exogenous administration of ACTH1-24 (Fig 2b) (Silver et al. 1987; Silver and Fowden 1995; O'Connor et al. 2005). The increment in plasma cortisol in response to insulin-induced hypoglycaemia is smaller and delayed relative to that evoked by direct administration of ACTH1-24 in both near-term fetuses and neonates (Fig 2b). However, by 7–14 days, there is a rapid, sustained increase in plasma cortisol concentrations in response to both hypoglycaemia and hypotension (Silver et al. 1987; O'Connor et al. 2005). The cortisol response to hypoglycaemia, therefore, rises progressively with postnatal age in contrast to that seen in response to exogenous ACTH administration (Fig 2a). This suggests that, during the perinatal period, there may be developmental changes occurring at the hypothalamic-pituitary level that determine the secretion of endogenous, bioactive ACTH. Certainly, there is little, if any, ACTH release in response to insulin-induced hypoglycaemia in the fetus, even close to term, yet by 3–4 days after birth this response is brisk and significantly greater in magnitude than that seen prenatally (Fig 3a). However, despite the greater increment in ACTH, adrenocortical sensitivity to endogenous ACTH appears to be less 3–4 days after birth than in newborn foals (Fig 3b), consistent with the findings with exogenous ACTH1-24 administration (Fig 2a). Rapid rises in ACTH and cortisol concentrations are also seen in 7-day-old foals in response to acute hypotension (O'Connor et al. 2005). However, by 14 days, the ACTH response to this physiological challenge is significantly less than at 7 days, despite a similar cortisol increment (O'Connor et al. 2005). There may, therefore, be changes in the bioactivity of pituitary ACTH during the neonatal period or, alternatively, other factors, such as adrenal innervation, may act to increase adrenocortical sensitivity to endogenous ACTH during stressful conditions in foals by age 14 days (Edwards 1997; Wood and Cudd 1997).
Figure 3. Effect of hypoglycaemia on HPA function shown as (a) mean ± s.e. increment (change) in plasma adrenocorticotropic hormone (ACTH concentrations from baseline (0 min) in response to insulin administration (0.5 u/kg bwt i.v.) (* significant increase from baseline, P<0.05, paired t test) and as (b) the relationship between the endogenous concentrations of ACTH and cortisol in pony fetuses before term (<300 days of gestation, open circles) and near term (>320 days of gestation, filled circles) and in newborn pony foals at 3–5 days after birth (filled triangles). Number of animals = 5–8. Data from Silver and Fowden (1994, 1995); Fowden and Silver (1995) and AL Fowden and AJ Forhead (unpublished observations).
Download figure to PowerPoint
The cellular and molecular mechanisms responsible for the perinatal changes in basal and stimulated HPA function in the foal remain largely unknown. At the pituitary level, there is little evidence for changes in the morphology or density of the corticotrophs observed in the fetal ovine pituitary during the prepartum period (Fowden and Silver 1995). However, the alterations in the pulsatile pattern of fetal ACTH concentrations with proximity to delivery suggest that there are prepartum changes in hypothalamic secretion of the ACTH releasing factors, corticotrophin releasing hormone (CRH) and arginine vasopressin, and/or in the abundance of the receptor for these neuropeptides on the corticotrophs of the foal (Cudd et al. 1995). At the adrenal level, production of cortisol depends on 3 key rate-limiting enzymes: cholesterol side chain cleavage (P450SCC), 3β-hydroxysteroid dehydrogenase (3βHSD) and 17α-hydroxylase (P450C17). Cytochrome P450SCC, which converts cholesterol to pregnenolone (P5) is present in the zona glomerulosa and putative zona reticularis of the equine adrenal gland from as early as 150 days of gestation and rises in abundance in these regions, and in the zona fasciculata, with increasing gestational age (Han et al. 1995). The enzyme 3βHSD synthesises progesterone from P5 and increases in abundance, predominantly in the zona fasciculata, from about 280 days of gestation onwards, in association with ultrastructural changes to this zone indicative of increased steroid synthesis (Webb and Stevens 1981; Han et al. 1995). In contrast, P450C17, required for cortisol production, is only detectable in the equine adrenal gland at low concentrations until very close to term when its expression increases in parallel with the prepartum rise in fetal cortisol concentrations (Fig 1b). By birth, all 3 enzymes are abundant throughout the zona fasciculata of the neonatal adrenal gland and show little further change in expression during the first few weeks of post natal life (Han et al. 1995). These perinatal changes in cytoarchitecture and enzyme content of the equine adrenal gland are, therefore, consistent with the increased responsiveness to exogenous ACTH1-24 between late gestation and birth (Fig 2a), and with the positive correlation observed between the cortisol response to ACTH1-24 of the newborn foal and its gestational age at delivery (Ousey et al. 2011). They also support the suggestion that other factors are involved in the subsequent post natal changes in adreno cortical ACTH responsiveness (Fig 2a). These factors include adrenal blood flow, ACTH receptor density, ACTH clearance and the release of adreno-medullary peptides with effects on the adrenal cortex (Fowden and Silver 1995; Wood and Cudd 1997).
In rat dams, environmental challenges during pregnancy influence HPA function in the newborn pups but little is known about the immediate neonatal consequences of suboptimal intrauterine conditions on this axis in more precocious species such as the horse (Lesage et al. 2001; Fowden et al. 2006). Dysphagia and chronic weight loss during TB pregnancy caused by infection with Streptococcus equi increases the basal cortisol concentrations of their growth restricted foals 15–24 h after birth but has little effect on the cortisol response to exogenous ACTH1-24 at this age, irrespective of the nutritional plane of the mother at the time of infection (Ousey et al. 2008). Neonatal HPA function is also altered when fetal growth is manipulated by embryo transfer between equine breeds of different sizes (Allen et al. 2002; Giussani et al. 2003; Ousey et al. 2004). Both intrauterine growth-restriction (IUGR) caused by transferring a TB embryo into a pony uterus and fetal macrosomia induced by the reciprocal transfer of a pony embryo into a TB mare alter cortisol responses to ACTH and physiological stimuli in the newborn foal. In TB foals growth restricted by embryo transfer into pony mares, basal cortisol concentrations are high 12 h after birth, despite normal ACTH concentrations, although the cortisol response to exogenous ACTH1-24 is small compared with that of normally grown TB foals during the first day post partum (Ousey et al. 2004). However, 4–5 days later, the cortisol response to ACTH1-24 and HPA sensitivity to acute hypotension are no different in IUGR and normally grown TB foals (Giussani et al. 2003; Ousey et al. 2004). In contrast, overgrown pony foals delivered by TB mares have normal basal concentrations of cortisol and ACTH for 10 days after birth and a normal cortisol response to exogenous ACTH1-24 at 1 and 5 days (Giussani et al. 2003; Ousey et al. 2004). However, these overgrown pony foals had a reduced ACTH response to acute hypotension on Day 6, which was coupled with a cortisol response similar to that seen in normal pony foals with greater ACTH responses (Giussani et al. 2003). Neonatal adrenocortical sensitivity to endogenous ACTH, therefore, appears to be enhanced during stressful conditions when intrauterine growth is enhanced above the genetic norm by increasing the surface area of the placenta (Allen et al. 2002; Giussani et al. 2003). Conditions during intrauterine development, therefore, appear to affect HPA function of the newborn foal in the 10-day period after birth, although the changes are subtle in some instances and may take several days to develop.
Integrated hypothalmic-pituitary-adrenal (HPA) function during the prepartum period
By combining the cortisol and progestagen data, a possible sequence of maturational changes can be described for the integrated function of the fetal HPA axis during the prepartum period as follows. Before 290 days of gestation (>30 days from delivery), the fetal HPA axis has a basal level of activity and is relatively unresponsive to stimuli. Circulating concentrations of ACTH are low and the adrenal appears to produce P5 primarily, due to a lack of P450C17 (Fig 4a). Much of the cortisol circulating in the fetus at this time may be of maternal origin derived by transplacental passage down the concentration gradient (Fowden et al. 2008). After 300 days of gestation (25–5 days before delivery), activity of the HPA axis increases (Fig 4b). The adrenal cortex grows and develops morphologically, possibly due to increased ACTH exposure caused by changes in the pattern and bioactivity of the ACTH released by the pituitary (Comline and Silver 1971; Webb and Stevens 1981; Cudd et al. 1995). With low P450C17 expression, this leads to increased adrenal P5 production, which, in turn, enhances uteroplacental production of several progestagens (Holtan et al. 1991; Rossdale et al. 1992; Ousey et al. 2003). These steroids maintain uterine quiescence in the face of increasing uterine stretch and lead to rising total progestagen concentrations in the mare during the last 20–25 days of gestation (Fig 4b). Once the adrenal gland expresses sufficient P450C17 in the last 5 days or so of gestation, it appears to switch from P5 to cortisol production with the result that fetal cortisol concentrations rise and uteroplacental synthesis and maternal concentrations of total progestagens fall (Fig 4c). Withdrawal of the progestagenic block on uterine contractility is, therefore, coordinated with cortisol-stimulated maturation of the fetal tissues in the horse through this putative switch in adrenal steroid synthesis. Certainly, when delivery occurs prematurely either naturally or by induction with oxytocin before 320 days, plasma P5 and other progestagen concentrations are high in the newborn foal in conjunction with low cortisol concentrations, consistent with a switch in adrenal steroidogenesis very close to term (Holtan et al. 1991; Panzani et al. 2009). Thus, paradoxically, the fetal HPA axis appears to act sequentially first to prevent and then to facilitate the onset of labour in the mare (Fig 4).
Figure 4. Schematic diagram showing the gestational changes in fetal adrenal steroidogenesis and their relationship to uteroplacental progestagen synthesis and total maternal progestagen concentrations in ponies (a) >30 days from spontaneous delivery at full- term (<300 days of gestation, (b) 5–25 days from delivery (≈310–330 days of gestation) and (c) <5 days from delivery (≈330–335 days of gestation, where term = 335 days). ACTH, adrenocorticotropic hormone. Data from Holtan et al. (1991); Silver (1994); Ousey et al. (2001, 2003); Ousey (2004).
Download figure to PowerPoint
The stimulus for the rise in adrenal P450C17 expression so close to term remains unknown but may depend on removal of an inhibitor or on sufficient exposure to bioactive ACTH or to cortisol itself. The rising fetal cortisol concentrations may also have direct effects on uteroplacental progestagen production as administration of synthetic glucocorticoids to either the mare or fetus increases total maternal progestagen concentrations and can induce early delivery depending on the gestational age at treatment (Alm et al. 1975; Rossdale et al. 1992; Ousey et al. 2011). However, the specific progestagen profile in the mare differs between maternal and fetal treatment with ACTH or synthetic glucocorticoids, despite transplacental passage of natural and exogenous glucocorticoids from the dam (Rossdale et al. 1992; Ousey et al. 1998, 2000b, 2011). Collectively, these findings suggest that maternal as well as fetal HPA function may influence the periparturient endocrine profiles, particularly during adverse conditions. The trigger for the final endocrine cascade that leads to parturition, therefore, appears to be multifactorial in the mare and not solely dependent on increased cortisol production by the fetal adrenal gland as occurs in ruminants (Silver 1994; Wood and Cudd 1997; Challis et al. 2000).