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Endocrinology Vol. 145, No. 9 4278-4285
Copyright © 2004 by The Endocrine Society

Periconceptional Undernutrition in Sheep Accelerates Maturation of the Fetal Hypothalamic-Pituitary-Adrenal Axis in Late Gestation

Frank H. Bloomfield, Mark H. Oliver, Paul Hawkins, Alison C. Holloway, Melanie Campbell, Peter D. Gluckman, Jane E. Harding and John R. G. Challis

Liggins Institute (F.H.B., M.H.O., P.H., P.D.G., J.E.H.), University of Auckland, Auckland 1020, New Zealand; and Departments of Physiology and Obstetrics and Gynaecology (F.H.B., A.C.H., M.C., J.R.G.C.), University of Toronto, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Dr. Frank Bloomfield, Liggins Institute, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail: f.bloomfield{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the effects of moderate maternal periconceptional undernutrition from 60 d before to 30 d after mating on fetal hypothalamic-pituitary-adrenal axis function in late gestation. Ewes were sampled regularly during the period of undernutrition for circulating hormone levels. Vascular catheters were inserted into ewes and their singleton fetuses at 112 d gestation (term, 145 d), and fetal ACTH1–24 and metyrapone challenge tests were performed at 127 and 128 d. Postmortems were performed at 132 d. Fetuses of undernourished ewes (UN, n = 12) had elevated baseline cortisol concentrations (P < 0.05), compared with fetuses of ad libitum-fed ewes (n = 10). There were no differences between groups in fetal responses to ACTH challenge, but only UN fetuses demonstrated ACTH and 11-deoxycortisol responses to metyrapone (P < 0.05). UN fetuses had increased mRNA levels for proopiomelanocortin and prohormone convertase-1, but not -2, in the pars intermedia of the pituitary gland (P < 0.05). Glucocorticoid receptor mRNA levels were not different between groups in pituitary or hypothalamus. Maternal cortisol and ACTH levels during undernutrition were profoundly suppressed (P < 0.001), rather than elevated, in UN ewes. Furthermore, the normal pregnancy rise in maternal serum progesterone concentrations was delayed in undernourished mothers. These data demonstrate that events around the time of conception have profound effects on fetal hypothalamic-pituitary-adrenal development in late gestation and that factors other than fetal exposure to excess glucocorticoids may be important.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FETAL HYPOTHALAMIC-PITUITARY-ADRENAL axis (HPAA) plays a vital role in fetal maturation during late gestation (1). In all species studied to date, there is a rise in circulating cortisol concentrations before birth (2), which is responsible for the maturation of organs and metabolic pathways in preparation for life ex utero. This prepartum cortisol surge is also important in the processes leading to parturition and, in some species such as the sheep, is essential for the onset of the labor (3, 4, 5). During the prepartum cortisol surge, rather than the classical feedback loop, there is a feed-forward loop between cortisol and ACTH, allowing cortisol and ACTH concentrations to rise concomitantly. The mechanism underlying this feed-forward loop is not understood.

Cortisol crosses the placenta, and the fetal HPAA is therefore also exposed to maternal glucocorticoids (6). Inappropriate exposure of the developing fetus to maternal glucocorticoids has been proposed as a mechanism for fetal programming (7, 8), and administration of synthetic glucocorticoids to pregnant ewes and guinea pigs results in altered HPAA function and hypertension in the offspring (9, 10, 11). Undernutrition also results in altered HPAA function in the offspring in several species (12, 13, 14), and has also been hypothesized to exert these effects via excessive fetal glucocorticoid exposure (15). This could occur as a result of the stress of undernutrition leading to elevated maternal glucocorticoids, which are then transferred across the placenta (16) or as a result of reduced placental conversion of active to inactive glucocorticoid (14). Undernourished rat dams have been reported to have elevated glucocorticoid levels along with growth restriction and altered HPAA function in the pups (15). Maternal adrenalectomy of undernourished dams, with corticosterone replacement at basal levels, resulted in pups that were still growth restricted but did not have altered HPAA function, suggesting that it may be fetal exposure to excessive glucocorticoid rather than growth restriction per se that results in fetal programming. However, many experimental paradigms of maternal undernutrition have not reported maternal glucocorticoid concentrations, and in undernourished sheep decreased maternal cortisol levels have also been reported (17).

We recently reported that moderate periconceptional undernutrition in the sheep, from 60 d before until 30 d after mating (term is 145 d in this breed), programs the fetal glucose/insulin axis, with altered insulin responses to glucose but not arginine (18). In a parallel cohort of animals allowed to deliver normally, we demonstrated precocious activation of the fetal HPAA and preterm birth (19). Here we report studies of dynamic fetal HPAA function testing in late gestation and measurements of maternal circulating hormone concentrations during the period of undernutrition, attempting to identify the level of the HPAA at which activation occurred and possible mechanisms underlying that activation.

To confirm the hypothesis that undernutrition exposes the fetus to excess maternal glucocorticoid, we measured maternal cortisol and ACTH concentrations before, during, and after undernutrition. The period of undernutrition encompassed the period when maintenance of pregnancy depends on progesterone production by the corpus luteum, the function of which has been reported to be altered in undernutrition (20), and also covered the period of initial placental development and attachment in the sheep (21). Elevated progesterone concentrations around the time of conception have also been reported to have profound effects on fetal and placental growth (22, 23). Luteal progesterone production is in turn partly regulated by ovine placental lactogen (oPL) (24, 25), a hormone produced by the binucleate cells of the ovine placenta. oPL levels are nutritionally regulated (26), with concentrations in the ewe falling with maternal food restriction (27). During the period of histiotrophic nutrition of the developing embryo, before the placenta has fully attached and developed, oPL levels correlate with uterine gland hypertrophy (the source of histiotrophic nutrients) (28, 29). In late gestation, oPL concentrations are correlated with both placental and fetal weights (30), and a role for oPL in nutrient partitioning between the mother and fetus has been postulated (31, 32, 33). We therefore also measured maternal progesterone and oPL concentrations throughout pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
After acclimatization to a concentrate feed designed by us for prolonged feeding of ruminants, nonpregnant Romney ewes were randomly assigned to maintenance feeds (N; concentrates at 3–4% of body weight per day) or undernutrition (UN; 2-d fast followed by concentrates at 1–2% of body weight per day) from 60 d before mating as described previously (18). The UN regimen was designed to reduce maternal body weight by 10–15% (19). A fortnight before mating with Dorset rams, the estrous cycle of all ewes was synchronized (34). UN was continued until 30 d after mating in UN ewes. Thereafter all ewes were fed maintenance feeds. Only singleton-bearing ewes were used in this experiment, and there were 10 N ewes and 12 UN ewes. From –60 d gestation until transport to the laboratory at 105 d gestation, maternal blood samples were withdrawn by jugular puncture at fortnightly intervals.

After acclimatization to laboratory conditions for 7 d, surgery was performed for the implantation of chronic indwelling vascular catheters in fetal and maternal arteries and veins as described previously (18). On d 127 of gestation, fetuses underwent an ACTH1–24 stimulation test, and on d 128 a metyrapone test was performed. Both the ACTH1–24 and the metyrapone tests were performed in the morning on all animals, with the baseline samples taken between 0800 and 1000 h before feeding. Metyrapone blocks 11ß-hydroxylase (P450c11ß1), the enzyme that catalyzes the conversion of 11-deoxycortisol to cortisol, the final step in cortisol biosynthesis. Thus, the normal response to metyrapone is a fall in circulating cortisol concentrations, followed by a rise in ACTH concentrations in response to a decrease in negative feedback and a secondary rise in 11-deoxycortisol because ACTH drives the adrenal gland to produce more cortisol, synthesis of which is blocked by metyrapone. For the ACTH stimulation test, paired fetal and maternal arterial blood samples were withdrawn –15, 0, 5, 10, 15, 30, 60, and 120 min after an iv bolus to the fetus of 0.3 µg ACTH1–24 (Sigma Chemical, St. Louis, MO). For the metyrapone test, similar paired samples were withdrawn –15, 0, 30, 60, and 120 min after fetal iv injection of 30 mg of metyrapone (Sigma) over 1 min. All samples were collected onto ice and processed for later hormone analysis as described previously (12). Cortisol and ACTH concentrations from the four baseline samples of these two tests were averaged to provide a basal cortisol and ACTH concentration for each fetus. However, in the analysis of each test, only the baseline samples from that test were used. At 131 d gestation, ewes were killed with an overdose of pentobarbitone. Fetal tissues were rapidly dissected, frozen over dry ice, and stored at –80 C until analyzed. Experiments were approved by the institutional animal ethics committee.

Hormone analysis
ACTH was measured by a commercial RIA kit that detects ACTH1–24 (Diasorin, Stillwater, MN) and that has been previously validated for use in the sheep (35). Intra- and interassay coefficients of variation (CVs) were 7.9 and 7.2%, respectively. Cortisol was measured by an in-house RIA (12). Briefly, cortisol was extracted from 100 µl plasma with 4 ml diethyl ether with an efficiency of more than 95%. After reconstitution with PBS and gelatin, samples were incubated in duplicate overnight at 4 C in the presence of cortisol antibody and 3H-cortisol (Amersham Life Sciences, Arlington Heights, IL). The cortisol antibody was generated in this laboratory and had cross-reactivities as follows: less than 0.1% with cortisone, 21-deoxycortisone, progesterone, 17{alpha}-hydroxypregnenolone, and pregnanediol; 1.6% with 11-deoxycortisol; and 0.2% with 11ß-hydroxyprogesterone. Bound and free cortisol were separated by incubation and centrifugation with dextran-coated neutral charcoal (Dextran T70; Pharmacia Fine Chemicals, Baie d’Urfe, Quebec, Canada; Norit A, Fisher Scientific, Toronto, Canada), and the supernatant mixed with 4 ml scintillation cocktail and counted. Two volumes of sample were assayed and parallelism was observed. Intra- and interassay CVs were 5.0 and 6.9%, respectively, and the detection limit was 0.2 ng/ml. 11-Deoxycortisol was measured using a commercial double-antibody RIA kits (ICN Pharmaceuticals Inc., Costa Mesa, CA) after extraction and 3-fold concentration of plasma samples with diethyl ether (intraassay CV 9.2%; detection limit 0.016 ng/ml). Progesterone was measured by an in-house RIA after extraction with diethyl ether as described previously (12) (intraassay CV 5.4%; detection limit 1.5 ng/ml). oPL was analyzed by RIA as described previously (26) (intra- and interassay CVs 6.7 and 9.4%, respectively).

In situ hybridization
We followed the method we described previously, with minor modifications (36). Briefly, 12-µm sections of frozen pituitaries and hypothalami were sectioned using a cryostat (Jung CM 300, Leica Instruments, Nussloch, Germany) and mounted onto precoated slides (Superfrost, Fisher Scientific, Nepean, Ontario, Canada), fixed in 4% paraformaldehyde for 5 min, rinsed in PBS (2 x 1 min) and dehydrated in an ascending alcohol series. The 45-mer oligonucleotide probes complementary to sequences of the porcine proopiomelanocortin (POMC) (37) and prohormone convertase (PC)-1 and -2 genes (38, 39), the ovine glucocorticoid receptor (GR) (40), bovine prolactin (PRL) (36), ovine arginine vasopressin (AVP) (41), and CRH genes (42) and a 42-mer oligonucleotide probe complementary to bases 17–58 of the partial ovine urocortin (UCN) gene (43) were labeled using terminal deoxynucleotidyl transferase (Pharmacia Biotech, Baie d’Urfe, Quebec, Canada) and [{alpha}-35S]dATP (NEN Life Science Products Du Pont Canada, Mississauga, Ontario, Canada). The sections were hybridized overnight in a moist chamber (42 C) with the radiolabeled probes. After hybridization, the sections were washed and exposed to autoradiographic film (Biomax; Kodak, Rochester, NY) with 14C standards (American Radiochemical, St. Louis, MO). Control 45-mer antisense or nonsense sequence oligonucleotide probes were included with each probe to assess nonspecific hybridization. The autoradiograms were then analyzed using computerized image analysis software (Imaging Research, St. Catherines, Ontario, Canada). The relative ODs of hybridized probes were assessed using a minimum of 12 sections for each animal. For pituitary sections, analysis of the pars intermedia and pars distalis was undertaken separately. For hypothalamic sections, values represent total paraventricular nucleus (PVN) signal.

Statistical analysis
Baseline fetal and maternal hormone concentrations were compared by an unpaired Student’s t test after confirming parametric distribution. Fetal cortisol area under the curve (AUC) after ACTH1–24 injection, and fetal ACTH, 11-deoxycortisol and cortisol AUC after metyrapone injection were compared by unpaired Student’s t test. The acute fetal cortisol response to metyrapone injection was analyzed by a paired Student’s t test. Maternal ACTH, cortisol, and progesterone levels during the period of nutritional manipulation (–60 to +30 d from conception) and from the end of nutritional manipulation onwards were analyzed separately with repeated-measures ANOVA. For the in situ hybridizations, relative ODs were compared by Student’s t test, if necessary after log transformation to achieve parametric distribution. We regarded P < 0.05 as statistically significant. Data are expressed as mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
There were no significant differences between groups in fetal weight (UN, 3.99 ± 0.09 kg; N, 4.28 ± 0.21 kg; P > 0.1) or adrenal weight (UN, 491 ± 5 mg; N, 448 ± 3 mg; P > 0.7). Adrenal weights expressed per unit body weight were also not significantly different (data not shown). Overall, there was a statistically significant, although not very strong, positive correlation between adrenal weight and baseline circulating cortisol concentration (r2 = 0.26, P = 0.03). The association was stronger in the UN fetuses than N fetuses but no longer statistically significant (UN, r2 = 0.3, P = 0.1; N, r2 = 0.1, P = 0.5).

Baseline HPAA function
Baseline fetal cortisol concentrations (the mean of the two baseline samples taken on d 127 and the two taken on d 128 for each animal) were significantly higher in fetuses of undernourished ewes (2.5 ± 0.4 vs. 1.3 ± 0.1 ng/ml, P < 0.02; Fig. 1Go). Fetal baseline ACTH concentrations were not significantly different between groups (UN, 29.5 ± 3.1 pg/ml; N, 29.0 ± 3.7 pg/ml; P > 0.9). In UN fetuses there was a significant positive correlation between plasma cortisol (F) and ACTH concentrations (F = 0.3 + 0.05*ACTH; r2 = 0.7, P < 0.001), whereas in N fetuses there was a nonsignificant negative correlation (F = 2.06–0.03*ACTH; r2 = 0.28, P = 0.11). Maternal cortisol concentrations at baseline were not significantly different between groups (UN, 3.0 ± 0.5 ng/ml; N, 2.1 ± 0.3 ng/ml; P > 0.1).



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  		FIG. 1. ACTH (top) and cortisol (bottom) responses to a synacthen (ACTH1–24) challenge. Values are mean ± SEM. The arrow denotes the time of ACTH injection. Inset graphs represent AUC for individual animals with the mean represented by a line. {bullet}, UN (n = 12); {square}, N (n = 10).

 
HPAA stimulation tests
After injection of ACTH1–24, there was no difference between groups in peak or AUC values for ACTH or cortisol (Fig. 1Go).

Before administration of metyrapone, fetal 11-deoxycortisol concentrations were below the detection limit of the assay and are reported as the minimum detectable dose (0.016 ng/ml). After metyrapone injection, only UN fetuses showed a significant fall in circulating cortisol levels and rises in ACTH and 11-deoxycortisol concentrations (Fig. 2Go), although two UN fetuses failed to respond to metyrapone with a fall in cortisol or rise in 11-deoxycortisol concentrations. The AUC for both ACTH and 11-deoxycortisol were significantly elevated in UN fetuses (Fig. 2Go). UN fetuses with the highest baseline cortisol concentrations had the greatest fall in cortisol after metyrapone administration and the greatest rise in 11-deoxycortisol concentrations (data not shown).



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  		FIG. 2. Cortisol (top), 11-deoxycortisol (middle), and ACTH (bottom) responses to a metyrapone challenge. Values are mean ± SEM. The arrow denotes the time of metyrapone injection. Inset graphs represent AUC for individual animals with the median represented by a line. {bullet}, UN (n = 12); {square}, N (n = 10). *, P < 0.05 vs. N fetuses, {dagger}, P < 0.05 vs. baseline cortisol value.

 
In situ hybridization
There were no differences between groups in CRH (P = 0.29) or AVP (P = 0.09) mRNA levels in the PVN of the hypothalamus (Fig. 3Go). In the pituitary gland, POMC mRNA levels were up-regulated in UN fetuses in the pars intermedia (P < 0.05) but not in the pars distalis (Fig. 3Go). PC-1 (P < 0.05), but not PC-2, mRNA levels were also up-regulated in the pars intermedia (Fig. 2Go). Regression analysis demonstrated a significant association between POMC and PC-1 mRNA levels in fetuses (R2 = 0.3, P < 0.05), suggesting that those fetuses that up-regulated POMC expression also up-regulated PC-1 expression. GR mRNA levels in the pars distalis were not significantly different between groups, and there was no significant correlation between GR mRNA levels and basal circulating cortisol concentration (r2 = 0.16 for N fetuses, P = 0.3; r2 = 0.05 for UN fetuses, P = 0.55). Interestingly, we did not find evidence of GR message in the pars intermedia (Fig. 3Go). We also investigated mRNA levels of other pituitary hormones that have corticotrophic action. UCN and PRL mRNA levels were not significantly different between groups (data not shown).



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  		FIG. 3. Representative in situ hybridization images with semiquantitative densitometric analysis (point graphs) of CRH and AVP mRNA levels in the PVN of the hypothalamus (top) and POMC, PC-1, PC-2, and GR in the pituitary (bottom). Signal in the pituitary was analyzed separately for the pars distalis (PD) and pars intermedia (PI). In point graphs individual animals are shown with medians. Left column and {square}, N (n = 9); middle column and {bullet}, UN (n = 9); right column, sense controls. *, P < 0.05 vs. N.

 
Maternal hormones during undernutrition
Before commencing nutritional manipulation, maternal plasma concentrations of cortisol, ACTH, and progesterone were not significantly different between groups (Fig. 4Go). During undernutrition, the maternal HPAA was profound-ly suppressed, with ACTH concentrations approximately half, and cortisol concentrations almost a quarter, of those in the control group (P < 0.001; Fig. 4Go). After cessation of undernutrition cortisol concentrations returned to control values and were subsequently not different between groups (Fig. 4Go). ACTH concentrations returned to values slightly, but significantly, above control values and remained elevated (Fig. 4Go).



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  		FIG. 4. Maternal circulating cortisol (top left), ACTH (top right), progesterone (bottom left), and oPL (bottom right) concentrations. Values are mean ± SEM. Black bar, Period of undernutrition; {bullet}, UN ewes (n = 12); {square}, N ewes (n = 10). ***, P < 0.001 vs. N ewes.

 
Maternal progesterone concentrations were not significantly different between groups during the period of undernutrition. In the ad libitum-fed ewes, progesterone concentrations rose steadily throughout the study (Fig. 4Go). However, in UN ewes progesterone concentrations plateaued after cessation of undernutrition and remained significantly lower than in control ewes until d 93 of gestation (Fig. 4Go).

Maternal plasma concentrations of oPL were not different for most of the study period but began to diverge in late gestation, with values in UN ewes falling and becoming significantly less than those in the N ewes as delivery approached (Fig. 4Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The fetal HPAA plays a crucial role in the onset of parturition in the sheep (3, 44). In the human, a prepartum glucocorticoid surge also occurs and is thought to be important both in the preparation of the fetus for extrauterine life and in the processes of parturition, although the precise role of glucocorticoids in the latter is less clear (5). The underlying mechanisms that determine when this cortisol surge takes place, and thus when parturition will occur, have not been elucidated.

During fetal life the ovine adrenal gland has three phases of activity. Early in gestation it is capable of secreting cortisol but then goes through a quiescent phase between approximately 90 and 120 d gestation (45, 46). This phase is characterized by low mRNA levels of P450c17 (47). From 120 d onward, there is a steady and gradual increase in cortisol production by the adrenal gland, and this is thought to be in response to increasing ACTH secretion from the pituitary (48).

The studies we report here provide evidence that the time of adrenal maturation may be regulated, in part, by maternal nutritional status in the periconceptional period, despite the fact that the moderate nutritional restriction ended almost 100 d before we tested HPAA function. We previously reported that this moderate nutritional restriction also resulted in preterm birth, probably as a direct consequence of the advanced fetal HPAA maturation (19). The results of the ACTH stimulation test demonstrate that the adrenal cortisol response was not altered by periconceptional undernutrition and suggest that the pathway for cortisol biosynthesis was equally responsive to ACTH1–24 stimulation in UN and N fetuses. However, only the UN fetuses demonstrated a significant response to metyrapone, as evidenced by a significant fall in fetal cortisol levels and a rise in 11-deoxycortisol and ACTH levels. A positive response to a metyrapone test necessitates successful inhibition of 11ß-hydroxylase, demonstrated by a fall in circulating cortisol concentrations and a rise in 11-deoxycortisol concentrations, and an intact negative feedback loop, demonstrated by a rise in ACTH concentrations.

We believe that the most consistent explanation for the findings of similar responses between groups to ACTH1–24 injection but different responses to the metyrapone challenge is an alteration in negative feedback sensitivity in UN fetuses. However, we cannot exclude the possibility that the differences in responses to the metyrapone test could be due to technical limitations at the very low glucocorticoid concentrations seen in the fetus, particularly when some of the circulating cortisol is of maternal origin. The cross-reactivity of the cortisol antibody used in the cortisol assay with 11-deoxycortisol could have led to some measurement error when cortisol concentrations are very low and 11-deoxycortisol concentrations are high after metyrapone. There is, however, no reason this should be the case in UN fetuses but not in N fetuses. It is also possible that there was inadequate inhibition of 11ß-hydroxylase in N fetuses but not in UN fetuses, although there is no reason to suppose that this was the case given that the metyrapone challenge was the same in all fetuses, that a lack of response was also seen in two of the UN fetuses, and that there were no significant differences between groups in fetal weight or adrenal weight.

Another explanation for the different responses between groups to the metyrapone test could be that the circulating cortisol in the N fetuses is largely of maternal origin and therefore would not fall after fetal metyrapone administration. This would not invalidate the conclusion that the fetal HPAA is more mature in UN fetuses and in fact may further support it, although the conclusion that the feedback axes are different would be more tenuous. Similarly, it is possible that in the N fetuses a larger proportion of steady-state circulating cortisol was derived from reduction of cortisone via the action of 11ß-hydroxysteroid dehydrogenase (HSD) type 1, rather than de novo synthesis dependent on 11ß-hydroxylase in the adrenal gland. 11ß-HSD-1 expression is present in the ovine placenta and liver from mid-gestation and has been proposed as a source of circulating cortisol (49, 50). Furthermore, activity of this enzyme decreases in late gestation (50). In the fetal-facing basement membrane of the baboon placenta, the ratio of 11ß-HSD-1, which acts primarily as a reductase, to 11ß-HSD-2, an isoform with exclusive oxidative action, has also been reported to fall in late gestation, resulting in a decrease in extraadrenal cortisol production (51, 52). This could result in a release of suppression of the fetal adrenal gland by extraadrenal cortisol, thus facilitating maturation of the fetal HPAA as term approaches (53). If this mechanism partly explains the advanced HPAA maturation in UN fetuses, it could also explain the different responses to inhibition of 11ß-hydroxylase with metyrapone between UN and N fetuses. The potential role of the regulation of local concentrations of active glucocorticoid by the 11ß-HSD enzymes, both peripherally and centrally (54), in the role of the maturation of the fetal HPAA warrants further investigation.

Before 120 d gestation in the fetal sheep, adrenalectomy does not influence fetal ACTH or cortisol concentrations. However, after this time, fetal ACTH concentrations increase markedly, suggesting that the fetal pituitary sensitivity to cortisol-negative feedback is increasing (55). Negative feedback of the HPAA occurs at pituitary level and above via the GR. Although we did not find a difference in GR mRNA levels in the pars distalis, we could not find evidence of GR expression in the pars intermedia. This is consistent with a previous report in late-gestation ovine fetuses (56) and may suggest that ACTH secretion from the pars intermedia in fetal life is not under normal negative feedback control. Furthermore, POMC mRNA levels were increased in the pars intermedia, although not in the pars distalis. Differential regulation of POMC expression in the pars intermedia and pars distalis after hypoxia in late-gestation fetal sheep has been reported previously (56, 57). We also report up-regulation of PC-1, but not PC-2, in the pars intermedia. If these changes in expression are matched with changes in enzyme activity, then this could be expected to result in increased ACTH production from the pars intermedia in UN fetuses. This would be consistent with previous studies reporting abundant expression of immunoreactive ACTH in the fetal pars intermedia, in contrast to very sparse staining in the adult pars intermedia (58). A recent study demonstrated that in placentally restricted, chronically hypoxemic ovine fetuses, a population of non-CRH target pituitary corticotrophs emerged in late gestation that secreted high basal levels of ACTH in vitro (59). We did not collect pituitary cells for culture in our experiment and are therefore unable to determine whether a similar change in pituitary corticotroph cell population may contribute to the findings in our study. We did, however, investigate other potential routes of corticotrophic stimulation in the pituitary. UCN, a CRH-like peptide, has been shown to stimulate ACTH release from the pituitary and placenta (60, 61), and UCN mRNA is expressed in the pituitary and human fetal membranes and endometrium (62, 63). We did not find any difference in UCN mRNA levels between groups. Similarly, mRNA levels of PRL, which, in common with POMC mRNA levels, is under dopaminergic control (64), were not different between groups.

We speculate that the concurrent rises in ACTH and cortisol seen in late gestation before parturition may be due to ACTH secretion from the pars intermedia, which is not under normal negative feedback control and may be the site of the feed-forward loop seen during the prepartum period. If so, this could explain how UN fetuses can demonstrate a negative feedback response to metyrapone, mediated by GR in the pars distalis but also demonstrate evidence of a feed-forward loop, mediated by the pars intermedia. The presence of a feed-forward loop in these fetuses is supported by a significant positive correlation between cortisol and ACTH concentrations in UN fetuses but a negative nonsignificant correlation in N fetuses. It is also consistent with our finding of an early prepartum surge in cortisol and ACTH, culminating in early delivery, in the cohort of UN fetuses that were allowed to proceed to normal delivery (19).

Contrary to the prevailing hypothesis that proposes fetal exposure to elevated maternal glucocorticoids as a fundamental mechanism for fetal programing (7, 15, 65), we report a profound suppression of the maternal HPAA in response to a chronic, moderate nutritional insult. The difference in maternal glucocorticoid response to undernutrition between our study and others may be a reflection of the duration of undernutrition, with short periods of more severe undernutrition, resulting in an increase in maternal, and fetal, glucocorticoid concentrations (16) but prolonged chronic moderate undernutrition (as in our study), resulting in adaptive down-regulation. Indeed, our results are consistent with studies from the 1940s and 1960s in fasted nonpregnant ewes and rats, in which adrenal and pituitary weights were reduced and morphology of the glands suggested atrophy (66, 67, 68). Those studies did not measure circulating hormone concentrations. However, a recent study in which pregnant ewes were undernourished from 28 to 80 d of gestation also reported reduced maternal circulating cortisol concentrations (17).

On cessation of undernutrition in our study, maternal cortisol concentrations returned to normal, although ACTH levels returned to a level slightly, but significantly, higher than baseline and remained elevated for several weeks. We do not have detailed hormonal profiles covering the period that undernourished ewes were refed at 30 d gestation. Administration of the synthetic glucocorticoid dexamethasone to pregnant ewes at a similar point in gestation (28 d) permanently altered blood pressure of the offspring, whereas dexamethasone given at 64 d had no effect (11, 69). These findings may suggest that the period around 30 d gestation, when the fetal adrenal gland is just beginning to develop glucocorticoid secretory capacity, may represent a critical window during development when the fetal HPAA is very sensitive to glucocorticoid concentrations and can be permanently reset by changes in those concentrations. We cannot exclude the possibility that the fetuses in our study may have been exposed to a change in the level of glucocorticoid exposure at a critical time, as a result of either a cortisol spike on maternal refeeding or simply the change from low to normal levels of maternal cortisol. However, our results do suggest that the maternal glucocorticoid response to undernutrition may differ with different experimental paradigms and that effects seen in the fetus cannot necessarily be assumed to be consequent on increased fetal glucocorticoid exposure without some measure of glucocorticoid concentrations in the mother and/or fetus.

An alternative possible explanation for the altered fetal development in late gestation that we report here and elsewhere (18, 19) may lie in altered placental function. We do not have direct measurements of placental function, but the lower circulating levels of oPL, a somatotrophic hormone produced by the placenta, in late gestation and the lower progesterone concentrations during the first half of gestation in UN ewes, at the time that the placenta is attaching, suggest that there may be alterations in placental function after periconceptional undernutrition. Various aspects of placental function could interact with the fetal HPAA, including placental POMC production, 11ß-HSD-II activity, and local prostaglandin concentrations (70, 71, 72).

It is also possible that the lower progesterone concentrations in early gestation in UN ewes could affect fetal development in other ways. Kleemann et al. (22, 23) demonstrated that only 3 d of progesterone treatment around the time of conception in sheep can dramatically alter local hormone production and thus embryonic, fetal, and placental growth. In human pregnancies the appearance of pinopodes, morphological structures thought to be important in blastocyst attachment and uterine recipiency, is associated with concentrations of progesterone and progesterone receptors (73, 74, 75). In early gestation progesterone is produced by the corpus luteum and, in sheep, this has shown to be regulated in part by oPL (24, 25, 76). Luteal progesterone production has also been shown to be reduced by nutritional restriction in pigs (20).

It is clear that there may be complex interactions between maternal nutrition and the maternal hormonal environment, both systemic and local, around the time of conception that could have profound effects on the development of the embryo and placenta. Our data have demonstrated that periconceptional undernutrition alters development of the fetal HPAA many months later in a manner that is consistent with accelerated maturation. Our previous finding of preterm birth after periconceptional undernutrition in a similar cohort of sheep (19) supports this conclusion. Further investigations are necessary to determine the mechanisms by which these changes occur and the stage of development at which they occur and also whether they have significance for health in adult life.


   Footnotes
 
This work was supported by the Health Research Council of New Zealand, the Canadian Institutes for Health Research, and the Basser Traveling Fellowship awarded by the Royal Australasian College of Physicians (to F.H.B.).

Current address for A.C.H.: Department of Obstetrics and Gynaecology, McMaster University, Hamilton, Ontario, Canada.

Abbreviations: AUC, Area under the curve; AVP, arginine vasopressin; CV, coefficient of variation; F, plasma cortisol; GR, glucocorticoid receptor; HPAA, hypothalamic-pituitary-adrenal axis; HSD, hydroxysteroid dehydrogenase; N, normal nutrition; oPL, ovine placental lactogen; PC, prohormone convertase; POMC, proopiomelanocortin; PRL, prolactin; PVN, paraventricular nucleus; UCN, urocortin; UN, undernutrition.

Received April 2, 2004.

Accepted for publication June 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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