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(The FASEB Journal. 2002;16:1017-1026.)
© 2002 FASEB

Programming effects of short prenatal exposure to cortisol

MIODRAG DODIC1, VICKY HANTZIS, JHODIE DUNCAN*, SANDRA REES*, IRENE KOUKOULAS, KELLI JOHNSON, E. MARELYN WINTOUR and KAREN MORITZ

Howard Florey Institute and
* Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3010, Melbourne, Victoria, Australia

1Correspondence: Howard Florey Institute, University of Melbourne, Parkville 3052, Victoria, Australia. E-mail: m.dodic{at}hfi.unimelb.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Recent studies have linked fetal exposure to a suboptimal intrauterine environment with adult hypertension. The aims of this study were twofold: 1) to see whether cortisol treatment administered to the ewe for 2 days at 27 days of gestation (term ~150 days) resulted in high blood pressure in offspring; 2) to study the effect of the same treatment on gene expression in the brain at 130 days of gestation and in lambs at 2 months of age. Mean arterial pressure was significantly higher in the adult female and male offspring of sheep treated with cortisol than in the control group (females: 89±2 mmHg vs. 81±2; P<0.05 and males: 102±4 mmHg vs. 91±3; P<0.05). Prenatal cortisol treatment led to up-regulation of angiotensinogen, AT1, MR, and GR mRNA in the hippocampus in fetuses at 130 days of gestation but not in the animals at 2 months of age. This is the first evidence that short prenatal exposure to cortisol programmed high blood pressure in the adult female and male offspring of sheep. Altered gene expression in the hippocampus could have a significant effect on the development of the hippocampus, and on postnatal behavior.—Dodic, M., Hantzis, V., Duncan, J., Rees, S., Koukoulas, I., Johnson, K., Wintour, E. M., Moritz, K. Programming effects of short prenatal exposure to cortisol.


Key Words: renin-angiotensin system • glucocorticoid • sheep • mineralocorticoid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
THERE IS SOLID epidemiological evidence to suggest that small size at birth (for gestational age) is associated with an increased incidence of adult-onset diseases or dysfunction, including syndrome X (hypertension, non-insulin-dependent diabetes mellitus, and hyperlipidemia) (1 2 3) . It is thought that an adverse intrauterine environment during a critical stage of development permanently alters or ‘programs’ the development of fetal tissues, which allows the fetus to survive but with adverse consequences in postnatal life. Two systems that could serve as a mechanism for programming are the hippocampal-hypothalamo-pituitary-adrenal axis (HHPA) and the renin-angiotensin system (RAS).

Low birth weight adults showed greater rates of urinary glucocorticoid excretion (4) , elevated basal plasma cortisol concentrations (5) , and greater adrenocortical responsiveness to adrenocorticotropic hormone (ACTH) (6 , 7) . These studies proposed that the link between the size at birth and altered HHPA axis function in later life might be a mechanism whereby programming of adult cardiovascular and metabolic diseases occur. Low birth weight infants have small kidneys, elevated cord blood renin and angiotensin II concentrations, and elevated renin gene expression in the kidney (8 9 10) .

Animal studies to test the ‘programming’ hypothesis have imposed perturbations such as moderate to severe maternal undernutrition, restriction in specific dietary components (iron, protein), or restricting normal placental growth either throughout pregnancy or during parts of gestation and confirmed that restriction of fetal growth leads to elevated blood pressure in the progeny of rats (11 , 12) . Other models to study the programming hypothesis used prenatal glucocorticoid exposure, such as elevating endogenous levels of glucocorticoids by using carbenoxolone (11ß-HSD inhibitor, which blocks placental inactivation of endogenous glucocorticoids) (13) . Adult rats exposed to a large dose of carbenoxolone throughout gestation were of low birth weight and had high blood pressure, increased basal corticosterone levels, increased corticotropin-releasing hormone levels, and reduced GR mRNA in the hypothalamic paraventricular nucleus (14) . Other studies to test the programming hypothesis by naturally occurring glucocorticoids used either prenatal exposure to stress or infusions of corticosterone or adrenocorticotrophin (15) . Administration of the synthetic glucocorticoid dexamethasone to rats throughout pregnancy led to increased blood pressure in male and female offspring (16) . If dexamethasone was administered only during the last week of pregnancy, plasma corticosterone levels were elevated in 16-wk-old offspring, whereas mineralocorticoid (MR) and glucocorticoid (GR) mRNA levels in the hippocampus were decreased (17) . Prenatal glucocorticoid exposure also had marked effects on postnatal behavior (18) ; in particular, late gestation exposure to dexamethasone impaired coping and learning in aversive situations, which was attributed to altered hippocampal corticosteroid receptor levels.

We were the first to show that exposure of pregnant ewes to high levels of dexamethasone for only 2 days very early in gestation (at a mean age of 27 days of the 150 day gestation period) results in hypertensive female offspring at 3–4 months of age (19) . This hypertension amplifies with age and is associated with increased cardiac output, left ventricular hypertrophy with reduced cardiac functional reserve, and increased insulin sensitivity of the inhibition of lipolysis (20 21 22) . Recently this finding of increased blood pressure in the prenatally treated offspring has been confirmed in a second cohort of animals in both females (23) and males (unpublished data). In late gestation, fetuses of ewes treated with dexamethasone in early pregnancy showed significant changes in gene expression in the brain for components of the RAS but no changes in MR and GR mRNAs in the hippocampus (23) .

The hypotheses tested in the current study were twofold: 1) that prenatal treatment with naturally occurring glucocorticoid cortisol administered to the ewe for only 2 days at a mean age of 27 days of gestation at high but still physiological levels would result in high blood pressure in male and female offspring at 1.5 years of age; 2) that this cortisol treatment would have effects on gene expression in the brain similar to dexamethasone treatment. The aim was to show that physiologically relevant concentrations of natural glucocorticoid could reproduce the programming effects of dexamethasone in this very early ‘window’ of time.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Animals
All experiments were approved by the Animal Ethics Committee of the Howard Florey Institute in accordance with National Health and Medical Research Council guidelines. Pregnant merino ewes (n=55) weighing 45–55 kg were used in this study. On day 22–23 of gestation, a silastic cannula (inner diameter 0.76 mm, outer diameter 1.65 mm) was inserted into a maternal jugular vein under local anesthesia (0.5 mL of 0.5% Xylocaine, Astra). Ewes were then infused with isotonic saline (0.19 mL/h, n=28) or cortisol (5 mg/h, n=27) for 48 h. In the cortisol-infused ewes carrying singleton fetuses (first cohort, see below), plasma cortisol concentrations increased from 51 ± 11 to 390 ± 23 nmol/L. Plasma cortisol was measured in the maternal plasma using a radioimmunoassay, which has been described in detail and validated (24) . Intra-assay and interassay coefficients of variation were 10.3 and 13%, respectively.

A first cohort of ewes carrying single fetuses was allowed to lamb. In these animals blood pressure was measured at ~1.5 years of age. There were 17 females (saline n=8 and cortisol n=9) and 16 males (saline n=8 and cortisol n=8). Female animals were oophorectomized and carotid artery loops constructed at 1 year of age (19) . In females, tail docking was performed at 2 months of age. In males, castration and tail docking was performed at 2 months of age.

A second cohort of ewes (n=5 saline, n=5 cortisol) was maintained until fetuses were at 130 days of gestation, at which time they were killed (100 mg/kg pentobarbitone, Lethabarb, Arnolds, Reading) and fetal organs (kidney, heart, lung, brain, adrenal) weighed and collected. All ewes killed at this stage carried twin fetuses. Thus, there were tissues from 10 fetuses in each treatment group at 130 days (saline group: seven female and three male fetuses; cortisol group: six female and four male fetuses). The brain was further dissected and hippocampus, hypothalamus, and medulla oblongata were frozen in liquid nitrogen for later extraction of mRNA. Hippocampus was taken and processed with paraffin for gene localization studies.

A third cohort of ewes, all carrying twins, was allowed to lamb and suckled their lambs until 2 months of age. From this cohort, brain tissues were collected from the saline (n=7; 4 females and 3 males) and cortisol (n=5; 3 females and 2 males) -exposed animals. One lamb in each group died between 1 and 2 months of age.

Placental samples collected from saline-treated ewes (n=3) at the end of the treatment (28 days of gestation) from an earlier study (25) were extracted to assess the presence of mRNA for 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). One whole fetus was sectioned and stained in hematoxylin and eosin (H&E) to illustrate the stage of brain development at the time of treatment (28 days of gestation) (Fig. 1 ).



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  		Figure 1. A sagittal section of the ovine embryo at 28 days of gestation. Original magnification: x40.

Blood pressure and heart rate measurement protocol
In each group of sheep, mean arterial pressure (MAP) and heart rate (HR) were measured at ~1.5 years of age every 10 min for 3 days, as described (19) . MAP and HR were measured via a Tygon cannula (1.0 mm i.d, 1.5 mm o.d) inserted 10 cm proximally into a carotid artery loop and connected to a pressure transducer (TD XIII, Cobe). The pressure was corrected to compensate for the height of transducer above the level of the heart (19) . The analog signal was digitally converted via a DT 301 Board Data Translation device (Marlboro, MA) and blood pressure and heart rate data were collected at 100 Hz (HEM 3.1; Notocord, Kent Scientific Corp, Litchfield, CT). Heart rate was calculated by software from dP/dt Max and took into account the pick of the dP/dt curve during systole vs. time.

Preparation of RNA
Total RNA was extracted from brain tissues (hippocampus, hypothalamus, and medulla oblongata) using the phenol-chloroform method (26) . As pure RNA is required for real-time PCR, samples were DNase treated to eliminate any residual genomic DNA. To 75 µL of RNA in water, the following were added: 10 µL 0.1M DTT, 10 µL 50 mM Mg Cl2, 3.3 µL 3M NaOAc, 0.5 µL RNase inhibitor (40 U/µL), and 1 µL DNase I (RNase free, 1 U/µl). The mixture was incubated at 37°C for 15–20 min, then purified using a phenol extraction (twice) and chloroform extraction. Samples were stored at -80°C until use.

Before use in real-time PCR, 1 µg of each sample was reverse transcribed in a 10 µL reaction containing 1x TaqMan® RT buffer, 5.5 mM MgCl2, 500 µM each 2'-deoxynucleoside 5'-triphosphate, 2.5 µM random hexamers, 0.4 U/µl RNase inhibitor, and 1.25 U/µl MultiScribeTM reverse transcriptase (PE Applied Biosystems, Foster City, CA). To ensure there was no contaminating genomic DNA, control reactions without reverse transcriptase were included in a separate reverse transcription reaction with all total RNA samples. Reverse transcription was performed in a GeneAmp PCR System 9600 (PE Applied Biosystems) at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min. Upon completion, all samples were stored at -80°C until use.

Real-time PCR
A comparative CT (cycle of threshold fluorescence) method was used to determine relative mRNA expression levels in the hippocampus, hypothalamus, and medulla oblongata of MR and GR receptor, angiotensin II type 1 and type 2 receptors (AT1 and AT2), angiotensinogen (A’ogen), 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) along with an endogenous reference gene, 18S ribosomal RNA, at 130 days of gestation. The expression of MR, GR, AT1, and A’ogen was studied in hippocampus from lambs killed at 2 months of age. 11ß-HSD2 was studied in the placenta of saline-treated ewes. This method has been described elsewhere (25 , 27) . All primers and TaqMan® probes were designed using Primer ExpressTM Version 1.0 (PE Biosystems). The primer and TaqMan® probe sequences for all genes are shown in Table 1 . The TaqMan® probe and primers for 18S were supplied by PE Applied Biosystems in a control reagents kit. PCR reactions were carried out in 25 µL volumes consisting of 1x TaqMan® Universal PCR Master Mix (including passive reference), 50 nM TaqMan® 18S probe, 20 nM 18S forward primer, 80 nM 18S reverse primer, and the appropriate concentration of primers and TaqMan® probe for the MR, GR, AT1, AT2, A’ogen, and 11ß-HSD2. These concentrations had been determined in preliminary experiments and are shown in Table 1 . Additional preliminary experiments had shown that there was no difference in CT values when we compared any of these genes in a nonmultiplex reaction to a multiplex reaction (containing 18S). The amplification efficiency of these genes was equal to that of 18S over a range of template concentrations (50 ng to 0.5 pg). cDNA (50 ng) and no reverse transcriptase preparations were amplified at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.


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  		Table 1. Primer and probe concentrations and sequences used in real time PCR a

Calculations for real-time analysis
The {Delta}CT value (obtained by subtracting the CT value for 18S from the CT value of the gene of interest) of the calibrator was subtracted from the {Delta}CT value of each sample to give a {Delta}{Delta}CT value. The equation of 2-{Delta}{Delta}CT was used to obtain a final value for each sample relative to the calibrator. Coefficients of variation for one sample run five times in one assay were 23%, 15%, 40%, 18%, and 23%, respectively, for MR, GR, AT1, A’ogen, and 11ß-HSD2. We have used the mean {Delta}CT value of the saline group for any particular gene as the calibrator. Five aliquots of adult kidney cDNA were also run in each assay to determine the relative fetal/adult levels of expression of each gene.

Hybridization histochemistry
A plasmid containing 574 bp of partial ovine MR cDNA sequence was kindly donated by Dr. Anthony Albiston. After the recombinant plasmid was linearized, antisense (T3 Promotor) and sense (T7 negative control) riboprobes were prepared by in vitro transcription using the Promega riboprobe kit (Promega, Madison, WI), where [{alpha}-35S] UTP (100CI mmol-1) was incorporated (Bresatec, Thebarton, Australia). The riboprobes were hydrolyzed, precipitated, and resuspended in 10 mM DTT before hybridization histochemistry. A plasmid containing a 900 bp partial ovine GR cDNA sequence was kindly donated by Dr. G. L. Hammond (28) . The same procedure as above was carried out for preparation of the riboprobe for hybridization histochemistry.

The riboprobe was used at a final concentration of 0.02 ng/µl in hybridization buffer consisting of 50% deionized formamide, 20% dextran sulfate, 10% 10x salts (3M Na Cl, 100 mM Na2HPO4, pH 6.8, 100 mM Tris-HCl pH 7.5, 50 mM EDTA pH 8.0, 0.2% BSA, 0.2% Ficoll 400, 0.2% polyvinyl pyrrolidone), 3.5% tRNA (20 mg/mL), and 1% DTT. All slides were hybridized in duplicate and sense probes were used as negative controls. In brief, 5 µm paraffin sections were cut and mounted on 2% silanized slides, dried overnight at 37°C, dewaxed, and rehydrated. Sections were prehybridized with Pronase E (125 µg/mL, Sigma, St. Louis, MO) at 37°C for 10 min, postfixed in 4% paraformaldehyde for 10 min, dehydrated, and air dried. Sections were treated with 80 µL of riboprobe, covered with a coverslip, and left overnight at 55°C in a humidified chamber. RNase A digestion (150 µg/mL, Sigma-Aldrich) was performed the next day for 2 h at 37°C. Slides were dehydrated, air dried and placed on a Fuji Phosphor Imaging plate (BASIII) overnight before scanning on a Fujix BAS 2000 scanner to determine possible sites of labeling. Autoradiography was achieved by dipping slides in liquid emulsion (Ilford, Essex, UK) and leaving the slides at room temperature for 10 days. Slides were developed for 2 min in filtered Kodak D19 developer before fixing in Ilford Hypam fixer (1:5 dilution) and staining in H&E.

Statistical analysis
The analysis of differences in gene expression and blood pressure of the two treatment groups was made by an unpaired t test. Where data were not normally distributed, the Mann-Whitney test was used and data presented as median, 25% and 75%; otherwise data are presented as mean ± SE. Statistical analysis was performed using Sigmastat software package. Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
A morphological appearance of the ovine fetus at 28 days of gestation
As shown in Fig. 1 , at the time of treatment (26–28 days of gestation) the ovine embryo has only the transient form of the kidney (mesonephros). The neural tube is closed; by 28 days of gestation the telencephalic vesicle from which the hippocampus will form, is present (Fig. 1) . The metencephalon, which gives rise to the cerebellum and pons, together with myelencephalon, which gives rise to the medulla oblongata, have formed from the rhombencephalon vesicle (Fig. 1) .

Effects of prenatal cortisol treatment on blood pressure in adult female and male lambs
Prenatal cortisol treatment had no effect on growth patterns in female and male group of animals. The birth weights were similar in the two groups of animals (males: 4.5±0.2 kg in saline group vs. 4.8±0.2 kg in cortisol group; females: 4.6±0.1 kg in saline group vs. 4.3±0.2 kg in cortisol group).

Blood pressure was examined in female lambs at 16 ± 1 (saline; n=8) and 18 ± 1 (cortisol group; n=9) months of age. Body weights were similar between the two groups of animals (38±1 kg in saline group vs. 37±1 in cortisol group). As shown in Fig. 2 , basal mean arterial pressure was significantly higher in the female offspring of sheep treated with prenatal cortisol than in females in the control group (89±2 mmHg vs. 81±2; P<0.05).



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  		Figure 2. The mean arterial pressure (MAP) and heart rate (HR) in 1.5-year-old female and male sheep treated at 26–28 days of gestation with either saline (S; open bars) or cortisol (F; 5 mg/h; shaded bars). The results are shown as mean ± SE. *Saline vs. cortisol P < 0.05; #male vs. female P < 0.01.

In males, blood pressure was examined when lambs were 17 ± 1 (saline; n=8) and 18 ± 1 (cortisol group; n=8) months old. Body weights were similar between the two groups of animals (39±1 kg in saline group vs. 40±1 in cortisol group). Basal mean arterial pressure was significantly higher in the offspring of sheep treated with prenatal cortisol than in the control group (102±4 mmHg vs. 91±3; P<0.05) (Fig. 2) . In female and male lambs, heart rate was similar between the two groups of animals (Fig. 2) . The basal mean arterial pressure in males from the control group was higher than basal mean arterial pressure in the respective group of females (91±3 mmHg vs. 81±2; P<0.01).

Effects of cortisol treatment at 130 days of gestation and 2 months of age
Body and placental weights and crown rump lengths measured at 130 days of gestation and in animals 2 months of age were not different between the two treatment groups. The twin fetuses of ewes treated with saline weighed 2.8 ± 0.1 kg, whereas those from ewes treated with cortisol were 3.1 ± 0.1 kg. As shown in Table 2 , there was no difference in organ weights between the saline and cortisol fetuses. Volumes and composition of fetal fluids (amniotic and allantoic) were similar in all treatment groups. The composition of fetal urine and plasma (sodium, potassium, chloride, osmolality, urea, creatinine) was similar in the two treatment groups (data not shown).


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  		Table 2. The weights of the organs of twin fetuses at 130 days of gestation after either saline or cortisol treatment at 26–28 days of gestation

When adult sheep kidney was used as the calibrator, it appeared that A’ogen mRNA expression in the hippocampus and hypothalamus was present at ~24 (or 20-fold) hematoxylin and eosin higher than the amount present in adult kidney. In the medulla oblongata the level of A’ogen mRNA expression is closer to that of the adult kidney (i.e., ~1). However, the AT1 mRNA expression level in all the brain areas studied is ~20-fold lower (i.e., 1/20th) that present in the adult kidney.

Figure 3 shows the relative expression levels of A’ogen, AT1, MR, and GR in medulla oblongata, hypothalamus, and hippocampus in saline and cortisol-treated fetuses at 130 days of gestation. Relative to the saline-treated animals, cortisol treatment did not cause any significant alteration in gene expression of A’ogen, AT1, MR, and GR in medulla oblongata and hypothalamus in fetuses at 130 days of gestation. However, all four genes were up-regulated in hippocampus: A’ogen from the median of 1.0 in the saline group to the median of 2.1 in the cortisol group (P<0.01), AT1 from the median of 1.1 in the saline group to the median of 11.4 in the cortisol group (P<0.001), MR from 1.6 ± 0.5 in the saline group to 4.3 ± 0.6 in the cortisol group (P<0.01), and GR from 1.1 ± 0.2 in the saline group to 4.9 ± 0.7 in the cortisol group (P<0.001). As shown in Table 3 , these changes in hippocampal gene expression were not seen in animals exposed to cortisol for 48 h at 26–28 days of gestation and killed at 2 months of age. AT2 was not detected in medulla oblongata, hypothalamus, and hippocampus. Although 11ß-HSD2 mRNA was not detected in any brain region studied in three placentas at 28 days, the levels were 39%, 42%, and 100% of that in the adult kidney.



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  		Figure 3. The effect of cortisol treatment on gene expression in the brain of twin fetuses at 130 days of gestation. The ratio of angiotensinogen (A’ogen), angiotensin II type 1 receptor (AT1), mineralocorticoid (MR), and glucocorticoid receptors (GR) in medulla oblongata, hypothalamus, and hippocampus of twin fetuses at 130 days of gestation treated with cortisol (5 mg/h for 48 h between 26 and 28 days of gestation; shaded bars; n=10) compared to saline control (open bars; n=10). Neither AT2 nor 11ß-HSD2 mRNA was detected in medulla oblongata, hypothalamus, and hippocampus. As a positive control, AT2 or 11ß-HSD2 mRNA were detected in fetal kidney (not shown). If the data were not equally distributed, results are shown as median (25%; 75% shown by square dots), otherwise results are presented as mean ± SE. **P < 0.01; ***P < 0.001.


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  		Table 3. The effect of cortisol treatment on gene expression in the hippocampus in lambs at 2 months of age

Figure 4 shows the hybridization histochemistry of hippocampus (CA3 and CA1 regions) for GR and CA3 for MR in 130 day ovine fetuses. The CA3 region of the saline-treated, 130 days of gestation ovine fetus (Fig. 4A, B ) shows weak labeling with the GR probe. The same region in the cortisol-treated, 130 days of gestation ovine fetus (Fig. 4C, D ) shows much stronger labeling. The inserts in Fig. 4A, C show no detectible specific labeling with the sense probe. The CA1 region of the cortisol-treated, 130 days of gestation ovine fetus (Fig. 4E, F ) shows weaker labeling compared with the CA3 region in the same fetus. The MR riboprobe shows stronger labeling in the CA3 region of the cortisol-treated, 130 days of gestation fetus (Fig. 4H ) than in the saline-treated, 130 days of gestation ovine fetus (Fig. 4G ). The expression of MR and GR was highest in the dentate gyrus > CA3 > CA1 (not shown).



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  		Figure 4. Bright- and dark-field photomicrographs of different regions of 130 days fetal hippocampus hybridized with the GR (A–F) (magnification x40) and MR (G, H) riboprobes (magnification x100). The CA3 region of the saline-treated, 130 days of gestation ovine fetus (A, B) shows weak labeling with the GR probe. The same region in the cortisol-treated, 130 days of gestation ovine fetus (C, D) shows much stronger labeling. The inserts (A, C) show no detectible specific labeling with the sense probe. The CA1 region of the cortisol-treated, 130 days of gestation ovine fetus (E, F) shows weaker labeling than the CA3 region in the same fetus. The MR riboprobe shows stronger labeling in the CA3 region of the cortisol-treated, 130 days of gestation fetus (H) vs. the saline-treated, 130 days of gestation ovine fetus (G).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
The most important finding of this study was that cortisol, administered to the ewe for only 48 h, at the end of the first month of gestation led to high blood pressure in the male and female offspring of sheep at 1.5 years of age. The significance of this finding lies in the fact that increased blood pressure was programmed with high but still physiological levels of the naturally occurring glucocorticoid cortisol. The cortisol treatment used in this study increased maternal plasma cortisol concentration to levels similar to those shown previously (25) . Also, this treatment resulted in a significant increase in maternal glucose concentrations and suppressed plasma ACTH concentrations to undetectable levels (all P<0.05).

Timing of the programming event
We were the first to show that exposure of pregnant ewes to pharmacological levels of dexamethasone for only 2 days very early in gestation (at a mean age of 27 days of the 150 day gestation period) results in hypertensive female offspring at 3–4 months of age (19) . Subsequently, we demonstrated that this type of hypertension amplifies with age and is associated with increased cardiac output, left ventricular hypertrophy with reduced cardiac functional reserve, and increased insulin sensitivity of the inhibition of lipolysis (20 21 22) . Taken together, it seems that in sheep there is a critical stage of development, a window, at the end of the first month of gestation when short exposure to either synthetic or natural glucocorticoid for only 48 h can program high blood pressure in offspring. There is some evidence that disturbance of the intrauterine environment, particularly early in pregnancy, can have profound effects on the health of the adult. Exposure to malnutrition during the Dutch famine, especially during the first gestational trimester, led to a greater occurrence of coronary heart disease, an atherogenic lipid profile, and obesity in 50-year-old adults (29 30 31) . An early origin for programming of cardiovascular disease was also documented in rats (32) . Undernutrition of the pregnant rat confined to the preimplantation period (0–4.5 days) produced hypertension, but only in male offspring (32) .

Other evidence for programming effects of natural steroids on blood pressure
Regardless of the experimental perturbation applied to the mother causing long-term programming of the fetus, there is a good evidence that one common factor that may mediate the effect is exposure of the fetus to excess glucocorticoids (33) . Adult rats exposed to a large dose of carbenoxolone (11ß-HSD inhibitor, which blocks placental inactivation of endogenous glucocorticoids) throughout gestation were of low birth weight and had high blood pressure, increased basal corticosterone levels, increased corticotropin-releasing hormone levels, and reduced GR mRNA in the hypothalamic paraventricular nucleus (14) . However, these effects were not reproduced when smaller doses of carbenoxolone (still sufficient to elevate maternal blood pressure by ~20 mmHg) were administered in rats throughout gestation (34) . It is known that the mechanism by which carbenoxolone, particularly when administered at high doses, increases blood pressure is more complex than simple inhibition of 11ß-HSD (35 36 37) . These studies were based on a proposal that placental 11ß-HSD2 serves as a mechanism that protects the developing fetus from the excess of maternal glucocorticoids (38) . In this study, we found that even though placental 11ß-HSD2 was present at the levels 40%–100% of adult kidney, it was not sufficient to block the long-term effects after high but still physiological levels of maternal cortisol. The absence of 11ß-HSD2 from the brain of the late gestation fetus supports the findings of others (39) . Undernutrition during early gestation in sheep (15% reduction in maternal food intake) led to a reduced pituitary and adrenal responsiveness in late gestation (40) , but switched to enhanced responsiveness postnatally (41) . In these animals, exaggerated responsiveness of the HPA axis was associated with higher blood pressure (41) . However, in our study the effect of cortisol appeared to be time dependent since no change in MR or GR expression levels was seen in 2-month-old lambs. We have recently reported that a brief exposure to dexamethasone at 26–28 days of gestation resulted in hypertensive offspring, which were allowed to survive for 7 years but showed no change in MR or GR mRNA in the hippocampus or hypothalamus (42) . Taken together, these findings suggest that in sheep the HPA may not be the mechanism whereby prenatal exposure to dexamethasone programmed hypertension in adult offspring. More studies are required to prove conclusively that such is the case in the adult hypertension programmed by cortisol.

Programming effects of natural steroids on behavior
Only a few studies have demonstrated programming of the HPA in rat offspring after prenatal infusions of either corticosterone or adrenocorticotrophin (15) . However, none of these studies report on the effect of such prenatal stress/steroid exposure on adult blood pressure. Offspring exposed in utero to repeated loud unanticipated noise (such as experienced by people living under flight paths of busy airports) or living in a country preparing and ultimately going to war (Arab-Israeli war) showed growth retardation and delays in attaining motor, verbal, and social skills (43) . Other studies to test the programming hypothesis by naturally occurring glucocorticoids studied the effects of prenatal exposure of adult rats to either restraint stress or uncontrollable electric shocks mainly on HPA axis and behavioral changes of the offspring (44) . Such prenatally stressed adult rats showed elevated plasma ACTH and corticosterone levels and lower MR and GR densities in the hippocampus (45 , 46) . Adult rats exposed to carbenoxolone throughout gestation had increased basal corticosterone levels, increased corticotropin-releasing hormone levels and reduced GR mRNA in the hypothalamic paraventricular nucleus (14) . Dexamethasone administration only during the last week of pregnancy led to elevated plasma corticosterone levels as well as decreasing significantly the MR and GR mRNA in the hippocampus in 16-wk-old rat (17) . In this study, we showed that brief cortisol exposure at the end of the first month of gestation resulted in high gene expression for MR and GR in hippocampus but not in the hypothalamus or medulla oblongata of the late gestational ovine fetus (~4 months after the treatment ceased). An increase in glucocorticoid sensitivity to negative feedback and a subsequently decreased stress reactivity in adulthood are seen in rats handled neonatally (18 , 47) . Such an effect has been linked to increased GR and MR in the hippocampus, as well as to increased GABAA and benzodiazepine receptor levels in the locus ceruleus and the nucleus tractus solitarius of the medulla oblongata. If decreased levels of GR and MR are associated with impaired coping and learning (18) , one might propose that the cortisol-pretreated sheep would have better skills. This remains to be tested. However, the transient increase in MR in the hippocampus may have had a significant effect on the development of the hippocampus, since genetic disruption of the MR but not GR in mice results in hippocampal granule cell degeneration (48) . At the time of treatment (26–28 days) in the current experiments, the central nervous system of the fetus is relatively underdeveloped, as illustrated in Fig. 1 . The neural tube closes over in the period from 23 to 26 days, in the sheep, which is comparable to E9.5-E10 in the mouse, and 27–29 days in the human fetus (49) . By 28 days, the primordial plexiform layer contains two cells of different sizes and occupies about one-third of the wall of the telencephalon (50) . In the posterior lateral wall, the subventricular zone is beginning to differentiate but the medial wall remains two-layered. Thus, the hippocampal cells that show the changed gene expression in late gestation are not yet present as such.

The RAS and programming
Another system shown to be programmed by prenatal undernutrition and potentially glucocorticoid exposure is the renin-angiotensin system (51) . Studies in the rat model have shown that early administration of ACE inhibitor captopril 2–4 wk postpartum prevents the development of hypertension, programmed by intrauterine exposure to a maternal low-protein diet (52) , suggesting these effects may have been mediated by local RASs. The renal AT1 receptor expression has been shown to be increased, at term, in the offspring of sheep after exposure to undernutrition from day 28 to day 77 of gestation (51) . There is no information in the literature concerning the effects that various maternal perturbations might have on the expression of the components of the RAS in the brain. We found that brief cortisol exposure at the end of the first month of gestation resulted in high gene expression for AT1 and A’ogen in hippocampus, but not in hypothalamus or medulla oblongata of the late gestational ovine fetus (~4 months after the treatment ceased). As it is widely known in the literature, Ang II exerts a variety of actions on the brain including central control of BP, modulation of drinking behavior, salt appetite and sensory functions, effects on memory and learning, and stimulation of pituitary hormone release (53) . It is unlikely that high gene expression for AT1 and A’ogen in hippocampus after brief prenatal exposure to cortisol is related to high blood pressure seen in these animals at 1.5 years of age. It is possible the development of the hippocampus may have been accelerated; when A’ogen is not present in KO mice, there is a decreased density in the granular cell layer (54) . In the adult rat, expression of AT1 binding is relatively low in the hippocampus compared to immature rat brain (55) , suggesting a role of Ang II in the maturation of hippocampal formation (54) . In the rat, pharmacological and anatomical evidence suggests that Ang II plays a role in hippocampal function, memory, and recognition (56 , 57) . Ang II in the hippocampus is also speculated to play a major role in long-term potentiation. Studies have shown that Ang II injection above the hippocampus inhibits long-term potentiation in dentate granule cells in rats (56 , 58) , which would affect learning and memory. For MR and GR, this effect of cortisol appeared to be time dependent since no change in AT1 and A’ogen in hippocampus was seen in 2-month-old lambs. It is possible that even a transient increase in AT1 and A’ogen in hippocampus could result in morphological and functional changes to the hippocampus.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
This is the first evidence in the literature where short exposure to the naturally occurring glucocorticoid cortisol at the end of the first month of gestation in levels seen at times of stress programmed high blood pressure in the adult offspring of sheep. Although the effects on gene expression in the hippocampus are probably not related to programmed high blood pressure, the effects could have a significant effect on the development of the hippocampus and behavior of the offspring.


   ACKNOWLEDGMENTS
 
This work was supported by a Block Grant from NH&MRC (983001) and a grant in aid from BHP. The Applied Biosystems PRISM sequence detector system was purchased with donations from the Philip Bushell Foundation, the Harold and Cora Brennen Benevolent Trust, the Viertel Foundation, and the Ramaciotti Foundation. We thank Jehan Jeyaseelan for help in designing the probes and primers used in real-time PCR.

Received for publication January 2, 2002. Revision received March 27, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 

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