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. 2013 Sep 30;154(12):4650–4662. doi: 10.1210/en.2013-1414

Maternal Dietary Restriction During the Periconceptional Period in Normal-Weight or Obese Ewes Results in Adrenocortical Hypertrophy, an Up-Regulation of the JAK/STAT and Down-Regulation of the IGF1R Signaling Pathways in the Adrenal of the Postnatal Lamb

Song Zhang 1, Janna L Morrison 1, Amreet Gill 1, Leewen Rattanatray 1, Severence M MacLaughlin 1, David Kleemann 1, Simon K Walker 1, I Caroline McMillen 1,✉
PMCID: PMC3836080  PMID: 24108072

Abstract

Maternal dietary restriction during the periconceptional period results in an increase in adrenal growth and in the cortisol stress response in the offspring. The intraadrenal mechanisms that result in the programming of these changes are not clear. Activation of the IGF and the signal transducer and activator of transcription (STAT)/suppressors of cytokine signaling (SOCS) pathways regulate adrenal growth. We have used an embryo transfer model in sheep to investigate the impact of exposure to either dietary restriction in normal or obese mothers or to maternal obesity during the periconceptional period on adrenal growth and function in the offspring. We assessed the adrenal abundance of key signaling molecules in the IGF-I and Janus kinase/STAT/SOCS pathways including IGF-I receptor, IGF-II receptor, Akt, mammalian target of rapamycin, ribosomal protein S6, eukaryotic translation initiation factor 4E-binding protein 1, eukaryotic translation initiation factor 4E, STAT1, STAT3, STAT5, SOCS1, and SOCS3 in female and male postnatal lambs. Maternal dietary restriction in the periconceptional period resulted in the hypertrophy of the adrenocortical cells in the zona fasciculata-reticularis and an up-regulation in STAT1, phospho-STAT1, and phospho-STAT3 (Ser727) abundance and a down-regulation in IGF-I receptor, Akt, and phospho-Akt abundance in the adrenal cortex of the postnatal lamb. These studies highlight that weight loss around the time of conception, independent of the starting maternal body weight, results in the activation of the adrenal Janus kinase/STAT pathway and adrenocortical hypertrophy. Thus, signals of adversity around the time of conception have a long-term impact on the mechanisms that regulate adrenocortical growth.

A range of experimental and clinical studies have demonstrated that exposure of the embryo, fetus, or neonate to adverse environmental stressors such as undernutrition, placental dysfunction, excess glucocorticoids, or poor maternal care increases the risk of poor cardiovascular and metabolic health in later life (1–5). It has also been shown that exposure to adversity in early life results in the programming of an increased stress responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis in the offspring and that this contributes to the poor long-term health outcomes (1–5).

In sheep, exposure to maternal dietary restriction during the periconceptional period and early gestation results in altered development of the fetal adrenal from as early as 55 days' gestation (term, 150 ± 3 d gestation) (6), an earlier prepartum activation of the fetal HPA axis (7, 8), an increased risk of premature delivery (9), and increased plasma cortisol concentrations in the lamb (10, 11).We have also previously reported that exposure to dietary restriction during the periconceptional period in either normal or obese ewes resulted in a larger adrenal in male and female lambs and a greater cortisol response to stress in female lambs at 4 months of age (12). These changes occurred, however, in the absence of an increase in the ACTH response to stress or in the expression of the adrenal melanocortin-2 receptor and key steroidogenic enzymes within the adrenal of the lambs exposed to maternal dietary restriction in the periconceptional period (12). We proposed that the early programming of the increased stress response may therefore be a result of an early programming of an increase in adrenocortical growth. Interestingly, we found that maternal dietary restriction in normal-weight or obese ewes resulted in decreased methylation in the proximal CTCF binding site in the differentially methylated region of the IGF-II (IGF2)/H19 promoter and decreased IGF2 mRNA expression in the lamb adrenals at 4 months of age (12). It is not clear, however, how the changes within the IGF signaling system in the adrenal after exposure of the oocyte and/or early embryo to maternal dietary restriction results in altered adrenal growth and cortisol production in later life.

IGF-I or IGF-II can each bind to the IGF-I receptor (IGF1R), which activates the phosphoinositide-3 kinase-protein kinase B/Akt pathway. This results in the stimulation of downstream signaling molecules including the mammalian target of rapamycin (mTOR), ribosomal protein S6 (RPS6), and eukaryotic translation initiation factor 4E (EIF4E)-binding protein 1 (4EBP1).

Alternatively, ligands such as ILs, interferons, GH, and prolactin (PRL), bind to their receptor and activate the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and stimulate cell proliferation, differentiation, migration, apoptosis, and cell survival. These cellular responses are critical to numerous developmental and homeostatic processes (13, 14). Suppressors of cytokine signaling (SOCS) proteins are in turn induced by STAT activation, and these then act as negative feedback regulators of the JAK/STAT pathway (15).

GH and PRL promote adrenal growth and steroidogenesis, mainly through the JAK/STAT/SOCS pathway (16–19). It has been shown that STAT5 is essential for GH-stimulated target gene expression in the mouse (20, 21). In mutant PRL receptor (PRLR)-deficient mice, PRL triggered STAT5-dependent transcription in adrenal cells in the absence of an impact on corticosterone synthesis or secretion (22). PRL administration also significantly increased adrenal SOCS3 mRNA expression in adult mice, fetal sheep, and fetal adrenocortical cells in culture (23, 24).

We have used a model in which donor ewes were either normally nourished or overnourished prior to a period of dietary restriction, before transfer of the embryo at 6–7 days after conception to a recipient ewe of normal weight. We hypothesized that maternal dietary restriction in either normal-weight or obese ewes during the periconceptional period would result in the hypertrophy of adrenocortical cells, an up-regulation of the adrenal IGF1R signaling pathway, and/or an up-regulation of the abundance of the STAT1/STAT3/STAT5 proteins in the adrenal of the postnatal lamb. We have therefore investigated the impact of periconceptional dietary restriction and/or obesity on the morphology of the adrenal, activation of the IGF1R downstream signaling pathway and the abundance of major STAT and SOCS molecules in the adrenal of the postnatal lamb.

Materials and Methods

Animals and nutritional feeding regimen

All procedures were approved by the University of Adelaide Animal Ethics Committee and the Institute for Medical and Veterinary Science Animals Ethics Committee. Briefly, South Australian Merino ewes were moved into an enclosed shed and housed in individual pens 2 weeks before the start of the feeding regimen. All ewes were weighed and body condition scores were assessed employing a 1.0–5.0 scale with 0.5 intervals by an experienced assessor (25, 26). Using this scale, a body condition score of 1 represents an extremely emaciated animal and a body condition score of 5 represents a morbidly obese animal. During this 2-week period, ewes were acclimatized to a diet containing cereal hay, lucerne hay, barley, oats, almond shells, lupins, oat bran, lime, and molasses (Johnsons & Sons Pty Ltd). The pellets provided 9.5 MJ/kg metabolizable energy and 120 g/kg crude protein and contained 90.6% dry matter. All ewes received 100% of nutritional requirements as defined by the Agricultural and Food Research Council (27).

Donor ewes

At the end of this acclimatization period, donor ewes (n = 23) of normal body condition were randomly assigned to one of four nutritional treatment groups: control-control (CC), control restricted (CR), high-high (HH), or high restricted (HR) (12, 28).

The CC ewes (n = 6) were a control group that were maintained at 100% metabolizable energy requirement (MER) for 5 months before and 1 week after conception; CR ewes (n = 6) were maintained at 100% MER for the first 4 months and then were placed on an energy restricted diet of 70% MER for 1 month before and 1 week after conception; HH ewes (n = 6) were fed an ad libitum diet (170%–190% MER) for 5 months before and 1 week after conception; and HR ewes (n = 5) were fed an ad libitum diet (170%–190% MER) for 4 months and then were placed on an energy-restricted diet of 70% MER for 1 month before and 1 week after conception.

There was no significant difference in weights of nonpregnant donor ewes in the CC, CR, HH, and HR treatment groups before the start of the nutritional regimen in prior work (12, 28). We have shown in prior studies, however, that the donor ewes in the HH and HR groups were significantly heavier at conception and at embryo transfer than ewes in the CC and CR groups (12, 28). Plasma insulin but not glucose concentrations were significantly higher in the HR donor ewes when compared with the CC and CR donors but were not different from those in the HH ewes (28).

Superovulation

The reproductive cycle of all experimental ewes was synchronized and superovulation was induced (29) using an intravaginal progestogen pessary (45 mg of flugestone acetate; Intervet) for 12 days, followed by the administration of FSH (follitropin, 160 mg HIH-FSH-P1 standard; Bioniche Animal Health Inc). Pregnant mare serum gonadotropin (Pregnecol, 500 IU; Bioniche Animal Health Inc) was administered at the time of the first FSH treatment. Synthetic GnRH (Fretagyl, 30 μg; Intervet) was administered to the donor ewes 27 hours after pessary removal.

Artificial insemination and embryo collection

Fresh semen was collected from a ram (29) and donor ewes were inseminated by laparoscopy with approximately 2 × 107 spermatozoa 36 hours after the pessary withdrawal. Embryos were collected by laparoscopy via flushing with saline (Baxter) 6–7 days after artificial insemination. Embryos were recovered within 5 minutes of collection, washed three times, and then held at 38.5°C in HEPES-buffered synthetic oviduct fluid supplemented with BSA and amino acids at oviduct fluid concentrations.

Recipient ewes

Donor embryos of good quality were recovered and transferred to synchronized recipient ewes. These ewes were maintained on a control diet (100% MER). As reported previously, there was no difference in the weights of the recipient ewes allocated to carry the CC, CR, HH, or HR embryos (12, 28). Each recipient ewe received only one embryo, which resulted in four treatment groups, ie, CC (n = 13); CR (n = 16); HH (n = 17); and HR (n = 16). These ewes were fed a control diet for the remainder of pregnancy, which provided 100% MER for the maintenance of a pregnant ewe bearing a singleton fetus.

Pregnancy was confirmed by ultrasound at 49 days' gestation [CC (n = 9); CR (n = 12); HH (n = 13); HR (n = 13)]. There was no difference in the pregnancy rates between the nutritional treatment groups (CC, 69%; CR, 75%; HH, 76%; HR, 81%). Lambs (CC: females, n = 5, males, n = 2; CR: females, n = 3, males, n = 7; HH: females, n = 5, males, n = 7; HR: females, n = 7, males, n = 5) were delivered naturally (term = 150 ± 3 d), and birth weights were taken within the first 12 hours of birth. There was no effect of nutritional treatment during the periconceptional period on either the birth weight or body weight of the lambs. As reported previously, male lambs were, however, heavier at birth and at 4 months compared with female lambs (12, 28).

Postmortem and tissue collection

At 4 months of age, lambs were killed with a lethal overdose (∼30 mg/kg) of sodium pentobarbitone (Virbac Pty Ltd). Adrenals were collected and weighed. The relative adrenal weight was calculated as a ratio of adrenal weight to lamb body weight. Half of the right adrenal was fixed in 4% paraformaldehyde and embedded in paraffin wax, and the remainder was snap frozen in liquid nitrogen and subsequently stored at −80°C.

Quantification of mRNA expression using quantitative real-time RT-PCR

Total RNA was extracted from adrenal samples (CC: females, n = 5, males, n = 2; CR: females, n = 3, males, n = 7; HH: females, n = 5, males, n = 7; HR: females, n = 7, males, n = 5) using the Trizol reagent (Invitrogen by Life Technologies) and purified using the RNeasy minikit (QIAGEN) (12). cDNA was synthesized by reverse transcription using Superscript III (Invitrogen). Negative controls containing no RNA or Superscript III were used to test for DNA contamination.

The relative expression of mRNA transcripts was measured by quantitative real-time PCR using the ViiA 7 real-time PCR system (Applied Biosystems by Life Technologies). Each amplicon was sequenced to ensure the authenticity of the DNA product and melt curve analysis performed to demonstrate amplicon homogeneity. A PCR consisted of 3 μL of Fast SYBR Green master mix (Applied Biosystems), 0.6 μL each of forward and reverse primers (GeneWorks), 1.2 μL of molecular-grade H2O and 0.6 μL of cDNA (50 ng/μL) (Supplemental Table 1, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org) (23, 30, 31). Three replicates of cDNA were performed for each gene, and controls with no cDNA were included on each plate. Amplification efficiencies were determined from the slope of a plot of cycle threshold (Ct; defined as the Ct with the lowest significant increase in fluorescence) against the log of a series of diluted cDNA concentrations (ranging from 1 to 100 ng/μL). The abundance of each transcript relative to the abundance of the three reference genes, peptidylprolyl isomerase A (PPIA), phosphoglycerate kinase 1 (PGK1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was calculated using DataAssist 3.0 analysis software (Applied Biosystems).

Morphometric analysis

Immunohistochemical localization of 3β-hydroxysteroid dehydrogenase (3βHSD) and morphometric determinations

To determine the proportion of the adrenal gland comprised of the adrenal cortex, sections of the fixed and paraffin-embedded adrenal tissues from the midglandular region of the right adrenal were cut (7 μm) and stained with rabbit antisera raised against human recombinant type II 3βHSD (32) using immunohistochemistry (CC: females, n = 3, males, n = 2; CR: females, n = 3, males, n = 6; HH: females, n = 4, males, n = 6; HR: females, n = 5, males, n = 5). The 3βHSD was localized using a Zymed histostain-plus kit (Invitrogen) with 3,3′ diaminobenzidine substrate (Thermo Fisher Scientific) as the chromogen. Immunohistochemical localization of 3βHSD was used to identify the interface of the zona glomerulosa and zona fasciculata-reticularis and medulla.

The photomicrographic images were captured on an Olympus BX53 research microscope (Olympus) using a DP72 digital camera (Olympus) connected to VisioPharm NewCast software (VisioPharm). The Nucleator program was used to measure the areas of total adrenal, adrenal cortex, adrenal medulla, zona glomerulosa, and zona fasciculata-reticularis of the adrenal cortex. The total adrenal area was determined from the edge of the adrenal capsule to the middle of the adrenal vein. The areas of the adrenal cortex, zona glomerulosa, zona fasciculata-reticularis, and adrenal medulla were also separately determined.

Determination of the density of cell nuclei in adrenal

Determination of the density of cell nuclei in adrenal was performed on midglandular sections (7 μm) of the lamb right adrenal (CC: females, n = 5, males, n = 2; CR: females, n = 3, males, n = 6; HH: females, n = 4, males, n = 5; HR: females, n = 4, males, n = 5). The density of cell nuclei in the adrenal cortex (zona glomerulosa and zona fasciculata-reticularis) and medulla was measured in a defined area at ×40 magnification on an Olympus VANOX-AHT microscope (Olympus) using a Colorview I camera (Olympus) with AnalySIS image analysis software (Soft Imaging Systems). The total number of cell nuclei was counted in a defined area (2215–4430 μm2) in 10 random fields of view at least 1 mm apart in the adrenal cortex (zona glomerulosa and zona fasciculata-reticularis) and medulla. For each animal, the number of cell nuclei per square micrometer was calculated by the total number of nuclei in 10 defined areas divided by the total area (33).

Quantification of protein abundance by Western blotting

The abundance of proteins was determined using Western blotting as described in detail elsewhere (34). Briefly, adrenal cortex samples (50 mg) (CC: females, n = 4, males, n = 2; CR: females, n = 3, males, n = 3; HH: females, n = 3, males, n = 3; HR: females, n = 3, males, n = 3) were sonicated in extraction buffer and centrifuged at 12 400 rpm at 4°C for 14 minutes to remove lipid and insoluble material. Protein content of the extracts was determined as previously described (35). Equal amount of protein (20 μg) was subjected to SDS-PAGE. The proteins were transferred to polyvinylidene diflouride membrane (Merck Millipore), blocked, and then incubated with primary antisera raised against the following: IGF1R, total Akt, Akt1, Akt2, phospho-Akt, mTOR, phospho-mTOR, RPS6, phospho-RPS6, 4EBP1, phospho-4EBP1, EIF4E, phospho-EIF4E, STAT1, phospho-STAT1, STAT3, phospho-STAT3, phospho-STAT5, SOCS1, and SOCS3, all from Cell Signaling Technology; and IGF-II receptor (IGF2R) and STAT5, both from BD Transduction Laboratories.

Membranes were washed and bound antibody detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents according to the manufacturer's instructions (Thermo Fisher Scientific). AlphaEaseFC software (Alpha Innotech Corporation) was used to quantify the density of specific bands, and 10 and 20 μg of the same protein sample were loaded onto each gel to confirm that the chemiluminescent signal changed in a linear manner. Prior to Western blotting analysis, samples (20 μg protein) were subjected to SDS-PAGE and gels stained with Coomassie Brilliant Blue (Thermo Fisher Scientific), and there were no differences in abundance of the major proteins between the different experimental groups.

Statistical analysis

Data are presented as mean ± SEM. Data were analyzed using IBM Statistical Package for Social Scientists Statistics version 19 (SPSS Inc) and STATA11 data analysis and statistical software for repeated measures (Stata Corp Ltd). The effects of periconceptional nutrition and sex on the mRNA expression, protein abundance, and morphometric measures in the adrenal of lambs were determined using a two-way ANOVA with the donor ewe number nested within nutritional treatment groups to identify the lambs from the same donor ewe. When there was an interaction between the effects of periconceptional nutrition and sex, the effect of periconceptional nutrition was determined in the females and males separately. When there was no interaction between nutritional treatment and sex, data from female and male lambs were pooled for analysis and presentation. Normal distribution of the values was tested using STAT11 (Stata Corp Ltd) and log transformation was performed for values that were not normally distributed. Where log transformation did not alter the statistical results, data are presented as nontransformed values. Duncan's post hoc test was used to determine significant differences between groups, and a probability level of 5% (P < .05) was taken to be significant.

Results

Adrenal weight and morphometry

Adrenal weight was significantly higher in lambs in the CR and HR groups compared with the CC group (P < .05; Table 1), and the relative weight of the adrenal was also significantly higher in the CR and HR groups compared with the CC and HH groups (P < .01; Table 1). There was no effect of lamb sex on adrenal weight.

Table 1.

Adrenal Weight and Morphometric Measures in lambs

CC (F: n = 3, M: n = 2) CR (F: n = 3, M: n = 6) HH (F: n = 4, M: n = 6) HR (F: n = 5, M: n = 5)
Adrenal weight, g 1.58 ± 0.09a 1.99 ± 0.08b 1.74 ± 0.08a,b 1.86 ± 0.09b
Relative adrenal weight, g/kg 0.052 ± 0.004a 0.063 ± 0.002b 0.053 ± 0.002a 0.060 ± 0.003b
Total adrenal area, mm2 32.60 ± 2.91 44.76 ± 3.21 38.02 ± 2.22 39.04 ± 2.43
Adrenal cortical area, mm2 24.29 ± 1.68a 34.84 ± 2.29b 27.46 ± 1.35a 29.81 ± 1.87a,b
Zona glomerulosa area, mm2 2.90 ± 0.25 3.58 ± 0.24 3.18 ± 0.16 3.00 ± 0.20
Zona fasciculata-reticularis area, mm2 21.39 ± 1.52a 31.26 ± 2.14b 24.28 ± 1.23a 26.81 ± 1.72a,b
Adrenal medullary area, mm2 8.31 ± 1.24 9.92 ± 1.31 10.56 ± 0.98 9.23 ± 0.93
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Abbreviations: F, females; M, males. Values are means ± SEM.

a and b

Different superscripts denote the treatment groups that are significantly different from each other (P < .05).

The area of the adrenal cortex (adrenal cortical area) but not the adrenal medulla (adrenal medullary area) was significantly larger in the CR group compared with the CC and HH groups in both the male and female lambs (P < .05; Table 1). There was no effect of nutritional treatment on the area of the zona glomerulosa, whereas the area of the zona fasciculata-reticularis in the adrenal was greater in the CR group compared with the CC and HH groups (P < .05; Table 1).

The number of cell nuclei per square micrometer in the zona fasciculata-reticularis was significantly lower in the CR group compared with the CC group and lower in the HR group compared with the HH group in both male and female lambs (P < .05; Figure 1). There was no difference, however, in the number of cell nuclei per square micrometer in either the zona glomerulosa or the adrenal medulla between the CC, CR, HH, and HR lambs.

Figure 1.

Figure 1.

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A, Number of nuclei per square micrometer in the zona glomerulosa and zona fasciculata of the adrenal cortex and the adrenal medulla in the CC, CR, HH, and HR groups (CC: open bar, females, n = 5, males, n = 2; CR: striped bar, females, n = 3, males, n = 6; HH: gray bar, females, n = 4, males, n = 5; HR: gray striped bar, females, n = 4, males, n = 5). Different superscripts (a, b, and c) denote the treatment groups that are significantly different from each other (P < .05). B, A representative photomicrograph stained with hematoxylin and eosin of the zona fasciculata of the adrenal cortex from a CC, CR, HH, and HR lamb. Bar, 25 μm.

There was no effect of lamb sex on the areas of the adrenal cortex and adrenal medulla or the number of cell nuclei per unit in the zona fasciculata-reticularis, the zona glomerulosa, or the adrenal medulla.

There was a positive relationship between adrenal weight (y) and the area of the adrenal cortex (x) in lambs from all diet groups (y = 0.03x + 0.82, r2 = 0.47, P < .01). Similarly, there was a positive relationship between adrenal weight (y) and the area of the zona fasciculata-reticularis (x) in lambs from all diet groups (y = 0.04x + 0.87, r2 = 0.49, P < .01). There was also an inverse relationship between the area of the zona fasciculata-reticularis (y) and the cell density in this zone (x) in lambs from all diet groups (y = −17.1x + 154.4, r2 = 0.56, P < .01).

Impact of periconceptional nutrition on the mRNA expression of the GH receptor, PRLR, SOCS1, and SOCS3 in the postnatal adrenal

There was no effect of nutritional treatment or lamb sex on adrenal GH receptor, SOCS1, and SOCS3 mRNA expression (Supplemental Table 2). There was also no effect of periconceptional nutrition on the adrenal mRNA expression of PRLR (all forms), PRLR-long form (PRLR-L), and PRLR-short form (PRLR-S) (Supplemental Table 2). The mRNA expression of PRLR (all forms), PRLR-L, and PRLR-S mRNA was lower, however, in the adrenals of male lambs compared with female lambs (P < .05).

Impact of periconceptional nutrition on the protein abundance of IGF1R, IGF2R, and IGF1R signaling molecules in the postnatal adrenal

The protein abundance of adrenal IGF1R, total Akt, and phospho-Akt (Ser473) was lower in male lambs compared with female lambs independent of nutritional treatment group (P < .01; Figure 2). The protein abundance of adrenal IGF1R and total Akt was also significantly lower in the CR, HH, and HR groups compared with the CC group in both male and female lambs (P < .01; Figure 2). Adrenal phospho-Akt (Ser473) abundance was significantly lower in the CR and HR groups compared with the CC group and was also lower in the HR compared with the HH lambs in both male and female lambs (P < .05; Figure 2).

Figure 2.

Figure 2.

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The protein abundance of IGF1R (A), total Akt (B), phospho-Akt (Ser473) (C), and mTOR (D) in the adrenal cortex in female and male lambs separately (CC: open bar, females, n = 4, males, n = 2; CR: striped bar, females, n = 3, males, n = 3; HH: gray bar, females, n = 3, males, n = 3; HR: gray striped bar, females, n = 3, males, n = 3). Different superscripts (a, b, and c) denote the treatment groups that are significantly different from each other (P < .05). Western blot images (E) are provided for each target protein in the CC, CR, HH, and HR groups in both female and male lambs. #, Representative of a CC female lamb.

There was an interaction between the effects of nutritional treatment and sex on adrenal mTOR abundance (P < .05; Figure 2). In females, adrenal mTOR abundance was significantly lower in the CR, HH, and HR groups compared with the CC group (P < .05), whereas in males there was no difference between the treatment groups.

There was no effect of nutritional treatment, sex, or an interaction between the effects of treatment and sex on the protein abundance of adrenal IGF2R and IGF1R downstream signaling molecules including Akt1, Akt2, phospho-mTOR (Ser2448), RPS6, phospho-RPS6 (Ser235/236), 4EBP1, phospho-4EBP1 (Ser65), phospho-4EBP1 (Thr70), EIF4E, and phospho-EIF4E (Ser209) (Supplemental Table 3).

Impact of periconceptional nutrition on adrenal protein abundance of STAT isoforms

Adrenal phospho-STAT1 (Tyr701) abundance was significantly higher in the CR group compared with the CC and HH groups in both the male and female lambs (P < .05; Figure 3). There was no effect of lamb sex on phospho-STAT1 (Tyr701) abundance. There was also no effect of nutritional treatment or sex on the protein abundance of adrenal STAT3, phospho-STAT3 (Tyr705), and phospho-STAT5 (Tyr694) (Figures 3 and 4). There was an interaction between the effects of nutritional treatment and sex on adrenal STAT1 (P < .055), phospho-STAT3 (Ser727) (P < .01), and STAT5 abundance (P < .01) (Figures 3 and 4). Adrenal STAT1 abundance was significantly higher in the CR group compared with the CC, HH, and HR groups in the female (P < .05) but not the male lambs (Figure 3). Adrenal phospho-STAT3 (Ser727) abundance was also significantly higher in the CR and HR groups compared with the CC and HH groups in the female (P < .01) but not the male lambs (Figure 3). Adrenal STAT5 abundance was significantly lower in the HH and HR groups compared with the CC and CR groups in the female (P < .05) but not the male lambs (Figure 4).

Figure 3.

Figure 3.

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The protein abundance of adrenocortical STAT1 (A), phospho-STAT1 (Tyr701) (B), STAT3 (C), phospho-STAT3 (Tyr705) (D), and phospho-STAT3 (Ser727) (E) (CC: open bar, females, n = 4, males, n = 2; CR: striped bar, females, n = 3, males, n = 3; HH: gray bar, females, n = 3, males, n = 3; HR: gray striped bar, females, n = 3, males, n = 3). Different superscripts (a, b, and c) denote the treatment groups that are significantly different from one another (P < .05). Western blot images (F) are provided for each target protein in the CC, CR, HH, and HR groups in both female and male lambs. #, Representative of a CC female lamb.

Figure 4.

Figure 4.

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The protein abundance of STAT5 (A) and phospho-STAT5 (Tyr694) (B) in the adrenal cortex of the CC, CR, HH, and HR lambs (CC: open bar, females, n = 4, males, n = 2; CR: striped bar, females, n = 3, males, n = 3; HH: gray bar, females, n = 3, males, n = 3; HR: gray striped bar, females, n = 3, males, n = 3). Different superscripts (a and b) denote the treatment groups that are significantly different from each other (P < .05). Western blot images (C) are provided for each target protein in the CC, CR, HH, and HR groups in both female and male lambs. #, Representative of a CC female lamb.

Impact of periconceptional nutrition on the protein abundance of adrenal SOCS isoforms

Adrenal SOCS1 abundance was significantly higher in the HR group compared with the HH group in male and female lambs (P < .05; Figure 5). There was no effect of lamb sex on SOCS1 abundance. There was an interaction between the effects of nutritional treatment and sex on adrenal SOCS3 abundance (P < .05; Figure 5). In female lambs, adrenal SOCS3 abundance was significantly lower in the HH group compared with the CC and CR groups and was also lower in the HR compared with the CR group (P < .01).

Figure 5.

Figure 5.

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The protein abundance of SOCS1 (A) and SOCS3 (B) in the adrenal cortex of the CC, CR, HH, and HR lambs (CC: open bar, females, n = 4, males, n = 2; CR: striped bar, females, n = 3, males, n = 3; HH: gray bar, females, n = 3, males, n = 3; HR: gray striped bar, females, n = 3, males, n = 3). Different superscripts (a, b, and c) denote the treatment groups that are significantly different from each other (P < .05). Western blot images (C) are provided for each target protein in the CC, CR, HH, and HR groups in both female and male lambs. #, Representative of a CC female lamb.

Discussion

Periconceptional undernutrition and adrenocortical growth

We have demonstrated that the increased adrenal mass in male and female lambs after exposure to maternal dietary restriction in normal-weight ewes during the periconceptional period is a result of increased growth of the adrenal cortex and specifically the zona fasciculata-reticularis region. There were significantly fewer cell nuclei per unit area present in the zona fasciculata-reticularis of adrenals in the CR lambs, suggesting that the increase in adrenocortical growth is a consequence of hypertrophy of cells in this region. Although the area of the zona fasciculata-reticularis was greater and the number of cell nuclei per unit area present in this zone was lower in the adrenal in the CR compared with the CC group, these values were intermediate in adrenal in the HR group between those of the CC and CR groups. There was, however, a positive correlation between the adrenal weight and the area of the adrenal cortex and a positive correlation between the adrenal weight and the area of the zona fasciculata-reticularis from all diet groups, which suggests that the increase in adrenal weight was directly related to an increase in the area of adrenal cortex and in particular to the area of the zona fasciculata-reticularis. These data highlight that the increase in adrenocortical growth is likely to be a consequence of hypertrophy of cells in this region and that the effects of maternal dietary restriction in the normal-weight ewe are relatively greater than in the obese ewe.

Periconceptional undernutrition and the adrenal JAK/STAT/SOCS signaling

Maternal dietary restriction in the normal-weight ewe during the periconceptional period resulted in the activation of components of the STAT signaling pathway including an increase in the adrenocortical abundance of phospho-STAT1 (Tyr701) in the CR lambs and STAT1 and phospho-STAT3 (Ser727) in the CR female lambs. Maternal dietary restriction in the obese ewe during the periconceptional period resulted in an increase in phospho-STAT3 (Ser727) and a decrease in STAT5 in the adrenal of the HR female lambs. These effects occurred in the absence of an effect of either periconceptional nutrition or lamb sex on plasma glucose, insulin, and free fatty acid concentrations in the postnatal lamb (28). Adrenal SOCS1 abundance was also higher in the HR group compared with the HH group.

Cytokines have been implicated in the regulation of the activation of the HPA axis and an increase in glucocorticoid secretion in the offspring exposed to early-life malnutrition through the JAK/STAT signaling (36). One possibility therefore is that maternal undernutrition during the periconceptional period results in a programmed up-regulation of intrafetal or intraadrenal cytokine production to result in the increase in the STAT1 and STAT3 isoforms in the adrenal and increased adrenal growth in lambs exposed to maternal dietary restriction. There is evidence that activation of the STAT1/STAT3 signaling cascade may play a role in cellular hypertrophy in tissues including the heart (37), but there is limited information on the role of the STAT1/STAT3 pathway in stimulating the growth of adrenocortical cells.

We have previously shown that periconceptional undernutrition resulted in a greater cortisol response to stress in the female but not male lamb at 4 months (12). The dissociation between the adrenal growth response, which is present in both males and females, and the increased cortisol stress response, which is present in female lambs only, may be a result of the previously reported effects of sex steroids on the adrenal responsiveness to ACTH (12, 38). Alternatively, the dissociation in the growth and cortisol responses in the offspring may be attributed to the effects of sex steroids on the adrenal STAT signaling pathways. There is evidence that estrogen plays a role in the JAK/STAT signaling pathways (39–41). It has been demonstrated that estrogen rapidly induces phosphorylation/activation of STAT3 and STAT5 in vitro and in estrogen-treated rat brains (40, 41). The precise mechanisms by which STAT isoforms cooperate with sex steroids and other transcription factors and signaling cascades to activate transcription of target genes are not fully understood. However, it is possible that the alteration of the abundance of the adrenal STAT1, phospho-STAT3, and STAT5 in female but not male lambs contributed to a greater cortisol response to stress in the CR and HR female lambs. The present study highlights that exposure of the oocyte/embryo to a short period of moderate undernutrition around the time of conception has a specific impact on the mechanisms that regulate adrenal signaling pathways and adrenocortical growth to result in the programming of an increased adrenocortical mass and a greater cortisol response to stress in the offspring.

GH and PRL have been demonstrated to promote adrenal growth and steroidogenesis, mainly through the STAT5 pathway (16–22). In the present study, however, there was no difference in adrenal mRNA expression of GH receptor and PRLR or in adrenal phospho-STAT5 (Tyr694) abundance between the treatment groups.

Periconceptional undernutrition and the adrenal IGF-I signaling pathway

Maternal dietary restriction in either normal-weight or obese ewes during the periconceptional period resulted in a decrease in the abundance of adrenal IGF1R, Akt, and phospho-Akt (Ser473) in both male and female lambs and mTOR in female lambs, consistent with our previous findings that periconceptional undernutrition results in a decrease in the level of adrenal IGF2 expression and methylation in the offspring (12). It has been shown that there is cross talk between the IGF1R and JAK/STAT/SOCS signaling pathways (42). Shalita-Chesner et al (43) have previously reported that IFN-γ activates STAT1, which in turn suppresses IGF1R promoter activity in a human osteosarcoma cell line. SOCS molecules have also been shown to negatively regulate the IGF1R signaling pathway (44). In the present study, there was a decrease in the abundance of molecules in the upstream, but not downstream, part of the IGF1R signaling pathway in the adrenal cortex of the CR and HR lambs. These changes may represent a partial compensatory response to limit the adrenocortical hypertrophy present in these offspring.

Periconceptional obesity and adrenal IGF-I and JAK/STAT/SOCS signaling pathways

Interestingly, maternal obesity during the periconceptional period also resulted in a decrease in the abundance of adrenal IGF1R and Akt in males and females and mTOR in female offspring, which was not consistent with the previously reported increase in adrenal IGF-I (IGF1) mRNA expression, which was present in male and female lambs in the HH group (12). Periconceptional overnutrition also resulted in a decrease in the abundance of adrenal STAT5 and SOCS3 in female offspring. The down-regulation of the components of the IGF1R and STAT/SOCS signaling pathways in the HH group may reflect an intraadrenal autoregulatory function to limit any impact of the increase of adrenal IGF1 mRNA expression on adrenal growth.

Summary

The novelty of the current study is that it identifies the impact of exposure to maternal undernutrition in the periconceptional period alone in both normal-weight and obese ewes on the growth and development of the adrenal in her offspring. In addition, the sheep represents a model in which it has been shown that, as in the human, an increase in fetal cortisol production plays a key role in the successful transition of the fetus to extrauterine life and that exposure to periconceptional undernutrition results in an increase in the cortisol stress response in postnatal life (12, 45).

We have demonstrated that the increase in adrenal mass in the offspring of mothers exposed to dietary restriction in the normal-weight ewe in the periconceptional period is a consequence of hypertrophy of the adrenocortical cells in the zona fasciculata-reticularis. Maternal dietary restriction in the normal-weight ewe during the periconceptional period also activated components of the STAT signaling pathway including an increase in the adrenocortical abundance of STAT1 and STAT3 (Figure 6). Thus, weight loss around the time of conception programs an increase in adrenocortical growth associated with an up-regulation in the abundance of STAT1 and STAT3 within the adrenal cortex of the postnatal lamb. We have also demonstrated that there was a decrease in the abundance of molecules in the upstream, but not downstream, part of the IGF1R signaling pathway in the adrenal cortex of the CR and HR lambs (Figure 6). These changes may represent a partial compensatory response to the adrenocortical hypertrophy and/or the activated STAT pathway present in these offspring.

Figure 6.

Figure 6.

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A summary of the protein changes in IGF1R downstream signaling molecules and STAT/SOCS isoforms in the adrenal of lambs in response to maternal dietary restriction in normal weight (CR) and obese ewes (HR) during the periconceptional period compared with the control group (CC). ↑, Increased protein abundance; ↓, decreased protein abundance; ↔, no change in protein abundance; females, a change in protein abundance occurred only in female lambs in both the CR and HR groups; CR, a change in protein abundance occurred only in the CR group; CR females, a change in protein abundance occurred only in CR female lambs; HR females, a change in protein abundance occurred only in HR female lambs.

The impact of maternal dietary restriction in the obese ewe is less when compared with the impact of maternal dietary restriction in the normal-weight ewe on the programming of adrenal growth and signaling pathways (Figure 6). The abundance of adrenal STAT5 significantly decreased in both HH and HR female lambs, which highlights that there are separate effects of maternal obesity and of maternal dietary restriction on the programming of the JAK/STAT pathway within the adrenal. There was a decrease in the abundance of adrenal IGF1R and STAT/SOCS signaling molecules in the HH group, which may also limit any impact of an up-regulation of IGF-I on adrenal growth (Figure 7).

Figure 7.

Figure 7.

Open in a new tab

A summary of the protein changes in IGF1R downstream signaling molecules and STAT/SOCS isoforms in the adrenal of lambs in response to maternal obesity (HH) during the periconceptional period compared with the control group (CC). ↓, Decreased protein abundance; ↔, no change in protein abundance; females, a change in protein abundance occurred only in female lambs.

It appears, therefore, that signals of early adversity, rather than nutritional excess, have a greater impact on the adrenal and result in the programming of increased growth of the adrenal cortex and the cortisol stress response in the offspring. It would be interesting in follow-up studies to determine the specific factors (systemic, ovarian, or uterine) that mediate the impact of maternal undernutrition on the development of the HPA axis in the offspring. The present study highlights that the periconceptional period is a critical period for the programming of mechanisms that regulate adrenocortical growth.

Acknowledgments

We gratefully acknowledge the experiment and research assistance provided by Laura O'Carroll and the Early Origins of Adult Health Research group during the course of this study.

This work was supported by the Brailsford Robertson Trust (to I.C.M.) and the National Health and Medical Research Council of Australia (to I.C.M. and J.L.M.). J.L.M. was supported by the Heart Foundation South Australian Cardiovascular Research Network.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
CC
control-control
CR
control restricted
4EBP1
EIF4E-binding protein 1
EIF4E
eukaryotic translation initiation factor
HH
high-high
HPA
hypothalamo-pituitary-adrenal
HR
high restricted
3βHSD
3β-hydroxysteroid dehydrogenase
IGF1R
IGF-I receptor
IGF2R
IGF-II receptor
JAK
Janus kinase
MER
metabolizable energy requirement
mTOR
mammalian target of rapamycin
PRL
prolactin
PRLR
PRL receptor
RPS6
ribosomal protein S6
SOCS
suppressors of cytokine signaling
STAT
signal transducer and activator of transcription.

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