Embryo number and periconceptional undernutrition in the sheep have differential effects on adrenal epigenotype, growth, and development

Abstract

Exposure to poor maternal nutrition around the time of conception results in an early prepartum activation of the fetal pituitary-adrenal axis and in increased adrenal growth and stress response after birth associated with epigenetic changes in a differentially methylated region (DMR) of adrenal IGF2/H19. We have determined the effects of maternal undernutrition during the periconceptional period (PCUN: 70% of control intake from 60 days before until 6 days after conception) and early preimplantation period (PIUN: 70% of control intake for 6 days after conception) on fetal plasma ACTH and cortisol concentrations and fetal adrenal ACTHR, StAR, 3βHSD, CYP11B, CYP17, TGFβ1, IGF1, IGF1R, IGF2, and IGF2R mRNA expression and the methylation level of sites within the DMRs of IGF2/H19 and IGF2R in the adrenal of twin and singleton fetuses at 136-138 days gestation. Being a twin resulted in a delayed prepartum increase in fetal ACTH and in a lower cortisol response to CRH in the control but not PCUN and PIUN groups. PCUN, but not PIUN, resulted in an increase in adrenal weight and CYP17 expression in singletons, a decrease in adrenal IGF2 expression in singletons, and an increase in adrenal IGF2R expression in both twins and singletons. IGF2/H19 and IGF2R DMR methylation levels and ACTHR expression were lower in the twin adrenal. Thus, exposure of the oocyte and embryo to maternal undernutrition or to the environment of a twin pregnancy have differential effects on epigenetic and other factors that regulate fetal adrenal growth and IGF2 and IGF2R expression.

Keywords: adrenal; epigenetic; fetus; periconceptional; twin.

Fig. 1.

Fetal plasma ACTH and cortisol concentrations in singleton and twin fetal sheep during late gestation. Fetal plasma ACTH concentrations significantly increased (P < 0.01) with gestational age in singletons (A) but not twins (B). There was no effect of periconceptional undernutrition (PCUN) or preimplantation undernutrition (PIUN) on fetal plasma ACTH concentration in either singletons or twins. Fetal plasma cortisol concentrations significantly increased with gestational age in both singleton (C) and twin (D) fetuses (P < 0.02 and P < 0.05, respectively). There was no effect of PCUN or PIUN on fetal plasma cortisol concentrations in either singletons or twins. Different letters (a, b) denote mean values that are significantly different within the singleton and twin groups.

Fig. 2.

Fetal plasma ACTH concentrations in response to CRH in singleton and twin fetal sheep. Plasma ACTH concentrations increased (P < 0.0001) in response to CRH (1 μg bolus) in singleton (A) and twin (B) fetuses in all nutritional groups. CRH injected at time 0. #Significant increase in plasma ACTH concentrations vs. preinfusion values. Area under the curve (AUC) for the ACTH response post-CRH, shown as inset histograms, was significantly lower (P < 0.05) in twins (B) than in singletons (A) independently of nutritional treatment, as denoted by #.

Fig. 3.

Fetal plasma cortisol concentration in response to CRH in singleton and twin fetal sheep in late gestation. Plasma cortisol concentrations increased (P < 0.0001) in response to CRH (1 μg bolus) in singleton (A) and twin (B) fetuses in all nutritional groups. CRH injected at time 0. #Significant increase in plasma cortisol concentration vs. preinfusion values.

Fig. 4.

Cortisol response denoted as area under the curve (AUC) after CRH in Control, PCUN, and PIUN groups. The AUC of the cortisol response post-CRH was significantly lower (P < 0.0001) in twins than in singletons in the Control group (A), as denoted by #. There was no difference, however, in cortisol AUC post-CRH between twin and singleton fetuses in the PCUN (B) and PIUN (C) groups.

Fig. 5.

IGF2 and CYP17 mRNA expression relative to RpPO in Control, PCUN, and PIUN groups. In singletons, there was a significant decrease (P < 0.03) in adrenal IGF2 mRNA expression (A) and a significant increase (P < 0.03) in CYP17 mRNA expression (C) in the PCUN group compared with the Control or the PIUN group. In twins, there was no effect of maternal nutritional treatment on adrenal IGF2 (B) or CYP17 (D) mRNA expression. Different letters (a, b) denote mean values that are significantly different within the singleton or twin groups.

Fig. 6.

IGF2R mRNA expression relative to RpPO in Control, PCUN, and PIUN groups. There was no difference in adrenal IGF2R mRNA responses to PCUN and PIUN between twins and singletons. Adrenal IGF2R expression was significantly higher (P < 0.03) in the PCUN group (n = 16) vs. Control (n = 15) or PIUN (n = 13) groups. Different letters (a, b) denote mean values that are significantly different from each other.

Fig. 7.

Adrenal IGF1R and IGF2R abundance in Control, PCUN, and PIUN groups. Adrenal IGF1R abundance was significantly decreased (P < 0.05) in the PCUN group compared with the Control group. There was no difference in adrenal IGF2R abundance between the treatment groups in twins and singletons. Different letters (a, b) denote mean values that are significantly different from each other.