Molecular regulation of the hypothalamic-pituitary-adrenal axis in adult male guinea pigs after prenatal stress at different stages of gestation

Abstract

Studies in humans and animals have demonstrated that maternal stress during fetal development can lead to altered hypothalamic-pituitary-adrenal (HPA) axis function and behaviour postnatally. We have previously shown adult male guinea pigs that were born to mothers exposed to a stressor during the phase of rapid fetal brain growth (gestational days (GD) 50, 51 and 52; prenatal stress (PS)50) exhibit significantly increased basal plasma cortisol levels. In contrast, male guinea pig offspring whose mothers were exposed to stress later in gestation (GD60, 61 and 62; PS60) exhibited a significantly higher plasma cortisol response to activation of the HPA axis. In the present study, we hypothesized that the endocrine changes in HPA axis function observed in male guinea pig offspring would be reflected by altered molecular regulation of the HPA axis. Corticosteroid receptors in the hippocampus, hypothalamus and pituitary were measured, as well as corticotropin-releasing hormone (CRH), pro-opiomelanocortin (POMC) and adrenal enzymes in the paraventricular nucleus, pituitary and adrenal cortex, respectively, by in situ hybridization and Western blot. PS50 male offspring exhibited a significant reduction in glucocorticoid receptor (GR) mRNA (P <0.01) in the CA3 region of the hippocampus and significantly increased POMC mRNA (P <0.05) in the pituitary, consistent with the increase in basal HPA axis activity observed. In line with elevated activity of the HPA axis, both PS50 and PS60 male offspring exhibited significantly higher steroidogenic factor (SF)-1 (P <0.001) and melanocortin 2 receptor (MC2-R) mRNA (P <0.001) in the adrenal cortex. This study demonstrates that short periods of prenatal stress during critical windows of neuroendocrine development affect the expression of key regulators of HPA axis activity leading to the changes in endocrine function observed in prenatally stressed male offspring. Further, these changes are dependent on the timing of the maternal stressor, a pattern that is emerging in human studies.

Figure 1. Hippocampal glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA and protein expression in male offspring whose mothers were exposed to a high frequency strobe light for 2 h on gestational days 50, 51 and 52 (PS50; black bars), gestational days 60, 61 and 62 (PS60; grey bars) or left undisturbed throughout pregnancy (control; open bars)

A, representative images of GR, MR and sense in situ hybridization. B, relative optical density (ROD) of GR mRNA. C, ROD of MR mRNA. D, GR protein levels with representative Western blot image above. E, MR protein levels with representative Western blot image above. Animal numbers are indicated within bars. *P < 0.05 compared to control.

Figure 2. Hypothalamic expression of glucocorticoid receptor (GR) in the paraventricular nucleus (PVN), arginine vasopressin (AVP) mRNA in the PVN, corticotropin-releasing hormone (CRH) mRNA in the PVN, and sense CRH mRNA in male offspring whose mothers were exposed to stress at PS50 and PS60 (as described in Fig. 1), or left undisturbed throughout pregnancy (control)

A, representative in situ hybridization images of AVP, CRH and GR mRNA in the PVN. Relative optical density (ROD) by in situ hybridization of GR mRNA (B), AVP mRNA (C) and CRH mRNA (D). Animal numbers are indicated within bars.

Figure 3. Pituitary pro-opiomelanocortin (POMC) mRNA expression in male offspring whose mothers were exposed to stress at PS50 and PS60 (as described in Fig. 1), or left undisturbed throughout pregnancy (control)

A, representative in situ hybridization images of POMC mRNA expression in control, PS50 and PS60 pituitary. Relative optical density (ROD) of glucocorticoid receptor (GR) mRNA in the anterior lobe (B), POMC mRNA in the inferior anterior lobe (C), POMC mRNA in the superior anterior lobe (D) and POMC mRNA in the intermediate lobe (E). Animal numbers are indicated within bars. *P < 0.05, **P < 0.01, compared to control. IL, intermediate lobe; AL, anterior lobe; Sup, superior anterior lobe; Inf, inferior anterior lobe.

Figure 4. SF-1, StAR, MC2-R, CYP11A1 and CYP17 mRNA expression in adrenal gland of male offspring whose mothers were exposed to stress at PS50 and PS60 (as described in Fig. 1), or left undisturbed throughout pregnancy (control)

A, representative in situ hybridization images of melanocortin 2 receptor (MC2-R) mRNA expression in the adrenal. Relative optical density (ROD) of steroidogenic factor (SF)-1 mRNA (B), steroidogenic acute regulatory protein (StAR) mRNA (C), MC2-R mRNA (D), CYP11A1 mRNA (E) and CYP17 mRNA (F). Animal numbers are indicated within bars. **P < 0.01, compared to control.