We previously reported that in the sheep fetus, long term hypoxia (LTH) resulted in elevated basal plasma ACTH1–39 while cortisol levels were not different from normoxic controls. We also showed that LTH enhances endothelial nitric oxide synthase (eNOS) expression in the fetal adrenal. This study was designed to determine the effect of nitric oxide on cortisol production in adrenocortical cells from LTH fetal sheep. Ewes were maintained at high altitude (3,820 m) from ~40 days’ gestation (dG) to near term. Between 138–141 dG, fetal adrenal glands were collected from LTH and age-matched normoxic control fetuses. Adrenal cortical cells were pre-treated with sodium nitroprusside (SNP), L-NAME, L-arginine, or Diethyleneamine nitric oxide (DETA-NO) then challenged with 10nM ACTH. Cortisol responses were compared after 1 h. ACTH induced cortisol secretion was significantly higher in LTH vs. control (p<0.01). Enhancement of nitric oxide with L-arginine resulted in a significant reduction of ACTH-mediated cortisol production in the LTH group. DETA-NO also caused a significant decrease in ACTH-mediated cortisol production (p<0.05). Inhibition of NOS with L-NAME significantly increased cortisol production in the LTH group (p<0.05 compared to ACTH alone) while the effect on the control group was not significant. NOS activity was significantly higher in the LTH group compared to control but this difference was eliminated following ACTH treatment. These data indicate that LTH enhances adrenal cortical sensitivity to the inhibitory effects of NO on cortisol production. NO may therefore play an important role in regulating ACTH-induced cortisol production in the LTH fetal adrenal.

This study was designed to test the hypothesis that fetal adrenal nitric oxide synthase (NOS) is elevated in response to long term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3,820 m) for approximately the last 100 days of gestation. Between days 138-141 of gestation, adrenal glands were collected from LTH fetuses and age-matched normoxic controls. qRT-PCR and Western analysis were used to quantify NOS expression and NOS distribution was examined by immunohistochemistry and double-staining immunofluorescence for eNOS and 17α hydroxylase (CYP17). nNOS was expressed at very low levels and with no differences between groups. Expression of eNOS was significantly greater in the LTH group compared with control. nNOS was distributed throughout the cortex while the greatest density of eNOS was observed in the zona fasciculata/reticularis area and eNOS co-localized with CYP17. We conclude that LTH enhances eNOS expression in the inner adrenal cortex which may play a role in regulation of cortisol biosynthesis in the LTH fetus.

This study was designed to determine the potential mechanism(s) of previously observed enhanced fetal cortisol secretion following exposure to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3,820 m) for approximately the last 100 days of gestation. Between days 138-141 of gestation, adrenal glands were collected from LTH and age-matched normoxic control fetuses. cAMP, cortisol and steroidogenic acute regulatory protein (StAR) were measured in response to ACTH stimulation. Cortisol responses to ACTH were also measured in the presence of either the PKA inhibitor H-89, POMC or 22kDa pro-ACTH. Cortisol output was higher in the LTH group compared to the control, (p< 0.05) following ACTH treatment while the cAMP response was similar in both groups. Although PKA inhibition decreased cortisol production in both groups, however no differences were observed between groups. Western analysis revealed a significant increase in protein expression for StAR in the LTH group (p<0.05, compared to control). POMC and 22 kDa proACTH did not alter the cortisol response to ACTH treatment. Results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in the LTH group is not the result of differences in cAMP generation or PKA. We conclude that enhanced cortisol production in LTH adrenals is the result of enhanced protein expression of StAR and potential downstream signaling pathways.

Vargas VE, Kaushal KM, Monau TR, Myers DA, Ducsay CA. Extracellular signal-regulated kinases (ERK1/2) signaling pathway plays a role in cortisol secretion in the long-term hypoxic ovine fetal adrenal near term. Am J Physiol Regul Integr Comp Physiol 304: R636 -R643, 2013. First published February 20, 2013 doi:10.1152/ajpregu.00318.2012.-This study assessed the role of the extracellular signal-regulated kinase (ERK) signaling pathway on the previously observed enhanced cortisol secretion in response to adrenocorticotropic hormone (ACTH) treatment in fetal adrenocortical cells (FACs) from long-term hypoxic (LTH) ovine fetuses. Ewes were maintained at high altitude (3,820 m) from ϳ40 to 138 -141 days gestation when FACs were collected and challenged with either ACTH (10 nM) or 8-bromoadenosine 3=,5=-cyclic monophosphate (8-bromo-cAMP, 10 mM) in the presence or absence of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MEK)/ERK inhibitor UO126 (10 M). FACs from age-matched normoxic fetuses served as controls. Media and FACs were collected at selected time intervals after ACTH or 8-bromocAMP stimulation for cortisol measurement and Western analysis of ERK1/2 and phospho-ERK1 and -2 (pERK1/2). After ACTH or 8-bromo-cAMP treatment, cortisol production was greater in the LTH group compared with control (P Ͻ 0.05). UO126 reduced ACTH and 8-bromo-cAMP-mediated cortisol output in both groups (P Ͻ 0.01 vs. ACTH or 8-bromo-cAMP alone). Under basal conditions, ERK1/2 and pERK1/2 were not different between LTH and normoxic fetuses. In response to ACTH or 8-bromo-cAMP treatment, ERK1/2 were not different between groups; however, pERK1/2 were elevated in the LTH FACs compared with normoxic control FACs. ERK1/2 phosphorylation declined following ACTH treatment in the control group, but UO126 had no effect on ERK1/2 compared with untreated levels. Both ACTH and 8-bromo-cAMP treatment resulted in a decline of protein levels. UO126 pretreatment virtually eliminated pERK1/2 expression. We conclude that basal ERK signaling in FACs is necessary for normal cortisol production and sustained pERK in LTH adrenals enhances cortisol production.ACTH; UO126; 8-bromo-cAMP; sheep; fetus THE CLASSICAL adrenocorticotropic hormone (ACTH) signaling pathway via 3,5-cAMP is generally accepted as the major mechanism regulating cortisol biosynthesis in the adrenal cortex (10, 36). The late gestation increase in fetal plasma cortisol resulting from adrenocortical maturation is critical for optimal fetal organ maturation, and in sheep, it plays an essential role in parturition (24,25). Hypoxia has clearly been shown to play a key role in the regulation of the fetal hypothalamic-pituitaryadrenal axis (3,17,11). Importantly, from a clinical perspective, not only women that live at high altitude, but also women that smoke, have heart/lung disease, or are anemic during pregnancy expose the fetus to hypoxia (41, 22, 31).Our laboratory has developed an ovine model of high altitude-induced long-term hypoxia (LTH). We have fo...

This study was designed to determine if long-term hypoxia (LTH) alters adrenal glucocorticoid receptor (GR) expression in the ovine fetal adrenal cortex. Ewes were maintained at 3820 m from approximately 30 to 138 to 140 days' gestation, and fetal adrenals were collected. Western analysis revealed two approximately 94-kDa GR-alpha isoforms and a lower molecular weight (45 kDa) form. A decreasing trend in the ratio of 94-kDa/45-kDa bands following LTH suggested an increase in GR turnover. Immunohistochemistry demonstrated dense GR staining in the zona glomerulosa with minimal staining in the zona fasciculata in the control group, while dense staining was observed throughout the cortex in LTH. Western analysis and reverse transcription polymerase chain reaction confirmed that the GR- beta isoform is not present or expressed at extremely low levels in the fetal adrenal, hypothalamus, pituitary, and placenta. These data indicate that LTH alters GR-alpha function in the fetal adrenal cortex and suggest that GR-beta is not expressed in sheep.