4 ACTH LTH ZG ZF NO Leptin Leptin LTH M Cortisol Figure 2: Eﬀects of development under conditions of long-term hypoxia (LTH) on adrenocortical function in the late gestation ovine fetus. LTH increases perirenal adipose expression and release of leptin as well as adrenocortical leptin receptor (OB-Rb) expression. LTH also increases zona fasciculate-speciﬁc expression and activity of eNOS. Combined, leptin and nitric oxide (NO) serve to limit the ability of the elevated fetal plasma ACTH to stimulate cortisol production thus allowing for the maintenance of the normal ontogenic maturation of cortisol production preserving the prepartum exponential rise of this key steroid. In response to an acute secondary stress, these mechanisms allow release of cortisol synthesis thus resulting in an enhanced acute phase production of cortisol. (ZG-zona glomerulosa, ZF-zona fasciculata, M-medulla). not with the decreased expression of the downstream target genes. We also examined expression of factors (pMEIS-1 and Pbx-1) that act in concert with SF-1 in the bovine adrenal cortex and thus presumably the highly conserved regulatory regions of the ovine CYP11A1 and CYP17 promoters. Similar to SF-1, these factors were also expressed to a similar degree in LTH compared to normoxic adrenal cortices (Ducsay and Myers [39], unpublished observations). Thus, the eﬀects of LTH on the transcription of the ACTH-receptor, CYP11A1, and CYP17 must likely be either through an unknown repressor or, more likely, via altered activation of these tran- scription factors. It has been demonstrated that SF-1 activity is regulated by phosphorylation state, dictated by either ACTH activated cAMP/PKA pathways or ERK1/2 pathways, and dependent on protein phosphatase activity [40]. To address the potential for a decreased second messen- ger response to ACTH, especially in light of the decreased ACTH receptor expression, we examined cAMP production in adrenocortical cells obtained from LTH fetuses. Surpris- ingly, LTH fetal adrenocortical cells generate a similar cAMP response compared to adrenocortical cells from normoxic fetuses [41]. In these in vitro studies, we also noted an additional perplexing ﬁnding: that contrary to the in vivo sit- uation, LTH fetal adrenocortical cells exhibited greater basal cortisol production, while retaining the noted enhanced ACTH stimulated cortisol output compared to cells from Journal of Pregnancy normoxic fetuses. A similar enhanced cortisol response to cAMP was also noted, thus emphasizing that the eﬀect was potentially downstream from second messenger generation. Thus, there do not appear deﬁciencies in ACTH signaling via the major second messenger pathway utilized by the ACTH receptor. Thus, the noted lower mRNA levels for the ACTH receptor may not be reﬂected by actual levels of the ACTH receptor protein or that levels of the ACTH receptor may not be limiting. Alternatively, lower ACTH receptor expression may be overcome through more eﬃcient coupling of the ACTH receptor to Gαs or increased adenylate cyclase activity. However, at present these possibilities remain unexplored. What the in vitro studies did highlight was that the capacity of these cells to respond to ACTH was decidedly diﬀerent in vitro compared to the in vivo situation— namely, in terms of basal versus ACTH stimulated cortisol production. This implicated additional intra- or extra- adrenal factors that in vivo act on the fetal adrenocortical cells limiting the capacity of low resting concentrations ACTH (5 to 10 pM) to stimulate cortisol production under basal conditions yet allow an enhanced cortisol production in response to a secondary stressor with stress levels of ACTH (50–100 pM). To address these possibilities, we explored two potential mechanisms that could potentially explain the noted adaptation of the fetal adrenal cortex to LTH: nitric oxide (NO) and leptin. 5. Role of Nitric Oxide in Mediating LTH Effects on Cortisol Biosynthesis While the lower expression of the ACTH receptor, coupled with decreased expression of two rate limiting enzymes mediating cortisol biosynthesis (CYP11A1 and CYP17), provides a means for the capacity of the LTH fetus to maintain the normal ontogenic rise in fetal plasma cortisol, these adaptive changes seem contradictory to the capacity of the LTH adrenal cortex to produce greater cortisol in response to a secondary stressor. Although the enhanced ACTH1−39 released in the LTH fetus in response to a secondary stressor likely contributes to this boost in cortisol production, the decreased expression of the ACTH receptor coupled with lower expression of key enzymes suggests that other intracellular mechanism are involved in the divergent capacity for basal cortisol and stress-stimulated cortisol pro- duction. We initiated investigations into nitric oxide (NO) as one potential mediator of the eﬀects of LTH on the adrenal gland. Nitric oxide has profound inhibitory eﬀects on steroidogenesis in a wide range of endocrine tissues including the adrenal cortex and NO synthases (NOSs) are subject to regulation by hypoxia (see recent review by Ducsay and Myers [39]). As such NO represents a potential mechanism in the adrenocortical adaptation to LTH (Figure 2). Further, it has been reported that NO concentrations in the plasma of the LTH fetus are elevated compared to normoxic fetuses [42]. While the levels in plasma are likely not high enough to be of physiological importance for regulating intracellular adrenocortical events, they are indicative that