Journal of Pregnancy NOS expression and/or activity may be elevated in target organs such as the adrenal gland. Indeed, we were the ﬁrst laboratory to demonstrate NOS expression in the fetal adrenal cortex and the eﬀects of NO on fetal adrenal steroidogenesis. Endothelial NOS (eNOS) is the predominant NOS isoform in the ovine fetal adrenal cortex, and more importantly, we demonstrated that LTH increases eNOS mRNA and protein expression [43] and that the increased expression was primarily in CYP17 expressing cells within the zona fasciculata indicating a selective increase in the cortisol producing adrenocortical layer. In vitro studies demonstrated that enhancement of NO production with L-arginine, a NOS substrate, resulted in a signiﬁcant reduction of ACTH-mediated cortisol production selectively in LTH-derived fetal adrenocortical cells [44]. The use of NO donors, DETA-NO, and Sodium Nitroprusside (SNP) similarly decreased ACTH-mediated cortisol production while inhibition of NOS activity with L-NAME signiﬁcantly increased cortisol production in the LTH group but was without eﬀect on normoxic fetus-derived fetal adrenocortical cells. In accord with the elevated expression on eNOS, nitric oxide synthase activity was signiﬁcantly higher in LTH- derived fetal adrenocortical cells compared to those from normoxic control fetuses, and 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. Nitric oxide may, therefore, play an important role in regulating ACTH-induced cortisol production in the LTH fetal adrenal [44]. How NO mediates its inhibitory actions on steroidogenic tissue, including the adrenal, is equivocal. In vascular tissue, the eﬀects of NO are well established and largely depend on cGMP pathways (reviewed in [45]). However, the eﬀects of NO on steroidogenesis in endocrine tissues appear to be independent of cGMP [46]. One potential mechanism by which NO inhibits steroidogenesis is via acting on the initial rate-limiting enzyme, CYP11A1. Nitric oxide competes with oxygen binding in the active site of CYP11A1 and CYP17 [46, 47]. Further, Peterson et al. [48] suggested that because these enzymes use several rounds of attack of the heme- oxygen complex on the steroid substrate, multistep enzymes (such as CYP11A1) would be more sensitive to NO inhibition than other steroidogenic enzymes. Overall, this competitive interaction makes NO a potentially eﬀective inhibitor of steroidogenesis. Thus, a model can be proposed where eNOS is upreg- ulated in the LTH fetus, and under basal conditions (low ACTH), NO decreases the activity of CYP11A1 and per- haps other cytochrome P450 enzymes helping to limit the production of cortisol despite the elevated basal ACTH1−39 (Figure 2). During a secondary stress-induced elevation of ACTH, high levels of ACTH signaling inhibit NOS activity removing this inhibition resulting in the observed enhanced cortisol production observed in these fetuses. These ﬁnd- ings implicate eNOS/NO as an intra-adrenal mechanism. Importantly the NO eﬀects are also retained in vitro [43, 44]. Therefore, although NO plays a role in providing a brake on basal cortisol production, it does not appear to provide a mechanism by which the expression of key genes 5 is decreased in the LTH adrenal gland. Taken together, these data implicate additional in vivo factors involved in the adaptation of the HPA axis to LTH. 6. Role of Leptin Mediating LTH Effects on Cortisol Biosynthesis A role for leptin in suppressing the HPA axis in adults has been established, both at the hypothalamic level as well as directly at the level of the adrenal cortex. The active or long form of the leptin receptor (OB-Rb) is found in the adrenal cortex in rodents, humans [49], and ruminants [50]. Leptin suppresses cortisol output in response to ACTH stimulation in adult bovine adrenocortical cells, and this eﬀect was mediated through an reduction in CYP17 expression [51]. Additional studies conﬁrmed this observation and extended the inhibitory eﬀect of leptin to expression of CYP11A [52]. In addition to downregulation of steroidogenic enzymes, leptin also appears to inhibit adrenal glucocorticoid secretion by an additional mechanism. Recent studies in the rat have clearly demonstrated that the leptin inhibition of corticosterone secretion occurs through a rapid reduction in expression of both StAR and PBR protein [53, 54]. Although the function(s) of leptin in the adult has been widely studied, the role of leptin in the fetus is not well deﬁned. Clearly, leptin is found in the fetal circulation and is expressed in fetal adipocytes [55, 56] and placental trophoblast tissue [57]. Intracerebral infusion of leptin blunted the size of increase that occurred in amplitude and mean value of plasma ACTH and cortisol pulses near term [58]. These data are consistent with adult mouse data where leptin attenuates restraint-induced activation of the HPA [59]. More recent studies from the McMillen group also demonstrated an inhibitory eﬀect of leptin on the fetal ovine HPA. Intravenous leptin infusions suppressed prepartum rise in both ACTH and cortisol [60]. Infusion after day 144 did not aﬀect ACTH but there was still marked suppression of cortisol up to three days prior to delivery. Based on this evidence, we initially addressed whether leptin, a hypoxia inducible gene, was elevated in LTH fetal sheep. We noted that plasma leptin as well as peri-renal adipose expression of leptin was signiﬁcantly elevated in the LTH fetus as was expression of the OB-Rb in both the adrenal cortex and hypothalamus [61]. While past studies in the ovine fetus, and largely in adults as well, have focused on the actions of exogenous leptin in regulating the HPA axis, we have addressed the role of endogenous leptin in the HPA adaptation to LTH using a speciﬁc ovine leptin receptor antagonist. As we have recently communicated [62], a 4- day infusion of the antagonist to LTH fetuses starting at 139 days of gestation restored expression of both CYP11A1 and CYP17 to levels similar to those of the normoxic control fetuses. Surprisingly, the infusion of the leptin antagonist had no eﬀect on expression of either enzyme in the normoxic controls and, similarly, did not alter fetal plasma ACTH or cortisol in these fetuses during the infusion period. In the LTH fetuses, although CYP11A1 and CYP17 expressions increased in response to the antagonist, fetal plasma cortisol