Journal of Pregnancy LTH To adrenal gland CRH and AVP Anterior pituitary PVN ACTH ACTH POMC ACTH Figure 1: Eﬀects of development under conditions of long-term hypoxia (LTH) on hypothalamic PVN and anterior pituitary cor- ticotrope function in the late gestation ovine fetus. LTH upregulates expression of both CRH and AVP in the parvocellular division of the PVN. At the level of the anterior pituitary corticotrope, this increased hypothalamic drive results in increased processing of POMC to ACTH as well as ACTH release from the gland into the circulation. The changes at the level of the PVN and anterior pituitary result in increased basal circulating ACTH as well as an enhanced ACTH release in response to a subsequent acute stressor. and thus the ratio of ACTH1−39 to precursors was increased in the LTH compared to normoxic fetuses, indicating that the bioactivity of fetal plasma IR-ACTH achieved in the LTH fetuses was greater than that in normoxic fetuses (see Figure 1, [37]). This is consistent with the enhanced cortisol response observed in LTH fetuses during this secondary stressor. Intriguingly, basal plasma ACTH1−39 concentrations were also elevated in the LTH fetal sheep. Despite the elevated plasma levels of ACTH1−39, these fetuses maintain normal basal cortisol concentrations compared to normoxic control fetuses. To support the elevated plasma ACTH1−39, we found enhanced processing of POMC to ACTH in the anterior pitu- itary of the LTH fetus, as well as signs of increased secretion (less stores) accompanied by sustained POMC expression with a trend toward greater POMC mRNA [36]. Considering our observations that lesion of the PVN prevents the enhanced processing of POMC to ACTH during late ges- tation [35], we hypothesized a hypothalamic origin for the increased processing of POMC in response to LTH. In our original report [36], we noted only a trend for hypothalamic CRH mRNA to be increased in the LTH fetuses using real- time PCR analysis of whole dissected hypothalami. However, recently (Ducsay and Myers, unpublished observations) using in situ hybridization techniques, we found a signiﬁcant upregulation of both CRH mRNA and AVP mRNA in the hypothalamic parvocellular division of the PVN. Initial morphometric analysis also suggests an expansion of the PVN reﬂecting an increase in the number of CRH neurons or neurons expressing CRH at detectable levels in response to LTH. At the level of the anterior pituitary, we noted increased mRNA for the CRH1 receptor with decreased CRH1 protein- the latter perhaps suggestive of a downregulation of this receptor in response to increased hypothalamic drive to the anterior pituitary. Of note, anterior pituitary AVP receptor (V1b) expression (mRNA and protein) was upregulated 3 in the LTH fetus perhaps indicative of a selective gain of responsiveness to this ACTH secretagogue [18]. In support of this potential gain of AVP function in response to LTH, we also found that ACTH release in response to AVP was greater in LTH fetal sheep, while the response to CRH was similar between LTH and normoxic controls. Thus, the hypothalamic and anterior pituitary corticotrope response to LTH appears complex and may represent a shift from CRH to AVP in the regulation of the HPA axis. It appears that LTH advances the normal ontogenic maturation of POMC processing to ACTH at the anterior pituitary via actions at the level of CRH and more likely, AVP neurons, in the parvocellular PVN (Figure 1). Upon casual observation, while this helps to explain the increased cortisol response to a secondary stressor in the LTH fetus, it is in conﬂict with the noted maintained normal basal plasma cortisol levels in these fetuses. Importantly, the fetal adrenal cortex seems to have developed an adaptive mechanism preventing an early maturation culminating with a premature rise in fetal plasma cortisol resulting in a growth restricted, preterm fetus. Thus, we shifted our focus to the adrenal cortex. 4. Effect of LTH on the Ovine Fetal Adrenal Cortex Based on the elevated basal plasma ACTH1−39 noted in the LTH fetuses and the capacity for greater cortisol production in response to a secondary stressor, we initially hypothesized that the LTH adrenal cortex would have responded corre- spondingly with an increase in expression of key enzymes regulating cortisol biosynthesis as well as supporting genes such as StAR and the ACTH receptor. However, despite the elevated basal plasma ACTH1−39, expression of two key enzymes mediating cortisol synthesis (CYP11A1 and CYP17) was approximately 50% lower in the LTH adrenal cortex as was the ACTH receptor [38]. Although we initially did not observe any change in StAR mRNA, we did note an increase in the 30 kDa spent form of the cholesterol transporter protein. An increased transport of cholesterol into the inner mitochondrial membrane, the site of the ﬁrst limiting step in cortisol biosynthesis via CYP11A1 could help maintain basal plasma levels of cortisol noted in LTH fetuses despite the noted lowered expression of the steroidogenic enzymes. The ﬁndings of decreased, not increased, levels of genes whose expression is exquisitely dependent upon ACTH in the LTH adrenal cortex led to a key fundamental question: namely, what LTH-stimulated mechanism(s) would provide essentially a brake on early activation and excessive produc- tion of cortisol, preventing early birth and growth restric- tion? Initially, we analyzed expression of a key transcription factor, SF-1, via which ACTH coordinates expression of its responsive genes in the adrenal cortex. Levels of SF-1 as well as DAX-1, a transcription factor repressing expression of CYP17 and CYP11A1, were not altered in the LTH adrenal cortex [38]. Since these factors also play a key role in adreno- cortical diﬀerentiation and growth, this is consistent with no diﬀerences in adrenal zonation or size in the LTH fetus, but