In human and nonhuman primate pregnancy, progesterone (P4) and estrogen synthesized within the placenta and cortisol (F) produced by the fetal adrenal late in gestation play major roles in pregnancy maintenance and maturation of the fetus. The applicants have shown that estrogen plays a central integrative role in regulating placental P4 synthesis and in initiating de novo F synthesis by the fetal adrenal near term by modulating trans-placental F and cortisone (E) metabolism, which we propose activates the fetal hypothalamic-hypophyseal-adrenocortical axis (HPAA). The proposed consortium study will employ in vivo experimental paradigms in pregnant baboons and molecular biological methodologies to test the hypotheses that: [1] a developmental regulation of P4 biosynthesis exists which involves enhanced expression/translation of the mRNAs for the low density lipoproteins (LDL) receptor and mitochondrial cytochrome P450 cholesterol side-chain cleavage (scc) enzyme system within the placenta by estrogen; [2] the ontogenesis of fetal adrenal F production elicited by estrogen-induced alterations in transplacental F and E metabolism results from activation of corticotrophin releasing hormone (CRH)-ACTH production by the fetal hypothalamus-pituitary; and [3] that the increase in activities of the enzymes 17alpha-hydroxylase/17-20 lyase (17alpha-OHase) and delta5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) catalyzing de novo F production by the fetal adrenal near term, which result from activation of the HPAA by estrogen-induced changes in placental F-E metabolism, is the result of enhanced expression/translation of the mRNAs for these enzymes by fetal pituitary ACTH. In Study I, placental mRNAs and levels of LDL receptor and P450scc/adrenodoxin will be quantified by Northern/slot hybridization and Western immunoblot analyses, respectively, in baboons of early, mid and late gestation when estrogen is normally increasing, late in gestation following suppression of estrogen production by fetal hypophysectomy to remove ACTH-dependent C19-steroid precursors, and at midgestation in animals in which estrogen has been elevated by maternal administration of estradiol. In Experiment II-1, pituitary ACTH production will be determined by in vitro perifusion and expression of CRH and POMC mRNAs measured by quantitative in situ hybridization of hypothalamic/pituitary tissue obtained from baboon fetuses in which estrogen has been elevated and de novo F production initiated prematurely at midgestation and confirmed by analysis of adrenal steroidogenesis by in vitro perifusion. The importance of fetal ACTH per se to the initiation of de novo F production will be determined by treating baboon fetuses in utero with ACTH at midgestation. In Experiment II-2, the mRNAs (Northern/slot blot) for and peptide levels (Western immunoblot) of 3beta-HSD and 17alpha- OHase will be quantified in fetal adrenals obtained at midgestation following administration of estrogen or treatment of the fetus with ACTH and at term following fetal hypophysectomy with/without supplementation with ACTH. Results from these studies are expected to provide new insight into the regulation, at the genomic level, of fetoplacental steroid hormone biosynthesis, and thus ultimately improve our knowledge of the regulation of pregnancy maintenance and development of fetal self-sufficiency.
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