Maturation of pituitary adrenal function is poorly understood. This system is vital in the fetus, subserving a diverse series of roles from stimulating pulmonary surfactant production to modulating enzymes involved with the synthesis of neurotransmitters. In the present application, we will test the unifying hypothesis that input from the hypothalamus begins a maturational change of corticotrophs, which results in stimulation of fetal cortisol secretion and that fetal cortisol then enhances the developmental change in corticotroph phenotype and acts, as well, to increase ACTH receptor number on the fetal adrenal; these effects of cortisol increase fetal adrenal responsiveness to ACTH in late gestation. To test this hypothesis, we propose to: a) measure the proportion of corticotrophs which bind CRH or AVP, the binding sites for CRH and AVP and the message for CRH and AVP receptors at three stages of fetal development; b) determine if alterations in corticotroph phenotype are associated with alterations in the secretory profile for POMC and ACTH1-39 from individual corticotrophs; c) establish if input from the hypothalamus or changes in cortisol affect the change in corticotroph phenotype; and, d) determine if a physiological consequence of blocking the change in corticotroph phenotype is attenuation of the increase in adrenal responsiveness ordinarily seen close to term and if the mechanism of this attenuation in responsiveness is related to a reduction in ACTH receptor expression on fetal adrenal cells in late gestation. Fetal sheep are used for these experiments because we have defined the period in gestation when the functional alterations in corticotroph populations occur and we can surgically manipulate and study this model in utero. To accomplish our goal, we will employ florescent labelled ligand binding for the measurement of corticotroph phenotype, RNase protection assays for the measurement of steady-state CRH, AVP, and ACTH receptor message levels and immunoblot techniques for the studies of POMC secretion from single cells. To our knowledge, these will be the first studies to examine the factors promoting the maturational change in corticotroph phenotype in the fetus from a molecular, cellular, and whole animal basis simultaneously. Thus, the proposed studies will provide important, new information on the role of the hypothalamus and adrenal in the developmental regulation of corticotroph phenotype, the physiological consequences of changes in corticotroph phenotype and the mechanisms by which cortisol may influence corticotroph phenotype expression and adrenal responsiveness to ACTH. This information, when combined with results of our experiments to define the model for phenotype change, will help explain how the fetus prepares itself for birth and may be useful for the development of treatments to minimize the consequences of premature birth.
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