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During the final weeks of gestation in sheep there is maturation of adrenocortical steroidogenesis resulting in the prepartum increase in plasma cortisol that induces parturition and organ maturation essential for neonatal survival. The fetal pituitary and hypothalamic paraventricular nucleus (PVN) are integral in initiating maturation of the adrenocortical stress response. Identifying the neurohumoral regulation of POMC gene expression and processing in the fetal pituitary is paramount in understanding neuroendocrine regulation the fetal HHAA. This proposal has three related goals: 1) to determine the role of corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) in stimulating POMC gene expression in the ovine fetal anterior pituitary (AP); 2) identify the roles of AVP and CRH in regulating the biosynthetic processing of POMC to ACTH; 3) to identify the potential of the fetal NIL to produce ACTH during the final weeks of gestation in sheep. The following hypotheses address my goals: I: CRH is the primary hypothalamic neuropeptide stimulating POMC gene expression in the fetal AP at the critical stage of gestation proposed. I hypothesize that AVP, while acting as a major ACTH-secretagogue in the ovine fetus, does not enhance POMC gene expression, either alone or in synergy with CRH; II: CRH and AVP both stimulate the processing of POMC to ACTH by enhancing the ratio of expression of PC1 to PC2 necessary for ACTH production in the corticotrope. I hypothesize that CRH and AVP stimulate expression of PC1 and suppress expression of PC2 in the AP corticotrope. I hypothesize that CRH and AVP stimulate expression of CPE, an enzyme critical for the final processing of ACTH containing peptides to ACTH; III: ACTH is a major POMC derived peptide synthesized in the late gestation fetal NIL. I hypothesize that enhanced expression of PC1 in the late gestation sheep fetus NIL compared to the adult pattern of enzyme expression favors ACTH production in the fetal NIL. Specific Aims (SA) 1 and 2 address Hypothesis I: To quantify CRH and AVP stimulated POMC gene transcription, POMC RNA splicing (heteronuclear [hn] RNA processing), and steady-state levels of cytoplasmic POMC mRNA in fetal AP. SA 2 addresses Hypothesis II: To quantify CRH and AVP induced changes in levels of PC1, PC2 and CPE mRNA, protein and enzymatic activities in corticotropes of the fetal AP. SA 3 addresses both Hypothesis I and II: During the final 30 days of gestation, I will quantify changes in PC1, PC2, an CPE mRNA, protein expression and POMC processing. I will also quantify the ratio of POMC hnRNA to POMC mRNA in the fetal anterior pituitary corticotrope. SA 4 and 5 address Hypothesis III: During the final 30 dGA, I will quantify POMC gene expression as a function of the ratio and quantity of POMC hnRNA processing intermediates to cytoplasmic mRNA in the fetal NIL to determine the capacity of the NIL to synthesize POMC. SA 5: During the final 30 dGA, I will analyze POMC processing to determine if ACTH is a major product of the fetal NIL. I will concurrently determine levels of PC1, PC2 and CPE gene expression (mRNA and protein) and enzymatic activities in the fetal NIL. Studies proposed to achieve Specific Aim 4 and 5 will determine the molecular identity of ACTH immunoreactive forms and the ACTH potential of the fetal NIL during a critical stage of gestation in the ovine fetus. The proposed experiment will establish for the first time in sheep the roles of the two major PVN neuropeptides regulating the adrenocortical stress axis (CRH and AVP) in stimulating POMC gene expression and the expression and activities of genes necessary for the post-translational processing of POMC to ACTH in the fetal corticotrope.
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