POMC EXPRESSION AND PROCESSING IN PITUITARY

在绵羊妊娠的最后几周，有成熟的 肾上腺皮质类固醇激素的生成导致围产期的增加 诱导分娩和器官成熟的血浆皮质醇 为了新生儿的存活。胎儿脑垂体和下丘脑 室旁核(PVN)是启动成熟期不可或缺的物质。 肾上腺皮质应激反应。辨别神经体液 胎儿期脑垂体POMC基因表达及加工的调控 对于了解胎儿HHAA的神经内分泌调节至关重要。 该提案有三个相关的目标：1)确定 血浆促肾上腺皮质激素释放激素和精氨酸加压素的变化 刺激绵羊胚胎垂体前叶POMC基因的表达 (AP)；2)确定AVP和CRH在调节生物合成中的作用 从POMC到ACTH的处理；3)识别胎儿Nil的潜力 在绵羊怀孕的最后几周产生促肾上腺皮质激素。这个 以下假设解决了我的目标：I：CRH是主要的下丘脑 神经肽刺激胎儿AP中POMC基因的表达 提出了孕育的关键阶段。我假设AVP，而 在绵羊胎儿中作为主要的促ACTH分泌剂，不会增强 POMC基因表达，单独或与CRH协同；II：CRH和 AVP均通过提高POMC与ACTH的比例来促进POMC/ACTH的加工 促肾上腺皮质激素产生所必需的PC1至PC2的表达 促肾上腺皮质激素。我假设CRH和AVP刺激PC1的表达 抑制促肾上腺皮质激素受体PC2的表达。我假设 促肾上腺皮质激素释放激素和血管紧张素转换酶刺激CPE的表达 ACTH含肽的最终加工为ACTH；III：ACTH是一种 妊娠晚期合成的主要POMC衍生肽为NIL。 我推测PC1在妊娠晚期绵羊体内的表达增强 与成人相比，胎儿NIL的酶表达模式有利于ACTH 在胎儿零的生产。特定目标(SA)1和2地址 假设一：为了量化CRH和AVP刺激的POMC基因转录， POMC RNA剪接(异核[HN]RNA处理)和稳定状态 胎儿AP胞浆POMC基因表达水平的研究SA 2解决了假设 II：量化CRH和AVP引起的PC1、PC2和CPE水平的变化 胎儿肾上腺皮质激素的信使核糖核酸、蛋白质和酶活性 SA 3解决了假设I和II的问题：在 妊娠，我将量化PC1，PC2，一种CPE mRNA，蛋白质的变化 表达和POMC处理。我还将量化POMC的比例 胎儿脑垂体前叶促肾上腺皮质激素中HNRNA与POMC mRNA的关系SA 4 和5解决假设III：在最后30个DGA期间，我将量化 POMC基因表达与POMC比例和数量的关系 在胎儿NIL-TO中，hnRNA加工为胞质mRNA的中间产物 测定NIL合成POMC的能力。SA 5：在 最后30 DGA，我将分析POMC处理以确定ACTH是否是 胎儿Nil的主要产品。我将同时确定 PC1、PC2和CPE基因表达(mRNA和蛋白)及酶促 胎儿无活动。为达到特定目标而建议进行的研究 4和5将决定ACTH免疫反应的分子同一性 胎儿近中性粒细胞形态和促肾上腺皮质激素潜能 在绵羊胎儿中怀孕。拟议的实验将为 首次在绵羊中发现两种主要的PVN神经肽的作用 刺激中调节肾上腺皮质应激轴(CRH和AVP) POMC基因表达与必需基因的表达和活性 胎儿POMC到ACTH的翻译后加工 促肾上腺皮质激素。

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.