POMC EXPRESSION AND PROCESSING IN PITUITARY

在绵羊妊娠的最后几周， 肾上腺皮质类固醇生成导致肾上腺皮质激素增加， 诱导分娩和器官成熟所必需的血浆皮质醇 对于新生儿的存活率。 胎儿垂体和下丘脑 室旁核（PVN）是启动成熟的组成部分， 肾上腺皮质应激反应 识别神经体液 胎儿垂体POMC基因表达和加工的调控 在了解胎儿HHAA的神经内分泌调节方面至关重要。 该提案有三个相关目标：1）确定 促肾上腺皮质激素释放激素（CRH）和精氨酸加压素（AVP）， 刺激绵羊胚胎垂体前叶POMC基因表达 (AP)（2）研究AVP和CRH在调节细胞生物合成中的作用。 将POMC加工成ACTH; 3）鉴定胎儿NIL的潜力 在绵羊妊娠的最后几周产生ACTH。 的 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POMC基因在胎儿AP中的表达 关键的怀孕阶段提出。 我假设AVP，而 作为绵羊胎儿的主要促ACTH分泌素， POMC基因表达，单独或与CRH协同; II：CRH和 AVP通过提高POMC向ACTH的转化率，促进POMC向ACTH的转化， 表达的PC 1到PC 2所需的ACTH生产中， 促皮质素 我推测CRH和AVP刺激PC 1的表达， 并抑制AP促肾上腺皮质激素中PC 2的表达。 我假设 CRH和AVP刺激CPE的表达，CPE是一种对 将含有ACTH的肽最终加工成ACTH; III：ACTH是一种 妊娠晚期胎儿NIL中合成的主要POMC衍生肽。 我推测在妊娠晚期绵羊中PC 1的表达增强 胎儿NIL与成人的酶表达模式相比，有利于ACTH 在胎儿NIL中产生。 具体目标（SA）1和2地址 假设I：为了定量CRH和AVP刺激的POMC基因转录， POMC RNA剪接（异源[hn] RNA加工）和稳态 胎儿AP细胞质POMC mRNA水平。 SA 2解决假设 II：定量CRH和AVP诱导的PC 1、PC 2和CPE水平的变化 胎儿AP促皮质激素的mRNA、蛋白质和酶活性。 SA 3解决了假设一和假设二：在最后30天， 在妊娠期，我将量化PC 1，PC 2，CPE mRNA，蛋白质 表达和POMC处理。 我还将量化POMC的比例 胎儿垂体前叶促皮质激素中hnRNA到POMC mRNA的表达 SA 4 假设三：在最后的30 dGA，我将量化 POMC基因表达作为POMC比例和数量的函数 在胎儿NIL中，hnRNA加工中间产物为细胞质mRNA， 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是否是一个 胎儿NIL的主要产物。 我将同时确定 PC 1、PC 2和CPE基因表达（mRNA和蛋白质）和酶 胎儿NIL中的活性。 为实现特定目标而建议的研究 图4和图5将确定ACTH免疫反应性的 形式和ACTH潜力的胎儿NIL在一个关键阶段， 绵羊胎儿的妊娠。 拟议的实验将建立 首次在绵羊中研究了PVN两种主要神经肽的作用 调节肾上腺皮质应激轴（CRH和AVP） POMC基因的表达和必需基因的表达和活性 在胎儿中将POMC翻译后加工为ACTH 促皮质素

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.