Induction of FSH-beta by TGF-Beta Family Members

描述（由申请方提供）：卵泡刺激素（FSH）是一种仅在垂体促性腺激素中产生的α/β异源二聚体，其协调哺乳动物的卵巢卵泡发育，导致卵子产生。它的合成和分泌主要取决于其β亚基（FSHB）的转录，该亚基受到严格调控，但在体内可以变化超过20倍。该建议的中心假设是激活素样诱导是调节FSHB的最重要的转录事件，其推论是这种诱导依赖于FSHB近端启动子中的离散响应元件。AIM 1将使用体外方法在绵羊和小鼠FSHB启动子上定位这些元件。突变的FSHB-启动子-荧光素酶（FSHBLuc）构建体的瞬时表达将用于LbetaT 2细胞（转化的小鼠促性腺激素细胞）中，以检测是否存在关键反应元件。缺失突变已经定位了激活素作用所需的绵羊FSHB启动子中的两个区域。目的2将确定这两个区域和目的1中确定的其他元素的生理相关性。具有突变的应答元件的FSHBLuc（5至7个构建体）将在转基因小鼠中表达，并且将针对由功能障碍的应答元件引起的偏差来检查它们的体内调节，以确定它们的生理重要性。在正常发情周期期间、性腺切除术后或GnRH激动剂或拮抗剂治疗后，将在体内监测调节。将在原代转基因垂体培养物中测试激活素、促卵泡素或卵泡抑素的作用。AIM 3将表征5'和3' FSHB序列，其允许FSHB近端启动子在促性腺激素体内正常发挥功能。将使用目的1中的DNA转移方法和DNA酶超敏反应试验来定位关键DNA序列。然后，转基因小鼠将携带在这些序列中具有突变的FSHBLuc（1至3个构建体），以确定5'和3'区域在体内的重要性。AIMS 1-3将定位最终影响哺乳动物卵子（和精子）产生的DNA元件。这些元素（或其结合蛋白）的有害突变将改变生育力。我们的研究将显示每种元素在体内的重要性，并预测每种元素的破坏在临床上是如何明显的。此外，AIM 3将开始表征控制促性腺激素特异性表达的FSHB基因的细胞靶向机制，并且它可能是表达大多数内分泌激素的机制的原型。

DESCRIPTION (provided by applicant): Follicle-stimulating homrone (FSH) is an alpha/beta heterodimer produced only in pituitary gonadotropes that orchestrates ovarian follicular development leading to egg production in mammals. Its synthesis and secretion depend primarily on transcription of its beta subunit (FSHB) which is tightly regulated, but can vary more than 20-fold in vivo. The central hypothesis of this proposal is that activin-like induction is the most important transcriptional event regulating FSHB, and its corollary is that this induction depends on discrete response elements in the FSHB proximal promoter. AIM 1 will use in vitro methods to locate these elements on ovine and mouse FSHB promoters. Transient expression of mutated FSHB-promoter-luciferase (FSHBLuc) constructs will be used in LbetaT2 cells (transformed mouse gonadotropes) to detect the presence (or absence) of critical response elements. Already deletion mutagenesis has located two regions in the ovine FSHB promoter required for activin action. AIM 2 will determine the physiological relevance of these two regions and other elements identified in aim 1. FSHBLuc (5 to 7 constructs) with mutated response elements will be expressed in transgenic mice, and their regulation in vivo will be examined for deviations caused by the dysfunctional response elements(s) to determine their physiological importance. Regulation will be monitored in vivo during the normal estrous cycle, after gonadectomy or following treatment with GnRH agonists or antagonists. The effects of activin, inhibin or follistatin will be tested in primary transgenic pituitary cultures. AIM 3 will characterize 5' and 3' FSHB sequences that allow the FSHB proximal promoter to function properly in vivo in gonadotropes. DNA transfer methods, as in aim 1, and DNAse hypersensitivity assays will be used to locate key DNA sequences. Then transgenic mice will carry FSHBLuc with mutations in these sequences (1 to 3 constructs) to determine the importance of the 5' and 3' regions in vivo. AIMS 1-3 will locate DNA elements that ultimately affect egg (and sperm) production in mammals. Deleterious mutations of these elements (or their binding proteins) will alter fertility. Our studies will show the importance of each element in vivo and predict how destruction of each element will be evident clinically. Moreover, AIM 3 will begin to characterize a cell-targeting mechanism on the FSHB gene that controls gonadotrope-specific express on and it likely to be prototypical of mechanisms that express most endocrine hormones.