Induction of FSH-beta by TGF-Beta Family Members

卵泡刺激素（FSH）是一种α/β异源二聚体，仅在垂体促性腺激素中产生， 在哺乳动物中协调卵巢卵泡发育导致卵子产生。其合成和 分泌主要依赖于其β亚基（FSHB）的转录，其受到严格调控，但可以变化 >20倍体内。这个提议的中心假设是激活素样诱导是最重要的 转录事件调控FSHB，其推论是这种诱导依赖于离散响应， FSHB近端启动子中的元件。AIM 1将使用体外方法在绵羊上定位这些元素 和小鼠FSHB启动子。突变的FSHB启动子荧光素酶（FSHBLuc）的瞬时表达 构建体将用于LbetaT 2细胞（转化的小鼠促性腺激素细胞）中以检测LbetaT 2细胞的存在（或 关键反应要素）。缺失诱变已经在绵羊中定位了两个区域 激活素作用所需的FSHB启动子。AIM 2将确定这两个生理相关性 区域和目标1中确定的其他要素。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，并将检查其体内调节是否存在由 功能失调的反应元件，以确定其生理重要性。规例将 在正常发情周期、性腺切除术后或GnRH治疗后进行体内监测 激动剂或拮抗剂。将在原代转基因小鼠中测试激活素、促卵泡素或卵泡抑素的作用。 垂体培养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。DNA转移方法，如目的1，和DNA酶超敏反应 分析将用于定位关键DNA序列。然后转基因小鼠将携带突变的FSHBLuc 在这些序列中（1至3个构建体），以确定5 ′和3 ′区域在体内的重要性。目标1-3 将定位最终影响哺乳动物卵子（和精子）产生的DNA元件。有害 这些元件（或其结合蛋白）的突变将改变生育力。我们的研究将表明 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FSHB基因的细胞靶向机制，该基因控制促性腺激素特异性 它可能是表达大多数内分泌激素的机制的原型。

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 >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.