Induction of FSH-beta by TGF-Beta Family Members

描述（由申请人提供）：促卵泡激素（FSH）是一种α / β异二聚体，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。它的合成和分泌主要依赖于其β亚基（FSHB）的转录，这是严格调控的，但在体内可以变化超过20倍。该提案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将使用体外方法在羊和小鼠FSHB启动子上定位这些元件。瞬时表达突变的fshb启动子-荧光素酶（fshblc）结构将用于LbetaT2细胞（转化的小鼠促性腺激素），以检测关键反应元件的存在（或不存在）。缺失诱变已经在羊FSHB启动子中找到了激活作用所需的两个区域。AIM 2将确定这两个区域和AIM 1中确定的其他元素的生理相关性。具有突变反应元件的fshblc（5 - 7个构建体）将在转基因小鼠中表达，并检查其在体内的调节是否因功能失调的反应元件引起偏差，以确定其生理重要性。将在体内监测正常发情周期、性腺切除术后或用GnRH激动剂或拮抗剂治疗后的调节情况。激活素、抑制素或卵泡抑素的作用将在原代转基因垂体培养物中进行测试。AIM 3将表征5‘和3’ FSHB序列，这些序列允许FSHB近端启动子在促性腺激素体内正常发挥作用。DNA转移方法，如目标1，和DNA酶超敏试验将用于定位关键的DNA序列。然后，转基因小鼠将携带这些序列（1到3个结构）发生突变的fshblc，以确定5‘和3’区域在体内的重要性。目的1-3将定位最终影响哺乳动物卵子（和精子）产生的DNA元素。这些元素（或它们的结合蛋白）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，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.