Steroidogenic Factor 1: Mediator of Gonadal Function

描述(申请人提供)：孤儿核受体类固醇生成因子1(SF-1)在内分泌发育和功能中发挥重要作用。SF-1基因敲除(KO)小鼠表现为肾上腺和性腺发育不全，垂体促性腺激素功能受损，下丘脑腹内侧核(VMH)结构明显异常。拟议的研究试图扩大我们对SF-1的这些多效性作用的理解，特别是它在性腺发育中的作用。首先，我们将使用Cre-loxP技术在睾丸和卵巢的特定细胞系中灭活SF-1。通过使用不同的启动子来指导Cre的表达，我们设想我们将在不同的发育阶段实现细胞类型的特异性失活以及失活。我们还将使用SF-1调控序列控制的转基因增强型绿色荧光蛋白报告基因从野生型和SF-1 KO小鼠性腺中纯化表达SF-L的细胞，使我们能够比较相同细胞群体在存在和不存在SF-I的情况下的基因表达谱。我们将对这些候选基因的5-侧翼区进行瞬时转染实验，以确定它们是SF-1的直接或间接靶标。为了开发一种机制来灭活性腺发生早期阶段感兴趣的基因的表达，我们将使用BAC转基因将Cre重组酶的表达靶向到表达SF-1的位置。最后，我们将尝试定义调控性腺中SF-1表达的序列，重点放在第6内含子中与DNA酶敏感部位相关的保守序列上。这些研究将为SF-1如何在性腺发育和功能中发挥多种作用提供新的见解。

DESCRIPTION (provided by applicant): The orphan nuclear receptor steroidogenic factor 1 (SF-1) plays essential roles in endocrine development and function. SF-1 knockout (KO) mice exhibit adrenal and gonadal agenesis, impaired pituitary gonadotrope function, and marked structural abnormalities of the ventromedial hypothalamic nucleus (VMH). The proposed studies seek to expand our understanding of these pleiotropic roles of SF-1, focusing specifically on its roles in gonadal development. First, we will use the Cre-loxP technology to inactivate SF-1 in specific cell lineages of the testes and ovaries. By using different promoters to direct Cre expression, we envision that we will achieve both cell-type specific inactivation as well as inactivation at different stages of development. We also will use a transgenic enhanced green fluorescent protein (eGFP) reporter gene directed by SF-1 regulatory sequences to purify SF-l-expressing cells from the gonads of wild-type and SF-1 KO mice, allowing us to compare gene expression profiles of the same cell populations in the presence or absence of SF-I. We will use transient transfection experiments with the 5-flanking regions of these candidate genes to determine if they are direct or indirect targets of SF-1. To develop a mechanism to inactivate the expression of genes of interest from early stages of gonadogenesis, we will use BAC transgenesis to target expression of Cre recombinase to the sites where SF-1 is expressed. Finally, we will attempt to define the sequences that regulate SF-1 expression in the gonads, focusing on a conserved sequence in the 6th intron that is associated with a DNase hypersensitive site. These studies will provide novel insights into how SF-1 exerts its multiple actions in gonadal development and function.