Steroidogenic Factor 1: Mediator of Gonadal Function

描述（由申请人提供）：孤儿核受体甾体生成因子1 （SF-1）在内分泌发育和功能中起重要作用。SF-1基因敲除（KO）小鼠表现为肾上腺和性腺发育不全，垂体促性腺功能受损，下丘脑腹内侧核（VMH）结构明显异常。拟议的研究旨在扩大我们对SF-1这些多效性作用的理解，特别关注其在性腺发育中的作用。首先，我们将使用Cre-loxP技术灭活睾丸和卵巢特定细胞系中的SF-1。通过使用不同的启动子来指导Cre表达，我们设想我们将实现细胞类型特异性失活以及不同发育阶段的失活。我们还将使用由SF-1调控序列引导的转基因增强型绿色荧光蛋白（eGFP）报告基因，从野生型和SF-1 KO小鼠的性腺中纯化表达SF-1的细胞，使我们能够比较在SF-1存在或不存在的情况下相同细胞群的基因表达谱。我们将对这些候选基因的5侧区域进行瞬时转染实验，以确定它们是SF-1的直接靶点还是间接靶点。为了开发一种机制，在促性腺激素发生的早期阶段灭活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的位点。最后，我们将尝试确定生殖腺中调节SF-1表达的序列，重点关注与dna酶超敏位点相关的第6内含子中的保守序列。这些研究将为SF-1如何在性腺发育和功能中发挥其多重作用提供新的见解。

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.