Mediators for dynamic regulation of Star transcription in Leydig cells

7.项目概要/摘要 睾丸内的间质细胞是促进胎儿和成年男性生殖力的雄激素的来源 阶段，但Leydig细胞群在每个年龄段都是不同的。因此，成人和胎儿间质细胞的功能 在不同的细胞和内分泌环境中合成睾酮。本提案的目标是 专注于调节星星，已知的看门人在控制胆固醇进入酶 转化为睾酮所需的一系列类固醇生成酶的活性。在这里，我们重点关注 新的调控事件，发挥动态相互作用与星星染色体位点，将解释如何 波动的星星转录物积累可能与雄激素合成的变化有关。此前我们 使用高分辨率荧光原位杂交（HR-FISH）来定位和量化一种独特的模式 与其他类固醇生成基因相比，星星的初级、剪接和mRNA种类积累 在单个Leydig细胞中。我们将使用这种方法和其他创新技术来比较 在体外研究单个成人和胎儿间质细胞的研究，在MA10细胞和原代 培养和体内，在整个睾丸内。脉冲LH主要刺激cAMP/PKA信号，以促进 睾丸激素在成年睾丸间质细胞中的合成。虽然我们有足够的手段来解释星星 当LH/PKA打开转录时，我们对脉冲被激活时发生的事情知之甚少。 移除，甚至更少关于脉冲间隔。在AIM 1中，我们将检验事件 在此期间发生在星星位点的运动对控制睾酮输出的重要性与最初的运动一样。 刺激。同时，维持胎儿睾丸间质细胞雄激素合成的外界刺激较少 这一点很清楚，但证据表明旁分泌信号，PKA活性发挥了作用。另一个旁分泌因子， 支持细胞来源的沙漠刺猬（Hh）是已知的启动胎儿Leydig细胞分化，但它的作用， 在维护方面还没有经过测试。一旦分化，胎儿Leydig细胞产生雄激素， 直到妊娠晚期，速率稳步增加。因此，在AIM 2中，我们将测试监管的假设， 星星基因座上的事件促进胎儿间质细胞内雄激素的受控增加， 与成年睾丸间质细胞脉冲间期发生的变化相比较。我们的发现有可能 解释类固醇控制的基础生物学，并将对我们的 能够探索睾酮合成障碍的机制，如内分泌失调， 破坏，导致男性从生命的各个阶段的重大临床分歧。

7. Project summary/Abstract Leydig cells within the testis are the source of androgens that promote virility at both fetal and adult stages, but Leydig cell populations are distinct at each age. As such, adult and fetal Leydig cells function to synthesize testosterone in distinct cellular and endocrine environments. The AIMs of this proposal are focused on regulation of Star, the known gatekeeper in controlling access of cholesterol to enzymatic activity of the series of steroidogenic enzymes required for conversion to testosterone. Here we focus on novel regulatory events that exert dynamic interactions with Star chromosomal loci that will explain how fluctuating Star transcript accumulation can relate to changes in androgen synthesis. Previously, we used high-resolution fluorescent in situ hybridization (HR-FISH) to localize and quantify a unique pattern for primary, spliced, and mRNA species accumulation for Star compared to other steroidogenic genes within single Leydig cells. We will use this and other innovative techniques to compare results from studies that investigate individual adult and fetal Leydig cells in vitro, within MA10 cells and primary cultures, and in vivo, within whole testes. Pulsatile LH stimulates primarily cAMP/PKA signals to promote testosterone synthesis in adult Leydig cells. While we have substantial means to explain how Star transcription is turned ON by LH/PKA, we understand little about what happens when the pulse is removed, and even less about the interpulse interval. In AIM 1, we will test the hypothesis that the events occurring at Star loci during this interval are just as critical to controlling testosterone output as the initial stimulus. Meanwhile, the external stimuli that maintain androgen synthesis in fetal Leydig cells are less clear, but evidence points to paracrine signals, with PKA activity playing a role. Another paracrine factor, Sertoli cell-derived Desert Hedgehog (Hh) is known to initiate fetal Leydig cell differentiation, but its role in their maintenance has not been tested. Once differentiated, fetal Leydig cells produce androgens at a steadily increasing rate until late gestation. Therefore, in AIM 2, we will test the hypothesis that regulatory events on Star loci facilitate a controlled increase in androgens within the fetal Leydig cell that compare to those that occur during the interpulse interval in adult Leydig cells. Our findings have the potential to explain fundamental biology underlying steroidogenic control and will have a profound impact on our ability to explore mechanisms by which disturbances in testosterone synthesis, as in endocrine disruption, cause significant clinical ramifications in males from all stages of life.