Mediators for dynamic regulation of Star transcription in Leydig cells

7.项目摘要/摘要 睾丸内的间质细胞是促进胎儿和成人生育能力的雄激素的来源。 但间质细胞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。这项提议的目的是 重点研究了控制胆固醇进入酶的已知守门人Star的调控 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 与恒星染色体基因发生动态相互作用的新调控事件将解释 波动的Star转录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)对独特的模式进行定位和定量 与其他类固醇生成基因相比，Star基因的初级、剪接和mRNA物种积累 在单个间质细胞内。我们将使用这项技术和其他创新技术来比较 研究单个成人和胎儿间质细胞在体外、MA10细胞和原代细胞内的情况 培养，在体内，在整个睾丸内。搏动性促黄体生成素主要刺激cAMP/PKA信号促进 成年间质细胞中睾酮的合成。虽然我们有实质性的手段来解释恒星是如何 转录是由黄体生成素/蛋白激酶A启动的，我们对脉冲被激活时会发生什么知之甚少 去除，甚至更少关于脉搏间的间隔。在目标1中，我们将检验以下假设 在这个时间间隔内发生在恒星基因座上的事件对于控制睾丸素的输出与最初的 刺激。与此同时，维持胎儿间质细胞雄激素合成的外部刺激较少 很清楚，但有证据表明有旁分泌信号，PKA活动也起到了作用。另一个旁分泌因素， 支持细胞来源的沙漠刺猬(HH)是已知的启动胎儿间质细胞分化的细胞，但它的作用 在它们的维护方面还没有经过测试。一旦分化，胎儿间质细胞就会在一年内产生雄激素 直到妊娠晚期，速度一直在稳步增加。因此，在AIM 2中，我们将检验监管的假设 恒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 而不是发生在成年间质细胞的脉搏间期。我们的发现有可能 解释类固醇生成控制的基础生物学，并将对我们的 探索睾酮合成紊乱的机制的能力，如内分泌 精神分裂，在男性生命的各个阶段都会造成重大的临床后果。

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.