Aging and Leydig Cell Function

MERIT扩展申请中提出的研究解决了以下分子机制： 年龄诱导的睾丸间质细胞微环境变化，睾丸间质细胞负责睾丸雄激素 生物合成，导致睾酮形成减少。我们建议如下： 活性氧积累和抗氧化防御系统之间的关系导致氧化能力增加， 这反过来又导致质膜上的信号转导级联损伤， 降低老年间质细胞对促黄体激素（LH）的反应性。(ii)因此，老年人 细胞产生的cAMP比年轻细胞少，导致细胞内胆固醇转运到 线粒体，因此降低了睾酮的底物可用性。第一个目标是确定 增加氧化应激引起cAMP和睾酮产生减少的机制， 老化的睾丸间质细胞我们将检验这一假设，即在对氧化应激增加的反应中， LH受体（LHR）与Gs蛋白偶联缺陷，导致对LH反应的cAMP减少 刺激.第二个目的是检验这一假设，即由于LH的能力减弱， 在老化的Leydig细胞中刺激cAMP产生，存在降低的转运蛋白（TSPO）水平， 类固醇生成急性调节蛋白（星星）动员的胆固醇，从而降低了 胆固醇进入线粒体内膜。我们进一步假设， 衰老细胞中的TSPO是由天然反义转录物（NAT）表达减少引起的。第三 目的是确定布朗挪威大鼠睾丸间质细胞与年龄相关的变化， 老化的人类睾丸有了这个目标，我们将把我们在大鼠身上的发现转化为人类。为此我们将 测量年轻和老年男性睾丸内液中的睾酮和ROS水平，捕获Leydig细胞， 从睾丸活检中分析类固醇生成酶的表达和相关蛋白的表达 在活性氧的产生和抗氧化防御系统，并检查蛋白质和脂质 由于亲/抗氧化剂的变化而受损。使用固定组织平衡。

The studies proposed in this MERIT extension application address the molecular mechanisms by which age-induced changes in the microenvironment of Leydig cells, the cells responsible for testicular androgen biosynthesis, lead to reduced testosterone formation. We propose the following: (i) Changes in the balance between reactive oxygen accumulation and the antioxidant defense system result in increased oxidative stress which, in turn, results in damage to the signal transduction cascade at the plasma membrane and thus in reduced responsiveness of aged Leydig cells to luteinizing hormone (LH). (ii) As a consequence, aged cells produce less cAMP than young cells, resulting in reduced intracellular cholesterol transport into the mitochondria and therefore reduced substrate availability for testosterone. The first aim is to determine the mechanism by which increased oxidative stress elicit reductions in cAMP and testosterone production in aged Leydig cells. We will test the hypothesis that in response to increased oxidative stress, there is defective coupling of LH receptors (LHR) to Gs proteins, resulting in reduced cAMP in response to LH stimulation. The second aim will test the hypothesis that as a consequence of the blunted ability of LH to stimulate cAMP production in aged Leydig cells, there are reduced levels of translocator protein (TSPO) and steroidogenic acute regualtory protein (STAR)-mobilized cholesterol, and thus less efficient transfer of cholesterol to the inner mitochondrial membrane. We further hypothesize that the reduced expression of TSPO in aged cells results from that reduced expression of Natural Antisense Transcripts (NATs). The third aim is to determine how the age-related changes in Brown Norway rat Leydig cells compare to those in aging human testes. With this aim, we will translate our findings in the rat to the human. To this end, we will measure testosterone and ROS levels in intratesticular fluid from young and aged men, capture Leydig cells from testicular biopsies to analyze steroidogenic enzyme expression and the expression of proteins involved in reactive oxygen production and in the antioxidant defense system, and examine proteins and lipids damaged by changes in the pro/antioxidant.balance using fixed tissue.