Mechanisms of Circadian Rhythmicity in CLOCK-Deficient Mice

描述(由申请人提供)：建议的研究将使用小鼠模型来调查两种情况的原因，不孕症和关节病。约10%的夫妇受到不孕不育的影响，其中约一半与男性不育有关。正常情况下未钙化(关节病)的组织中骨样物质的异常形成会干扰组织功能，限制运动，并且通常是疼痛的。异位钙化的形式从异位骨化(随着年龄的增长、创伤后的遗传和特发性)，到年龄相关的主动脉狭窄和主动脉瓣钙化(这是一个显著的并发症限制)，不一而足。 心脏和心脏瓣膜的外科修复)。因此，了解异位钙化的过程具有显著的潜在治疗益处。两个基因缺失的小鼠 参与昼夜节律发生的Clock和NPAS2，不仅失去了昼夜节律，而且还具有不孕症，并发展成严重的、与年龄相关的进行性异位钙化。Clock和NPAS2通常与BMAL1一起调节昼夜节律；BMAL1缺陷小鼠也有生育力下降和进行性关节病。由于其他遗传损伤，缺乏昼夜节律的动物不会出现这些病理表型，这表明可能是与生物钟功能无关的Clock/NPAS2：BMal1转录复合体的破坏导致了这些病理的发生。对全身基因缺失动物的研究因这些基因的多个潜在作用部位而变得复杂，从对生殖内分泌学和行为的潜在影响到精子发生的缺陷(对于不育症)，以及肌肉骨骼系统的多种潜在细胞类型(对于关节病)。因此，我们将使用先进的遗传学方法来产生组织特异性的遗传损伤，以识别和分离参与受精调节和关节疾病预防的细胞类型，从而能够评估导致病理状况的分子事件。我们的总体假设是，缺乏Clock和NPAS2(或BMAL1)的动物的局部基因表达失调导致了这些病理条件(软骨交界处特殊但定义不明确的细胞类型的钙化 肌腱到骨骼和不孕不育)。我们的总体目标是在体内使用一种无偏见的遗传方法来定义所涉及的细胞类型，然后使用这些相同的方法来标记所涉及的细胞类型，并在基因对于防止这些表型至关重要的时候将它们分离出来进行分子分析。识别在这种非常可重复性的小鼠遗传模型中发生的细胞和分子事件应该有助于理解并最终调节生育能力，并有助于理解并最终预防人类病理性异位钙化。

DESCRIPTION (provided by applicant): The proposed studies will use a mouse model to investigate causes for two conditions, infertility and arthropathy. Infertility affects ~ 10% of couples, with about half of this related to male infertility. Abnormal formation of bone-like substances in tissues that are normally not calcified (arthropathy) interferes with tissue function limits motion, and is often painful. Forms of ectopic calcification range from heterotopic ossification (occurs with aging, following trauma, and in genetic and idiopathic forms), to age-related aortic stenosis, and aortic valve calcification (which is a prominent complication limiting surgical repair of the heart and cardiac valves). Understanding the process of ectopic calcification thus has significant potential therapeutic benefit. Mice with disruption of two genes involved in circadian rhythm generation, CLOCK and NPAS2, not only lose their circadian rhythms, but they are also infertile and developed a severe, age-related and progressive ectopic calcification. CLOCK and NPAS2 normally partner with BMAL1 in regulating circadian rhythms; BMAL1- deficient mice are also have reduced fertility and progressive arthropathy. These pathological phenotypes do not occur in animals lacking circadian rhythms due to other genetic lesions, indicating that it is disruption of the CLOCK/NPAS2:BMAL1 transcriptional complexes, likely unrelated to circadian clock function, that allows these pathologies to develop. Studies of animals with gene disruption throughout the body are complicated by the multiple potential sites of action of these genes, ranging from potential effects on reproductive endocrinology and behavior to defects in spermatogenesis (for infertility), and multiple potential cell types of the musculo-skeletal system (for arthropathy). We will thus use advanced genetics approaches to produce tissue-specific genetic lesions, to identify and isolate the cell types involved in fertiliy regulation and in arthropathy prevention, allowing assessment of the molecular events that lead to the pathological conditions. Our overall hypothesis is that local dysregulation of gene expression in animals lacking CLOCK and NPAS2 (or BMAL1) allows these pathological conditions (calcification in specialized but poorly defined cell types at the junction of cartilage and tendon to bone, and infertility). Our overall objective is to define the cell types involved using an unbiased, genetic approach in vivo, and then use these same approaches to label the cell types involved and to isolate them for molecular analysis at times when the genes are critically important for preventing these phenotypes. Identification of the cellular and molecular events occurring in this very reproducible, genetic model in mice should contribute to understanding and ultimately regulating fertility, and for understanding and ultimately preventing pathological ectopic calcification in humans.