Role of HES/HEY family of proteins in mammalian spermatogenesis

描述（由申请人提供）：在哺乳动物睾丸中，精子发生始于一小群精原干细胞（ssc），这些细胞自我更新和分化最终产生精子。精子发生的任何分子事件的失败都会导致男性非阻塞性无精子症。大约1%的男性患有非阻塞性无精子症。在睾丸活检中，无精子症表现为仅支持细胞模式，成熟阻滞（MA）或低精子发生。在检查非阻塞性无精子症患者的研究中，大约30%的病例被确定为MA。在两项仅检查MA患者的研究中，发现所有患者都缺乏Notch-1受体或其配体Jagged。因此，每300-400名男性中就有1人可能由于Notch信号缺乏而导致睾丸功能衰竭。然而，迄今为止，Notch信号在睾丸中的确切作用尚不清楚。Notch蛋白（Notch1-4）是一种大的细胞表面受体，通过与邻近细胞（如Jagged和Delta）的膜结合配体接触而激活。激活后，Notch细胞内结构域（NICD）被切割并易位到细胞核，在那里它与dna结合蛋白结合并上调靶基因的表达，尤其是he /Hey转录抑制因子家族。Hes/Hey蛋白是bHLH转录因子，形成同源或异源二聚体以正常发挥作用。除了与其他bHLH蛋白（主要是共抑制因子）相互作用并结合特定的DNA启动子序列外，它们还可以募集染色质修饰剂，如组蛋白去乙酰化酶（hdac）。睾丸中存在许多Notch受体和配体，但迄今为止还没有功能性研究试图确定它们的确切作用。我们有证据表明Jagged1在未分化精原细胞中触发Notch1激活并上调Hes1，而在粗线精母细胞中，特别是在XY小体中，它触发Notch3激活并上调HeyL。因此，Jagged1的作用取决于目标细胞。在本应用中，我们将验证Notch信号通过上调Hes1和HeyL在精子发生的两个关键步骤(1)精原细胞分化和2)减数分裂时XY沉默)中起关键作用的假设。

DESCRIPTION (provided by applicant): In the mammalian testis, spermatogenesis starts from a small population of spermatogonial stem cells (SSCs) that self-renew and differentiate to ultimately produce sperm. Failure of any of the molecular events underlying spermatogenesis will result in non-obstructive azoospermia in men. Non-obstructive azoospermia affects approximately 1% of men. Upon testis biopsy, azoospermia presents with Sertoli cell-only pattern, maturation arrest (MA), or hypospermatogenesis. In studies examining patients with non obstructive azoospermia, approximately 30% of the cases were identified with MA. In two studies that examined only patients with MA, all were found to be deficient in either the Notch-1 receptor or its ligand Jagged. Thus, 1 in 300-400 men may have a form of testicular failure resulting from a deficiency in Notch signaling. However to date, the exact role of Notch signaling in the testis is unknown. The Notch proteins (Notch1-4) are large cell-surface receptors that are activated by contact with membrane-bound ligands on neighboring cells, such as Jagged and Delta (Dll). Upon activation, the Notch intracellular domain (NICD) is cleaved and translocates to the nucleus where it associates with DNA-binding proteins and upregulates the expression of target genes, most notably the Hes/Hey family of transcriptional repressors. Hes/Hey proteins are bHLH transcription factors that form homo or heterodimers to function properly. In addition to interacting with other bHLH proteins, mainly co-repressors, and binding to specific DNA promoter sequences, they also can recruit chromatin modifiers such as histone deacetylases (HDACs). The presence of a number of Notch receptors and ligands in the testis has been established, but to date no functional studies have been attempted to identify their exact role. We have evidence that Jagged1 triggers Notch1 activation and upregulates Hes1 in undifferentiated spermatogonia, while it triggers Notch3 activation and upregulation of HeyL in pachytene spermatocytes, specifically at the XY body. Therefore, the effects of Jagged1 depend on the target cell. In this application, we will test the hypothesis that Notch signaling, through up- regulation of Hes1 and HeyL, plays a crucial role at 2 critical steps of spermatogenesis, 1) spermatogonial differentiation and 2) XY silencing at meiosis.