Role of HES/HEY family of proteins in mammalian spermatogenesis

描述(申请人提供)：在哺乳动物的睾丸中，精子发生始于一小部分精原干细胞(SSCs)，它们自我更新和分化，最终产生精子。精子发生的任何分子事件的失败都将导致男性的非梗阻性无精子症。非梗阻性无精子症影响大约1%的男性。在睾丸活检中，无精子症表现为仅支持细胞模式、成熟停滞(MA)或生精不足。在检查非梗阻性无精子症患者的研究中，大约30%的病例被确定为MA。在两项只检查MA患者的研究中，所有患者都被发现Notch-1受体或其配体锯齿状缺陷。因此，每300-400名男性中就有一人可能因Notch信号缺乏而导致某种形式的睾丸衰竭。然而，到目前为止，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，通过与邻近细胞上的膜结合配体接触而被激活，如锯齿状和Delta(Dll)。一旦激活，Notch胞内域(NICD)被切割并移位到细胞核，在那里它与DNA结合蛋白结合，并上调靶基因的表达，最著名的是Hes/Hey转录抑制因子家族。HES/HY蛋白是bHLH转录因子，可以形成同源或异源二聚体来正常发挥功能。除了与其他bHLH蛋白相互作用，主要是共抑制物，并结合特定的DNA启动子序列，它们还可以招募染色质修饰物，如组蛋白脱乙酰酶(HDAC)。睾丸中存在许多Notch受体和配体已被证实，但到目前为止还没有功能研究试图确定它们的确切作用。我们有证据表明，Jagged1在未分化的精原细胞中触发Notch1激活并上调Hes1，而在粗线期精母细胞中触发Notch3激活并上调Heyl，特别是在XY小体。因此，Jagged1的效果取决于目标细胞。在这一应用中，我们将检验Notch信号，通过上调Hes1和Heyl，在精子发生的两个关键步骤中发挥关键作用，1)精原分化和2)减数分裂时XY沉默。 公共卫生相关性：精子发生是在睾丸中形成精子的过程。它始于精原干细胞，它可以自我更新或分化为更成熟的精原细胞。这些细胞发育成精母细胞，再经过减数分裂成为单倍体精子细胞和精子细胞。精子发生的任何分子事件的失败都会导致不育。最近的研究表明，许多不孕症病例可能与Notch信号通路的缺陷有关。Notch是一种位于生殖细胞表面的大型跨膜受体，它接受来自躯体支持细胞的信号。这项建议试图了解Notch及其下游细胞内靶点在精子发生的两个关键步骤中的功能：精原细胞分化和减数分裂。)

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. PUBLIC HEALTH RELEVANCE: Spermatogenesis is the process of sperm formation in the testis. It starts with a spermatogonial stem cell that self-renew or differentiate into more mature spermatogonia. These cells develop into spermatocytes that will undergo meiosis to become haploid spermatids and sperm cells. Failure of any of the molecular events underlying spermatogenesis will result in infertility. Recent studies indicate that a number of infertility cases might be linked to a defect in the Notch signaling pathway. Notch is a large transmembrane receptor at the surface of germ cells, which receives signals from the somatic nursing Sertoli cells. This proposal seeks to understand the function of Notch and its downstream intracellular targets at 2 critical steps of spermatogenesis: spermatogonial differentiation, and meiosis. )