Novel Centrosomal Proteins in Spermatogenesis and Sperm

描述(由申请人提供)：目的是确定一种新的中心体蛋白--蛇毒蛋白在精子发生和受精过程中的作用。除了在两极纺锤体和精子鞭毛的形成中发挥作用外，中心体还在调节细胞周期的蛋白质的泛素依赖的蛋白分解中发挥核心作用。后期促进复合体(APC)是一种多亚基泛素连接酶，定位于中心体。我们确定蛇毒蛋白是一种生精细胞特异的中心体蛋白，它与APC的激活剂Cdc20相关。蛇床子素定位于小鼠精母细胞和精子细胞的中心体，并存在于精子鞭毛的连接处。我们推测，蛇床子素调节APC活性，参与减数分裂的准确完成和精子连接件的形成或重塑。我们对蛇毒蛋白(SPM)基因敲除小鼠的初步研究表明，精子形态异常，男性生育能力受损。本研究的具体目的是：1)确定蛇床子素在精子发生中的作用。我们将确认基因敲除表型，并评估蛇床草素缺失对精子发生的影响。2)确定精子是否需要蛇床草素才能发育。我们将确定在Sprn/小鼠的精子中发生了什么结构和功能的变化，如果缺乏蛇毒蛋白导致非整倍体精子频率增加，以及突变的蛇毒蛋白等位基因的减少和非孟德尔遗传是否是由于染色体异常所致。3)确定蛇床子素是否调节精子细胞中APC依赖的泛素化。我们将a)从睾丸和精子细胞中分离APC并测定它们的泛素化能力，以及b)在体外泛素化实验中确定蛇床子素是否与分离的APC相关并能调节APC的活性。4)确定蛇床子素与其结合蛋白的功能关系。我们鉴定TZIP1是一种在生精细胞中有限制性表达的蛇毒蛋白结合蛋白。为了进一步定义有助于确定Speriolin功能的蛋白质相互作用，我们将：a)鉴定TZIP1的表达和亚细胞定位，并确定这种新的蛋白质是否是精子发生所必需的，以及b)鉴定和鉴定与Speriolin或TZIP1相关的其他蛋白质。这些研究有可能确定精子非整倍体和/或精子鞭毛颈部结构和功能缺陷的遗传原因。

DESCRIPTION (provided by applicant): The objective is to determine the role of speriolin, a novel centrosomal protein, during spermatogenesis and fertilization. In addition to its role in the formation of bipolar spindles and the sperm flagellum, the centrosome plays a central role in ubiquitin-dependent proteolysis of proteins that regulate the cell cycle. Components of the anaphase-promoting complex (APC), a multi-subunit ubiquitin ligase, are localized in the centrosome. We identified speriolin as a spermatogenic cell-specific centrosomal protein that associates with Cdc20, an activator of the APC. Speriolin is localized in the centrosome in mouse spermatocytes and spermatids and is present in the connecting piece of the sperm flagellum. We hypothesize that speriolin regulates APC activity, participating in the accurate completion of meiosis and in the formation or remodeling of the sperm connecting piece. Our initial studies of speriolin (Spm) knockout mice indicate that sperm morphology is abnormal and male fertility is impaired. The specific aims of this study are to: 1) Determine the role of speriolin in spermatogenesis. We will confirm the knockout phenotype and assess the effects speriolin loss on spermatogenesis. 2) Determine if sperm require speriolin to be developmentally competent. We will determine what structural and functional changes occur in sperm of Sprn / mice, if the absence of speriolin results in an increased frequency of aneuploid sperm, and if reduced litter sizes and non-Mendelian inheritance of the mutant speriolin allele are due to chromosomal abnormalities. 3) Determine if speriolin regulates APC-dependent ubiquitination in spermatids. We will a) isolate APCs from the testis and spermatids and assay their capacity for ubiquitination, and b) determine if speriolin is associated with isolated APCs and can modulate APC activity in in vitro ubiquitination assays. 4) Determine the functional relationship between speriolin and its associating proteins. We identified TZIP1 as a speriolin-binding protein with restricted expression in spermatogenic cells. To further define protein interactions that will help determine the function of speriolin, we will: a) characterize the expression and subcellular localization of TZIP1 and determine if this novel protein is required for spermatogenesis, and b) identify and characterize additional proteins that associate with speriolin or TZIP1. These studies have the potential for identifying a genetic cause of sperm aneuploidy and/or defects in the structure and function of the neck region of the sperm flagellum.