Novel Centrosomal Proteins in Spermatogenesis and Sperm

描述（由申请人提供）：目的是确定精子素（一种新型中心体蛋白）在精子发生和受精过程中的作用。除了在双极纺锤体和精子鞭毛的形成中起作用外，中心体在调节细胞周期的泛素依赖性蛋白质水解中起核心作用。后期促进复合体（APC）是一种多亚基泛素连接酶，其组分位于中心体中。我们发现精蛋白是一种生精细胞特异性中心体蛋白，与APC的激活剂Cdc20相关。精素定位于小鼠精母细胞和精子的中心体中，存在于精子鞭毛的连接处。我们假设精子素调节APC活性，参与减数分裂的准确完成和精子连接片的形成或重塑。我们对精子素（Spm）敲除小鼠的初步研究表明，精子形态异常，雄性生育能力受损。本研究的具体目的是：1)确定精素在精子发生中的作用。我们将确认敲除表型并评估精子素损失对精子发生的影响。2)确定精子是否需要精素才能发育。我们将确定Sprn /小鼠精子中发生的结构和功能变化，如果精子素的缺失导致非整倍体精子的频率增加，如果产仔数减少和突变精子素等位基因的非孟德尔遗传是由于染色体异常。3)确定精子素是否调控精子中apc依赖性泛素化。我们将a)从睾丸和精子中分离APC，并测定其泛素化能力，b)确定精素是否与分离的APC相关，并在体外泛素化实验中调节APC的活性。4)确定精磷脂与其相关蛋白之间的功能关系。我们发现TZIP1是一种在生精细胞中表达受限的精磷脂结合蛋白。为了进一步确定有助于确定精子素功能的蛋白相互作用，我们将：a)表征TZIP1的表达和亚细胞定位，并确定这种新蛋白是否为精子发生所必需，b)鉴定和表征与精子素或TZIP1相关的其他蛋白。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.