Novel Centrosomal Proteins in Spermatogenesis and Sperm

描述（由申请人提供）：目的是确定speriolin（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性蛋白水解中发挥核心作用。后期促进复合物（APC）是一种多亚基泛素连接酶，其组分位于中心体中。我们确定speriolin作为生精细胞特异性中心体蛋白，与Cdc 20，APC的激活剂。Speriolin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段。我们推测speriolin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speriolin（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speriolin在精子发生中的作用。我们将确认敲除表型和评估speriolin损失对精子发生的影响。2)确定精子是否需要speriolin才能发育。我们将确定什么样的结构和功能的变化发生在精子的Sprn /小鼠，如果speriolin的情况下，在非整倍体精子的频率增加，如果减少产仔数和非孟德尔遗传的突变speriolin等位基因是由于染色体异常。3)确定speriolin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，和B）在体外泛素化测定中确定speriolin是否与分离的APC相关并可以调节APC活性。4)确定speriolin与其相关蛋白的功能关系。我们确定TZIP 1作为一个speriolin结合蛋白的限制性表达在生精细胞。为了进一步确定有助于确定speriolin功能的蛋白质相互作用，我们将：a）表征TZIP 1的表达和亚细胞定位，并确定这种新蛋白质是否是精子发生所需的，以及B）鉴定和表征与speriolin或TZIP 1相关的其他蛋白质。这些研究有可能确定精子非整倍性和/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.