Novel Centrosomal Proteins in Spermatogenesis and Sperm

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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.