PARENT SESSION

(85) PROTEOMIC PROFILING OF SDS-RESISTANT STRUCTURES FROM THE SPERM FLAGELLUM.

Cao, Wenlei¹, Gerton, George¹, Moss, Stuart¹, ¹ Center for Research on Reproduction and Women’s Health, Philadelphia, PA

ABSTRACT- The flagellum of a mammalian spermatozoon contains a fibrous sheath (FS), outer dense fibers (ODFs), and a mitochondrial sheath (MS), all of them surrounding the axoneme in distinct subregions of the tail. While the characterization of individual proteins has provided clues about the roles of these accessory structures, a more complete understanding of flagellar function requires the identification of all the polypeptides in these structures. Epididymal sperm were treated with SDS to dissociate sperm heads from tails and the accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal-Coomasie blue staining showed a highly reproducible pattern of >200 spots. Individual spots were picked, digested with trypsin and identified by mass spectrometry and peptide microsequencing. Approximately 50 individual proteins were identified which could be separated into general categories: 1) Proteins previously reported to localize to these structures, e.g., ODF1-2 in the ODFs, AKAP3 and GAPD-S in the FS, glutathione peroxidase in the MS, validating this proteomic approach, 2) Proteins that had not been shown to localize to any accessory structure, but would be predicted to be present, e.g., glycolytic enzymes, 3) Proteins known to be part of the flagellum but not localized to a specific site, e.g., adenylate kinase, 4) Proteins not expected to be part of the accessory structures, based on their previously-reported locations, e.g., tektins, and 5) Unknown proteins for which no information is available to make a determination as to location, e.g., Riken clones. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This study is the first proteomic analysis of the accessory structures of the mammalian sperm and identified a number of unexpected and novel proteins within these structures.

KEY WORDS: Mus musculus, sperm tail, proteomics, two dimensional gel electrophoresis