Proteomic Profiling of Accessory Structures from the Mouse Sperm Flagellum

Abstract

The flagellum of a mammalian spermatozoon consists of an axoneme surrounded in distinct regions by accessory structures known as the fibrous sheath, outer dense fibers, and the mitochondrial sheath. Although 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 assemblies. Epididymal mouse sperm were treated with SDS to dislodge sperm heads and to extract the axoneme and membranous elements. The remaining flagellar accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal Coomassie 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 that could be assigned to five general categories: 1) proteins previously reported to localize to the accessory structures, e.g. ODF2 in the outer dense fibers, the spermspecific glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath, and glutathione peroxidase in the mitochondrial sheath, 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. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This proteomic analysis identified a number of unexpected and novel proteins in the accessory structures of the mammalian flagellum. The mammalian sperm flagellum is a complex structure whose proper assembly and function are critical for sperm motility and subsequent fertilization of the egg. Morphologically the flagellum is characterized by three distinct regions: the midpiece, the principal piece, and the end piece. Common to these three segments is the axoneme or "motor" of the flagellum that runs the length of this structure and is composed of the typical "9 ϩ 2" array of microtubules (1, 2). In the different segments, the axoneme may be surrounded by accessory structures: the mitochondrial sheath, outer dense fibers (ODFs), 1 and/or the fibrous sheath (FS). The midpiece is proximal to the sperm head and contains nine ODFs paired with the nine peripheral doublet microtubules, forming a "9 ϩ 9 ϩ 2" pattern. The mitochondrial sheath contains all the mitochondria of the sperm and is helically arranged around the ODFs in this region. Proximal to the midpiece is the principal piece. Seven of t...

TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa

“…3D). However, these results demonstrate that TCTE1 is associated with the axoneme (40). Confocal microscopy reveals the FLAG signal along the flagella of mouse spermatozoa in agreement with other work that shows that DRC5 is located along the axoneme (Fig.…”

A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility

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A proteomic analysis on human sperm tail: comparison between normozoospermia and asthenozoospermia

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