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Multiple Glycolytic Enzymes Are Tightly Bound to the Fibrous Sheath of Mouse Spermatozoa¹

Laboratories for Reproductive Biology,³ Departments of Pediatrics⁴ and Cell and Developmental Biology,⁵ University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

ABSTRACT

The fibrous sheath is a cytoskeletal structure located in the principal piece of mammalian sperm flagella. Previous studies showed that glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS), a germ cell-specific glycolytic isozyme that is required for sperm motility, is tightly bound to the fibrous sheath. To determine if other glycolytic enzymes are also bound to this cytoskeletal structure, we isolated highly purified fibrous sheath preparations from mouse epididymal sperm using a sequential extraction procedure. The isolated fibrous sheaths retain typical ultrastructural features and exhibit little contamination by axonemal or outer dense fiber proteins in Western blot analyses. Proteomic analysis using peptide-mass fingerprinting and MS/MS peptide fragment ion matching identified GAPDHS and two additional glycolytic enzyme subunits, the A isoform of aldolase 1 (ALDOA) and lactate dehydrogenase A (LDHA), in isolated fibrous sheaths. The presence of glycolytic enzymes in the fibrous sheath was also examined by Western blotting. In addition to GAPDHS, ALDOA, and LDHA, this method determined that pyruvate kinase is also tightly bound to the fibrous sheath. These data support a role for the fibrous sheath as a scaffold for anchoring multiple glycolytic enzymes along the length of the flagellum to provide a localized source of ATP that is essential for sperm motility.

fertilization, gamete biology, sperm, sperm motility and transport

¹ Supported by NICHD/NIH through U01 HD45982 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproductive Research and by the Andrew W. Mellon Foundation.

² Correspondence: Deborah A. O'Brien, Department of Cell and Developmental Biology, CB# 7090, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7090. FAX: 919 966 1856; dao{at}med.unc.edu

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