ISOLATION OF THE FIBROUS SHEATH OF MAMMALIAN SPERM FLAGELLA

Axoneme is found along the axis of the mammalian sperm tail. It has outer dense fibers, mitochondrial sheath, and fibrous sheath. Three segments of the sperm tail have been defined. The cross-sectional pattern of the middle segment contains nine outer dense fibers in close proximity with the central axoneme. On the outside, this complex is encircled by the mitochondrial sheath; the annulus marks the caudal end of this segment. In most species, the main segment, or key piece, is the longer flagellar segment. The axoneme is in contact with seven outer dense fibers, and on the outside, the complex is surrounded by the fibrous sheath. This structure, and the dense fibers, poses a challenging problem in reproductive biology with regard to both their function in sperm motility and the synthesis and assembly of their corresponding polypeptides during spermiogenesis. Incubation of sperm with a solution containing 6 M urea and 2-mercaptoethanol or dithiothreitol dissolves most of the structure, leaving the fibrous sheath and the sperm heads. The methods discussed in this chapter are sperm isolation, fibrous sheath isolation, and purification of the fibrous sheath polypeptides. Rat sperm are obtained from the caput and cauda epididymis of adult Holtzman male rats. The animals are sacrificed by cervical dislocation and the epididymides freed of fatty tissue, minced with scissors, and suspended in cold PBS. Treatment of rat sperm for 12 hours with solution B containing 6 M urea plus dithiothreitol induces the dissolution of most cell structures with the exception of the fibrous sheath and the sperm head. The sucrose gradient centrifugation step yields a pure fraction of fibrous sheaths at the interface between the 0.9 and 1.8 M layers. After centrifugation in the discontinuous sucrose gradient no opalescent layer at the interface between the 0.9 and 1.8 M sucrose layers was noticed. © 1995 Academic Press Inc.