Chapter 6 Isolation of Ram Sperm Flagella

Abstract

Publisher Summary This chapter describes a method for obtaining ram sperm tails by sonication. This is a modified version of that developed by Calvin (1976). Calvin described a sonication procedure for the separation of heads and tails of rat sperm that was applied to rabbit sperm. Adjustment of the energy input and time of sonication enabled the fractionation of bovine, porcine, and murine sperm into substructures such as heads and tails. The method also yields nearly pure sperm heads. Included in the chapter is a demembranation procedure for sperm tails that may be employed in studies, for example, where direct access to the sperm axoneme is desired. It has been used to study the CAMP-dependent phosphorylation of ram sperm axonemal proteins and the effects of various inhibitors of sperm motility on the phosphorylation of these proteins. It is expected that these methods could be applied, with minor modifications, to the sperm of many other mammalian species. The chapter discusses the various solutions and materials required for preparation of ram sperm flagella and how to wash the sperms and separate the flagella from sperm heads. Triton X-100 is employed to demembranate the sperm flagella. Centrifugation steps move the sperm flagella into and out of the demembranating solution. This treatment ensures that the flagella are exposed to the detergent for only a short period, and is virtually identical to that used to prepare cytosol-free sperm models for reactivation. The solutions needed for demembration and the demembranation procedure have been provided in detail in the chapter.