Reactivation of Motility of Demembranated Sea Urchin Sperm Flagella

Abstract

This chapter describes methods for spermatozoa from two sea urchin species that are readily available in the United States: Lytechinus pictus and Strongylocentrotus purpurutus. Both are cold water species, and the spermatozoa are damaged by high temperatures. Of the two species described here, L. pictus spermatozoa are less temperature sensitive and probably are not seriously damaged until the temperature exceeds 22°C. Iron contamination is a particularly serious problem with light sources that transmit significant power in the UV spectrum. The use of a UV filter is recommended. Solutions containing labile components should be made fresh for each day's work; the basic buffer solutions can safely be made up and refrigerated for use over 2 to 3 days. In the following recipes, Tris buffer refers either to trishydroxymethylaminomethane, in base form, with the solution pH lowered to 8.2 with acetic acid, or to an equimolar mixture of Tris base and Tris-HC1 that will give the desired pH with minimal adjustment. The procedures described here are designed to prepare “potentially symmetric” sperm flagella that generate reasonably symmetric bending waves at low free Ca^(2+) concentrations. The low-frequency reactivation solutions produce movements with frequencies of 1 to 2 cycles per second, and are particularly useful when observing with continuous illumination. The high-frequency reactivation solutions produce movement with frequencies of 30 to 40 cycles per second, comparable to the frequencies of live spermatozoa. When a drop of sperm suspension is placed on a microscope slide, the rapidly swimming spermatozoa quickly leave the interior of the drop and accumulate near surfaces, either sticking to the surface or swimming close to the surface with the plane of flagellar bending parallel to the surface. The chapter discusses the processes of reactivation of spermatozoa—Lytechinus pictus and strongylocentrotus purpuratus—, the required materials and detailed procedure.