A METHOD FOR THE FRACTIONATION AND PURIFICATION OF SEA URCHIN SPERM

Procedures for fractionation and purification of sea urchin sperm subunits were studied. Fragmentation of cells was observed rather than fractionation of cell subunits when classic sonication or homogenization techniques were used. Calcium shock in alcohol solution which successfully removes Protozoa cilia was not effective in removing sperm flagella. Alcohol was found to induce cohesion among whole sperm rendering separation of isolated subunits by centrifugation impossible. Calcium shock without alcohol in the medium was not effective in achieving dissociation of sperm subunits. Successful fractionation of sperm subunits was achieved by application of a stepwise vortex-shearing action in a 0.22 micron filtered calcium-free medium. Analysis by Nomarski optics indicated little or no cellular fragmentation. Centrifugation with various discontinuous sucrose-sea water solutions proved to be a successful technique to purify the fractionated heads, midpieces, and tails. The procedures reported here can be used to obtain fractionated and purified sperm cell heads, midpieces, and tails. These procedures do not require enzyme, homogenization, or sonication treatment of the material.