Reactivation of demembranated, cytosol-free ram spermatozoa

Jovenal T. San Agustin, University of Massachusetts Medical School
George B. Witman, University of Massachusetts Medical School


A procedure for preparing cytosol-free ram sperm models was developed. Sperm are introduced to a Triton X-100-containing demembranation medium layered on top of a discontinuous Percoll gradient. After brief exposure to the demembranating solution, the sperm are separated from the detergent-soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they are recovered. Optimum conditions consisted of Triton X-100 at 0.20% and a demembranation time of 35 sec. Cross-sections of midpieces and principal pieces of the demembranated sperm were examined by electron microscopy. With 0.20% Triton X-100 in the demembranation medium, 86% of the cross-sections showed no plasma membranes and the rest had broken plasma membranes. The remaining tail structures appeared to be morphologically intact. Assay of phosphoglucose isomerase as a marker enzyme confirmed that at least 98% of the cytosolic protein was removed. Ram sperm models obtained by this procedure could be reactivated, and the percent motility and beat parameters were similar to those of the intact sperm. Reconstitution with the detergent-soluble components was neither required for, nor enhanced, reactivation. Therefore, demembranated ram sperm do not require a detergent-soluble protein factor for reactivation.