Preparation of a plasma membrane-rich fraction from rat spermatozoa

Monoclonal antibodies to antigens located on surface and intracellular membranes of spermatozoa from the rat cauda epididymidis have been used as probes to assess the purity of putative plasma membrane fractions. Spermatozoa were demembranated by shearing forces generated on a vortex-mixer. Immunofluorescence and ultrastructural analysis of vortex-mixed spermatozoa showed that they were denuded of approximately 90% of surface membrane. Areas of acrosomal membranes were also removed. Crude plasma membranes were recovered in low-speed wash fluids and fractionated on a 13-23% Nycodenz density gradient. Three bands containing membrane vesicles were resolved. Absorption curves and direct binding assays using monoclonal antibodies specific for acrosomal membranes, mitochondrial membranes and fibrous sheath showed relatively strong binding to bands 1 and 2 but weak binding to band 3. Conversely a monoclonal antibody specific for a surface membrane antigen bound strongly to band 3 and weakly to bands 1 and 2. Identification on immunoblots of the antigens recognized by the monoclonal antibodies revealed that band 3 was positive for surface membrane antigens but gave no reaction for intracellular antigens. However, bands 1 and 2 were strongly positive for intracellular components. The results suggest that vortex-mixing is a simple and efficient means of removing the plasma membrane from spermatozoa and that a membrane fraction can be recovered from a Nycodenz density gradient that is enriched 40- to 50-fold in surface antigens.