Lateral regionalization and diffusion of a maturation-dependent antigen in the ram sperm plasma membrane

Abstract

We have used a monoclonal antibody ESA152 in fluorescence recovery after photobleaching (FPR) studies of a maturation-dependent surface antigen of ram sperm. The antibody is an immunoglobulin G secreted by a hybridoma derived from NS1 mouse myeloma cells. The ESA152 antigen is not detectable in testicular sperm. It is localized on the surface of ejaculated sperm where it is present on all regions of the surface, but tends to be concentrated on the posterior region of the head. The ESA152 antigen can be extracted by detergents or chloroform-methanol. The extracted antigen is sensitive to proteases and migrates with an apparent M(r) ~ 30,000 in SDS-containing 10-20% polyacrylamide gradient gels. FPR measurements of ESA152 lateral mobility in the membrane yield diffusion coefficients in the range 10-9-10-8 cm2/s, values typical of lipids but observed for proteins only at the fluid dynamic limit where diffusion is controlled by lipid fluidity. Immobile fractions, typical of membrane proteins, are observed on all regions. When the antigen is stained by a fluoresceinated F(ab) fragment of the ESA152 antibody, the diffusibility is highly regionalized, with particularly low, but rapid, recovery on the midpiece. Cross-linking of the antigen with the intact ESA152 antibody induces a redistribution in which the antigen is excluded from the posterior head region. This cross-linking is accompanied by increases in ESA152 diffusibility on both the anterior head and the midpiece.