Isolation of plasma membranes from purified mouse spermatogenic cells

Abstract

Plasma membranes have been prepared from purified pachytene spermatocytes, round spermatids and residual bodies of the adult mouse testis using procedures modified from other authors'. Isolated membranes have been examined using electron microscopy, lectin binding and enzymic assays. Ultrastructural observation reveals smooth unit-membrane vesicles from 0.4-1.7 micrometer diameter. No contamination by nuclei, mitochondria or lysosomes is detected microscopically. Radiolabelled lectin-binding experiments [125I-RCAI, 125I-green pea lectin] indicate that cell surface label cofractionates with material identified morphologically as plasma membrane. Estimates of total recovery of membrane, based upn the lectin data, average 33%. Biochemical analysis of subcellular markers reveal that no detectable DNA and only 1.2% of the total cellular RNA cofractionate with membranes. A variety of enzyme assays suggests little contamination by cytosol enzymes, Golgi material or mitochondria. Assays of 5′-nucleotidase (E.C. 3.1.3.5) indicate that this enzyme is not a major component of developing mouse spermatogenic cell membranes. Instead, Sertoli cells represent the most important source of this enzyme in the adult seminiferous tubule. Polyacrylamide gel analysis of membranes isolated from purified germ cells reveals significant differences in the protein compositions of pachytene spermatocyte and round spermatid membranes. The preparation of highly purified plasma membranes from homogeneous populations of spermatogenic cells should facilitate the biochemical characterization of cell surface antigens specific to developing male germ cells.