2-DIMENSIONAL ELECTROPHORETIC PROFILE OF HUMAN SPERM MEMBRANE-PROTEINS

The purpose of this study was to characterize highly enriched human sp ermatozoa membrane proteins by two-dimensional electrophoresis and com puter image analysis. Sperm membrane proteins were extracted by deterg ent solubilization from three different preparations: 1) washed semen cells following centrifugation and three wash steps in Ham's F-10 medi um (the standard sperm preparation, which is contaminated with seminal immature germ cells, white blood cells, and acellular material), 2) t he motile sperm fraction following centrifugation of diluted semen cel ls through a Percoll density gradient to enrich (>98%) the viable matu re sperm population, and 3) sperm membrane vesicles isolated from Perc oll-purified motile mature sperm by nitrogen cavitation followed by di fferential centrifugation. The two-dimensional gel profiles of extract s of washed semen cells and motile spermatozoa contained more than 600 protein spots between pH 4 and 7 and apparent molecular mass ranging from 7.9 to 93.5 kDa. Only 73% of the major proteins in these two samp les matched by computer image analysis. The highly enriched sperm memb rane vesicle extract showed a much simpler protein pattern, with only 64 major protein spots, 61 of which could be matched with proteins det ected in extracts from purified motile sperm. The isoelectric point an d molecular weight coordinates of these major human sperm membrane pro teins could serve as a foundation for systematic isolation and further characterization of human sperm antigens for studies of mechanisms of fertilization and the development of contraceptive vaccines.