ELECTRON-MICROSCOPIC STUDY OF HUMAN SPERM MEMBRANE ISOLATION

Object: Our purpose was to isolated pure, homogeneous human sperm memb ranes, free of cellular contaminants. Methods: Donor semen samples col lected after masturbation were stored at -70 degrees C and eventually pooled. Each attempt at sperm membrane isolation required 800 x 10(6) spermatozoa which were sonicated by ultrasound (40% output; Vibra Cell ). The effect of sonication time (3 x 5, 3 x 15, and 180 sec) on membr ane isolation was investigated. Sonicated samples were centrifuged (50 0g, 5 min) and the supernatant was pipetted off. The supernatant of th e centrifuged sample was layered on either a sucrose cushion (supernat ant on 1.6 M sucrose) or a discontinuous sucrose gradient and centrifu ged (100,000g, 1 hr). Contents of supernatants of sonicated samples an d fractions (sucrose interfaces) were then fixed in 1.0% tannic acid a nd 2.5% buffered glutaraldehyde and examined electron microscopically using standard procedures. Results: (1) The optimal sonification time was found to be 3 x 15 sec. (2) Membrane isolation using a sucrose cus hion was found to be inadequate, showing significant cellular contamin ation. (3) Sperm membrane isolation from the sucrose interface between 0.75 and 1.05 M sucrose was found to be most effective. Conclusion: T he advantage of this method is its simplicity. The drawback of this me thod is the large number of spermatozoa required for membrane purifica tion.