OBJECT: Our purpose was to isolated pure, homogeneous human sperm membranes, free of cellular contaminants. METHODS: Donor semen samples collected 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 membrane isolation was investigated. Sonicated samples were centrifuged (500 g, 5 min) and the supernatant was pipetted off. The supernatant of the centrifuged sample was layered on either a sucrose cushion (supernatant on 1.6 M sucrose) or a discontinuous sucrose gradient and centrifuged (100,000 g, 1 hr). Contents of supernatants of sonicated samples and fractions (sucrose interfaces) were then fixed in 1.0% tannic acid and 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 cushion was found to be inadequate, showing significant cellular contamination. (3) Sperm membrane isolation from the sucrose interface between 0.75 and 1.05 M sucrose was found to be most effective. CONCLUSION: The advantage of this method is its simplicity. The drawback of this method is the large number of spermatozoa required for membrane purification.
OBJECT: Our purpose was to isolated pure, homogeneous human sperm membranes, free of cellular contaminants. METHODS:Donor semen samples collected 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 membrane isolation was investigated. Sonicated samples were centrifuged (500 g, 5 min) and the supernatant was pipetted off. The supernatant of the centrifuged sample was layered on either a sucrose cushion (supernatant on 1.6 M sucrose) or a discontinuous sucrose gradient and centrifuged (100,000 g, 1 hr). Contents of supernatants of sonicated samples and fractions (sucrose interfaces) were then fixed in 1.0% tannic acid and 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 cushion was found to be inadequate, showing significant cellular contamination. (3) Sperm membrane isolation from the sucrose interface between 0.75 and 1.05 M sucrose was found to be most effective. CONCLUSION: The advantage of this method is its simplicity. The drawback of this method is the large number of spermatozoa required for membrane purification.
Authors: S Mathur; L Chao; J M Goust; G T Milroy; C Woodley-Miller; J Z Caldwell; J Daru; H O Williamson Journal: Am J Reprod Immunol Microbiol Date: 1988-05