ArticleJournal of assisted reproduction and genetics1994
Electron microscopic study of human sperm membrane isolation.
Article in Journal of assisted reproduction and genetics, 1994. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Authors and funding
4 authors.
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Abstract
objectOur purpose was to isolated pure, homogeneous human sperm membranes, free of cellular contaminants.
methodsDonor 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.
conclusionThe advantage of this method is its simplicity. The drawback of this method is the large number of spermatozoa required for membrane purification.
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