Use of lectins to characterize plasma membrane preparations from boar spermatozoa: a novel technique for monitoring membrane purity and quantity.
The object of this study was to develop a method to quantify the amount of outer acrosomal membrane material in isolated plasma membranes from boar sperm cells. The cells were fractionated by nitrogen cavitation, and plasma membranes were isolated by subsequent differential centrifugation steps. Marker enzyme measurement showed that the plasma membrane isolates were enriched in plasma membrane markers and did not contain nuclei, inner acrosomal membranes, or mitochondria. Since there is no marker enzyme known for the outer acrosomal membrane, lectins were used for the detection of this membrane. The membrane specificity of a number of lectin conjugates was tested with fluorescence microscopy and transmission electron microscopy. Membrane binding of these lectin conjugates was quantified with flow-cytometry and an enzyme-linked lectin binding assay. Wheat germ agglutinin was specific for the plasma membrane while peanut agglutinin was specific for the outer acrosomal membrane. The use of these lectins made it possible for the first time to discriminate between these two membranes. The isolated plasma membrane fraction was enriched more than 10-fold (17-fold after further purification by a sucrose gradient) in plasma membrane material compared to outer acrosomal membrane material. Highly purified sperm plasma membranes should prove to be useful for research on primary sperm-zona interactions.
Flesch
F M
FM
Department of Basic Sciences, Division of Biochemistry, Utrecht University, The Netherlands. f.slesch@vet.uu.nl
Voorhout
W F
WF
Colenbrander
B
B
van Golde
L M
LM
Gadella
B M
BM
D000177:Acrosome / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure*; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D005434:Flow Cytometry; D037102:Lectins / Q000378:metabolism*; D008297:Male; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009584:Nitrogen; D019887:Peanut Agglutinin / Q000378:metabolism; D013094:Spermatozoa / Q000648:ultrastructure*; D013552:Swine; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D015415:Biomarkers; D037102:Lectins; D019887:Peanut Agglutinin; D014909:Wheat Germ Agglutinins; D009584:Nitrogen