A simple technique for the purification of plasma membranes from ejaculated boar spermatozoa.
Spermatozoa were initially separated from fresh boar ejaculates using a 1.0 M sucrose density gradient. Spermatozoa (1 x 10(8) cells/ml) were subjected to gas cavitation (650 psi, 10 minutes), followed by a 4-step centrifugation technique to yield the final plasma membrane preparation. Purity of the plasma membrane isolate was determined using microscopic techniques (i.e. differential interference contrast and transmission electron microscopy) and marker enzymes for biochemical characterization. Plasma membranes were found to be removed primarily from the periacrosomal region of the sperm. Acrosomes appeared to remain intact on the cavitated spermatozoa. Transmission electron microscopy yielded a homogenous population of 100-200 microns unilamellar vesicles. Enzyme markers specific for plasma, acrosome and mitochondrial membranes substantial the purity observed under visual examination.
Althouse
G C
GC
Department of Veterinary Clinical Sciences, Iowa State University, Ames 50011, USA.
Bruns
K A
KA
Evans
L E
LE
Hopkins
S M
SM
Hsu
W H
WH
D015720:5'-Nucleotidase / Q000378:metabolism; D000176:Acrosin / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D008297:Male; D008854:Microscopy, Electron; D013094:Spermatozoa / Q000201:enzymology / Q000648:ultrastructure*; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013552:Swine
D013385:Succinate Dehydrogenase; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D000176:Acrosin