Isolation and partial purification of plasma membrane from porcine oocytes.
Egg plasma membrane (EPM) was isolated in comparatively large amounts from porcine slaughterhouse ovaries. Ovaries were minced, and the oocyte containing fluid was filtered to retrieve zona pellucidae-intact oocytes. The oocytes were homogenized and filtered again to remove zona pellucidae. The egg filtrate was subjected to differential centrifugation to remove membrane bound organelles and the remaining plasma membrane containing material was pelleted by ultracentrifugation. Plasma membranes were further separated from cellular material by sucrose density gradient centrifugation and were collected from portions of the gradient that correspond to the densities of plasma membrane. The purity of isolated plasma membranes was assessed by membrane marker enzyme analysis and transmission electron microscopy. Activities of the plasma membrane marker enzymes 5' nucleotidase and alkaline phosphatase increased from nondetectable levels in the egg filtrate to relatively high levels in the plasma membrane preparation. Marker enzymes for mitochondrial and lysosomal membranes fell from detectable levels in the egg filtrate to levels that were at the lower limits of the assays to detect in the final preparation. Evidence provided by binding of biotin-labeled EPM to capacitated sperm suggests that the isolated EPM retains its biological activity. The procedure presented here represents a novel method of isolating porcine egg plasma membranes for further study involving sperm-egg interaction.
Ash
K
K
Department of Animal Science, University of California, Davis 95616-8521.
Berger
T
T
Munn
R J
RJ
Horner
C M
CM
D015720:5'-Nucleotidase / Q000378:metabolism; D000135:Acid Phosphatase / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D005260:Female; D066298:In Vitro Techniques; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology; D009865:Oocytes / Q000201:enzymology / Q000648:ultrastructure*; D013094:Spermatozoa / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013552:Swine
D015415:Biomarkers; D013385:Succinate Dehydrogenase; D000469:Alkaline Phosphatase; D000135:Acid Phosphatase; D015720:5'-Nucleotidase