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 sp erm cells. The cells were fractionated by nitrogen cavitation, and plasma m embranes were isolated by subsequent differential centrifugation steps. Mar ker enzyme measurement showed that the plasma membrane isolates were enrich ed in plasma membrane markers and did not contain nuclei, inner acrosomal m embranes, or mitochondria, Since there is no marker enzyme known for the ou ter acrosomal membrane, lectins were used for the detection of this membran e, The membrane specificity of a number of lectin conjugates was tested with f luorescence microscopy and transmission electron microscopy, Membrane bindi ng of these lectin conjugates was quantified with flow-cytometry and an enz yme-linked lectin binding assay. Wheat germ agglutinin was specific for the plasma membrane while peanut agglutinin was specific for the outer acrosom al membrane. The use of these lectins made it possible for the first time t o discriminate between these two membranes. The isolated plasma membrane fr action was enriched more than 10-fold (17-fold after further purification b y a sucrose gradient) in plasma membrane material compared to outer acrosom al membrane material. Highly purified sperm plasma membranes should prove to be useful for resear ch on primary sperm-zone interactions.