A flow cytometric technique using peanut agglutinin for evaluating acrosomal loss from human spermatozoa.
Flow cytometric methods for the quantification of acrosome-reacted ejaculated human spermatozoa are described in which fluorescence-labeled peanut agglutinin (Arachis hypogaea) binding to the outer acrosomal membrane is used after incubation with ethidium homodimer (EHD) as vital dye and membrane permeation using methanol. Fluorescein-labeled fucoidan (F-fucoidin) was shown to bind to sperm that were also stained by propidium iodide (PI) and EHD; therefore, the use of F-fucoidin as vital dye was incorporated into the study. F-fucoidin and EHD can withstand the acrosome-staining procedure, unlike PI, which is leached out during cell processing. During incubation of spermatozoa, nonviable cells, as indicated by PI or F-fucoidin staining, appear in parallel with acrosome-reacted cells regardless of whether they are induced by the calcium ionophore A23187. The window of time in which viable, acrosome-reacted sperm cells can be detected is narrow and could depend on the vital dye as well as the acrosomal marker used.
Cooper
T G
TG
Institute of Reproductive Medicine of the University, Münster, Germany. cooper@uni-muenster.de
Yeung
C H
CH
D020101:Acrosome Reaction; D004996:Ethidium / Q000031:analogs & derivatives; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D037102:Lectins / Q000378:metabolism; D008297:Male; D019887:Peanut Agglutinin; D011134:Polysaccharides / Q000378:metabolism; D011419:Propidium; D013094:Spermatozoa / Q000502:physiology*; D013194:Staining and Labeling; D013997:Time Factors
D007202:Indicators and Reagents; D037102:Lectins; D019887:Peanut Agglutinin; D011134:Polysaccharides; D011419:Propidium; C018533:ethidium homodimer; C007789:fucoidan; D004996:Ethidium