Detection of human sperm acrosome reaction: comparison between methods using double staining, Pisum sativum agglutinin, concanavalin A and transmission electron microscopy

The acrosome reaction is an important marker for human sperm function.Since different laboratory techniques may be used for the detection of thisexocytotic process, the purpose of the present study was to compare threecommon markers [Pisum sativum agglutinin (PSA), concanavalin A (ConA),double staining] and transmission electron microscopy for identification ofacrosomal changes. Preliminary findings had demonstrated that similarresults were achieved with Trypan Blue and Hoechst 33258 staining.Therefore, supravital stainings were omitted. In various experiments, humanspermatozoa were treated with two concentrations (10 and 3.3 microM) ofcalcium ionophore A23187 for 15, 30 and 60 min after capacitation for 3 and6 h at 37 degrees C. The percentages of spermatozoa with acrosomal lossdetected by fluorescein isothiocyanate (FITC)-ConA were consistently lowerthan those obtained by double staining or FITC-PSA, which showed comparableresults. Following 6 h of capacitation and incubation with 10 microMionophore for 1 h at 37 degrees C, 25.9 +/- 15.7% of all spermatozoa showedalmost complete loss of the acrosomal content. Binding of FITC- ConA to theacrosomal region was observed in 27.0 +/- 13.2% of spermatozoa obtainedfrom the same sample. FITC-ConA and double staining or FITC-PSA detectdifferent stages of the acrosome reaction and may be helpful for adifferentiated evaluation of this sperm function.