DETECTION OF HUMAN SPERM ACROSOME REACTION - COMPARISON BETWEEN METHODS USING DOUBLE STAINING, PISUM-SATIVUM AGGLUTININ, CONCANAVALIN-A ANDTRANSMISSION ELECTRON-MICROSCOPY

The acrosome reaction is an important marker for human sperm function. Since different laboratory techniques may be used for the detection o f this exocytotic process, the purpose of the present study was to com pare three common markers [Pisum sativum agglutinin (PSA), concanavali n A (ConA), double staining] and transmission electron microscopy for identification of acrosomal changes. Preliminary findings had demonstr ated that similar results were achieved with Trypan Blue and Hoechst 3 3258 staining. Therefore, supravital stainings were omitted. Zn variou s experiments, human spermatozoa were treated with two concentrations (10 and 3.3 mu M) of calcium ionophore A23187 for 15, 30 and 60 min af ter capacitation for 3 and 6 h at 37 degrees C. The percentages of spe rmatozoa with acrosomal loss detected by fluorescein isothiocyanate (F ITC)-ConA were consistently lower than those obtained by double staini ng or FITC-PSA, which showed comparable results. Following 6 h of capa citation and incubation with 10 mu M ionophore for 1 h at 37 degrees C , 25.9 +/- 15.7% of all spermatozoa showed almost complete loss of the acrosomal content. Binding of FITC-ConA to the acrosomal region was o bserved in 27.0 +/- 13.2% of spermatozoa obtained from the same sample . FITC-ConA and double staining or FITC-PSA detect different stages of the acrosome reaction and may be helpful for a differentiated evaluat ion of this sperm function.