Fluorescence-labeled fucolectins are superior markers for flow cytometric quantitation of the human sperm acrosome reaction

Objective: To evaluate the binding of three fluorescein (FITC)-labeled fucose-specific lectins, Anguilla anguilla agglutinin (AAA), Tetragonolobus purpureas agglutinin (TPA), and Ulex europaeus-1 agglutinin (UEA-1), to unfixed, acrosome-intact and acrosome-reacted (AR) human sperm by flow cytometry and to compare the results with those found using FITC-labeled Pisum sativum agglutinin (PSA) and Arachis hypogaea agglutinin (PNA). Design: The binding of five FITC-labeled lectins (PSA, PNA, AAA, TPA, and UEA-1) to capacitated, calcium ionophore A23187 (CaI)-treated, or solvent-treated human sperm was quantitated by fluorescence flow cytometry. The binding of FITC-labeled lectins was compared with the binding of anti-CD46 monoclonal antibody (mAb), a marker for the human sperm AR. The effect of fucose alpha-1-->2-, alpha-1-->3-, and alpha-1-->4-linked oligosaccharides to inhibit the binding of FITC-fucolectins to AR sperm also was tested. Setting: University of Oklahoma Health Sciences Center, a tertiary care referral center. Results: The average percentage of fluorescing, solvent-treated sperm labeled with PSA, PNA, AAA, TPA, or UEA-1 was 98%, 97%, 15%, 13%, and 17%, respectively. The corresponding values for CaI-treated, lectin-labeled sperm were 98%, 98%, 89%, 91%, and 92%, respectively. The increase in mean fluorescence intensity for the binding of the five lectins to CaI-treated versus solvent-treated sperm was 2.9-, 6.4-, 34.5-, 22-, and 36.7-fold, respectively. The binding site of the fucolectins was confined to the equatorial segment of the AR sperm. High positive correlations were observed between the percentage of AR sperm detected using three FITC-labeled fucolectins (AAA, TPA, and UEA-1) and anti-CD4G mAb (r(2) = 0.87, 0.99, and 0.99, respectively). Fucolectin binding to AR sperm was sensitive to competitive inhibition by fucose alpha-1-->2-linked lacto-N-fucopentaose I and fucoidan. Conclusions: Fucosylated glycans are expressed on AR human sperm. Fluorescence-labeled fucolectins markedly improved the signal:noise ratio in the detection of acrosomal loss of human sperm when compared with FITC-PSA or FITC-PNA. Fluorescence-labeled fucolectins can be used as specific markers for flow cytometric quantitation of unfixed AR sperm in suspension.