A) F-actin staining using FITC-conjugated phalloidin (green) of NAGly-stimulated (3 μM) spermatozoa and control cells (0.1% DMSO). The cells were incubated for 3 h. DAPI staining (blue) was used to determine the number and localization of cell nuclei. Scale bars: 10 μm. ( B) Zoomed section of NAGly-stimulated (3 μM) and control spermatozoa stained with phalloidin-FITC (Phalloidin). Scale bars: 1 μm. ( C) Quantification of relative fluorescence intensities revealed a significant reduction of F-actin levels upon NAGly stimulation in human sperm. Shown are the mean ± SEM (DMSO: n = 95 cells; NAGly: n = 95 cells; from three independent experiments; Mann-Whitney-U-Test: ***p < 0.001). ( D) Representative pictures of the PNA-FITC (PNA) staining of control and NAGly-stimulated cells. Marked are the different categories of the acrosomal status (I-IV). DAPI staining (blue) was used to determine the number and localization of cell nuclei. Scale bars: 10 μm. ( E) Quantification of acrosomal exocytosis (categories III-IV) upon NAGly stimulation using PNA-FITC. Stimulated cells (NAGly) were compared to control cells (0.1% DMSO); Three to five independent samples with a sperm population ranging from 97 to 364 cells per single experimental condition were analyzed. Values show the mean ± SEM (*p < 0.05, ***p < 0.001, Student’s t-test).