Analysis of sperm capacitation progress and response to a compound triggering acrosome reaction (calcium ionophore A23187).

(A) Capacitation progress analysed by the flow cytometry of sperm recovered from WT (green bars) and NWC-KO (red bars) mice stained with anti-phospho-tyrosine antibody (anti-pY antibody). Capacitation progress was analysed at three time points (5, 30 and 60 min) in live sperm, as discriminated by fixable viability stain. Data are presented as mean values with SD from a single, representative experiment (WT, n = 4 mice, NWC-KO n = 5 mice). (B) Representative dot-plot showing flow cytometry analysis of WT sperm stained with PI and PNA-FITC after triggering acrosome reaction. The dot-plot shows four distinct populations: acrosome reacted and acrosome intact (right and left side), and PI positive and PI negative populations (upper and lower side). Two gates are shown: R-1 (PI-negative population) and R-2 (PI negative and acrosome reacted). (C) Sizes of populations of PI negative sperm. (D) Acrosome reaction analysis after the normalization of R-1 content of a given mouse to 100%. (C) and (D) present mean values obtained from n = 11 WT (green bars) mice and n = 12 NWC-KO mice (red bars).