FIGURE 8.

Analysis of sperm beat frequency on a single-cell level. A and B, sperm of WT, single CAII^−/−, and CAII^−/− CAIV^−/− animals were stimulated with 15 mm HCO₃⁻ (A) or 2% CO₂ (B). With both protocols, CAII^−/− sperm display the strongest delayed and reduced beat frequency increase compared with CAII^−/− CAIV^−/− and WT sperm. C and D, biochemical CAIV protein elimination by PLC treatment of WT sperm was tested successfully by DAB staining (C, scale bar = 4 μm) and Western blot analysis (D). E, in single-cell analysis, PLC-treated WT (CAIV^BC−) and genetic CAIV^−/− sperm (CAIV^{−/−/BC−}) display the same response upon HCO₃⁻ stimulation. F and G, shown is the response of genetic CAII^−/− and CAII^−/− CAIV^BC− double knockout sperm to HCO₃⁻ (F) and CO₂ (G). The beat frequency of CAII^−/− CAIV^BC− sperm increases only to a maximum of 4.16 ± 0.15 Hz with HCO₃⁻ and of 3.87 ± 0.19 Hz with CO₂ application (t = 80 s) and displays the weakest response. Data are mean ± S.E. of ≥17 single sperm.