Fig. 5. PKA is involved in hOR17-4 desensitization. (A) Immunostaining of human sperm using anti-PKA antibodies (green) shows, that PKA is localized to the midpiece and the tail in varying amounts, some cells also show ring-like staining around the head; nuclei are counterstained with DAPI. Bar, 10 µm. (B) Western blot analysis of human sperm lysate with anti-PKA phospho-substrate antibodies. Whereas non-stimulated cells (0) showed no staining with this antibody, cells that were treated with bourgeonal for 30 minutes showed labeling of a strong band of 40 kDa. (C) Representative ratiofluorometric recordings of the head and midpiece of individual human spermatozoa showing that PKA might be involved in hOR17-4 desensitization. The cytosolic Ca²⁺ level of Fura-2-loaded cells is depicted as the integrated fluorescence ratio (f₃₄₀/f₃₈₀) and viewed as a function of time. Cells were pre-incubated with PKA inhibitor H-89 in varying concentrations, bourgeonal was applied for 5 seconds in each case.