Figure 3.

Evaluation of capacitation-associated relocalization of ATP1A4 in bovine sperm. Bovine sperm preparations were incubated at 39°C in 5% CO₂ and under high humidity with heparin (10 μg/ml) or ouabain (100 μM) for 4 hr. A: Sperm preparations incubated with heparin at 0, 2, and 4 hr of incubation were processed for immunolocalization of ATP1A4, using a custom anti-ATP1A4. B: The acrosomal integrity of these sperm was concurrently evaluated using a fluorescent probe (FITC-PSA). Similarly, distribution patterns of ATP1A4 following incubation of sperm with ouabain at 0, 2, and 4 hr and the acrosomal integrity of these sperm were shown in Panels C and D, respectively. Fluorescence and phase contrast views of sperm incubated with pre-immune serum (E and F, respectively) or secondary antibody alone (G and H, respectively) were shown as negative controls.