Detection of anionic sites on the cytoplasmic surface of the guinea pig acrosomal membrane

The sperm acrosome reaction is an example of exocytosis, accomplished through the fusion of the acrosomal and plasma membranes. As in other examples of exocytosis, the acrosome reaction is initiated by an influx of Ca++, which may promote fusion by binding to anionic sites on adjacent bilayers. In this study we used ruthenium red (RR) and cationic ferritin (CF) to detect anionic sites on the surfaces of acrosomal and plasma membranes of guinea pig spermatozoa. These probes indicate a dense concentration of anionic sites on the cytoplasmic surface of the acrosomal membrane. Higher concentrations of salt (NaCl) were required to inhibit cationic probe labeling of the cytoplasmic surface of the acrosomal membrane compared to the concentration needed to inhibit the plasma membrane binding. The added NaCl also increased the separation of the plasma from the acrosomal membrane. Low-pH buffers stop cationic probe labeling of both membranes. Sections tangential to the acrosomal membrane revealed that the cation probes bound in a linear pattern, similar to the periodicity and distribution of intramembraneous particles observed in freeze-fracture replicas. Following fusion of the plasma and acrosomal membrane during the acrosome reaction, we could no longer detect a dense concentration of anionic sites on the cytoplasmic surface of the fused vesicles. The results indicate that the dense concentrations of anionic sites are either masked or lost following fusion with the overlying plasma membrane.