A GUANINE NUCLEOTIDE-BINDING REGULATORY PROTEIN IN HUMAN SPERM MEDIATES ACROSOMAL EXOCYTOSIS INDUCED BY THE HUMAN ZONA-PELLUCIDA

Guanine nucleotide-binding regulatory proteins play key intermediary roles in regulating zona pellucida-mediated acrosomal exocytosis in mouse and bull sperm. Since human sperm possess a G(i)-like protein and undergo the acrosome reaction in response to the human zona pellucida, we investigated whether this G protein plays a regulatory role in this exocytotic process. Zonae pellucidae isolated from eggs that had been inseminated but had shown no signs of fertilization after retrieval for in vitro fertilization and embryo transfer were pooled into groups of greater-than-or-equal-to 50 in order to reduce variability in biological responses due to the possible presence of ZP that had undergone modifications associated with the polyspermy block. Acid-solubilized zonae pellucidae were incubated with capacitated sperm, and the sperm then assessed for the acrosome reaction using both the P sativum agglutinin and chlortetracycline fluorescence assays; both assays gave similar results. Sperm incubated with solubilized zonae pellucidae at a final concentration of 2, 4, or 6 ZP/mu-l underwent acrosomal exocytosis to a similar extent as compared with A-23187. Sperm were incubated with 1-mu-g/ml pertussis toxin during capacitation to functionally inactivate the G(i)-like protein. Pertussis toxin treatment of sperm did not affect sperm motility and the ability of the cells to bind to structurally intact zonae pellucidae. Pertussis toxin, however, completely inhibited the percentage acrosome reactions induced by solubilized zonae pellucidae. By contrast, the A-23187-induced acrosome reaction was insensitive to PT treatment. Pertussis toxin inhibition of the zona pellucida-induced acrosome reaction occurred in a concentration-dependent manner with maximal effects observed at 100 ng/ml PT. These data suggest that the pertussis toxin-sensitive G(i)-like protein in human sperm plays an important regulatory role in the acrosome reaction induced by the human zona pellucida.