Characterization of an islet-activating protein-sensitive site in mouse sperm that is involved in the zona pellucida-induced acrosome reaction

Abstract

Recently, it has been suggested that a mouse sperm-associated protein with properties similar to the inhibitory guanine nucleotide-binding regulatory protein Gi plays an intermediary role in the zona pellucida-induced acrosome reaction (Endo, Y., Lee, M. A., and Kopf, G. S. 1987. Dev. Biol. 119, 210-216). In the present study experiments were designed to characterize further the role of this Gi-like protein in mediating this physiological event. Sperm capacitated in the presence of islet-activating protein (IAP) were inhibited from undergoing the acrosome reaction induced by either solubilized zonae pellucidae or purified ZP3. This inhibitory effect was observed with IAP concentrations previously shown to ADP-ribosylate the alpha-subunit of the Gi-like protein and inhibit the acrosome reaction induced by mechanically isolated, structurally intact zonae pellucidae. Inhibition occurred as a consequence of the inability of the sperm to progress from a capacitated, acrosome-intact state (B pattern) to an intermediate stage prior to the completion of the acrosome reaction (S pattern), as assessed with a chlortetracycline fluorescence assay. These inhibitory effects of IAP on the sperm acrosome reaction were confirmed by electron microscopy. In contrast, the acrosome reaction induced either spontaneously or nonspecifically by A-23187 was completely insensitive to IAP. Since the inhibitory effects of IAP on the acrosome reaction were at the level of the B to S transition, a transition normally accompanied by a loss in a transmembrane pH gradient, the effects of IAP on the loss of this gradient was assessed with the pH-sensitive fluorescent probe, 9-amino-3-chloro-7-methoxyacridine. The IAP-induced inhibition of the B to S transition was accompanied by a parallel retention of the transmembrane pH gradient. 3-Quinuclidinyl benzilate (QNB), a muscarinic cholinergic antagonist which has been demonstrated to specifically inhibit the zona-induced acrosome reaction at the B to S transition, also prevented the loss of this transmembrane pH gradient. The similarity of the effects of both IAP and QNB on sperm-zona interactions suggested that the IAP- and QNB-sensitive sites were either closely related or identical. Experiments designed to test these possibilities suggested that both sites were not identical. Finally, it was demonstrated that the acceleration of the zona-induced B to S transition observed with biologically active phorbol esters was insensitive to IAP, suggesting that the site of phorbol ester action is downstream from or independent of the IAP-sensitive site.(ABSTRACT TRUNCATED AT 400 WORDS)