Endpoint of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa characterized by acrosomal H+ and Ca2+ permeability: Population and single cell kinetics

The acrosome reaction induced by the mouse egg's zona pcllucida in mouse sperm has been shown to proceed in two stages as characterized empirically by sequential changes in patterns of chlorletracycline fluorescence on the sperm plasma membrane surfaces. The chlortctracy-cline fluorescence pattern characteristic of fully intact sperm is designated B:in sperm bound to structurally intact zonae that induce the acrosome reaction, the B pattern changes first to an intermediate pattern S and then to a terminal pattern AR characteristic of the completed acrosome reaction. In the same study, it was shown, using a 9-amino acridine fluorescent pH probe, that completion of the first stage was characterized by increase in H⁺ permeability such that the H⁺ gradient between sperm head and medium was dissipated. In this study, we show that the fluorescent pH probe 9-N-dodecylamino acridine and the intracellular Ca2⁺ fluores cent probe fura-2 are both localized to the anterior part of the sperm head encompassing the acrosomal compartment in intact sperm, and the fluorescence associated with each probe is lost as the first stage of the acrosome reaction is completed. Loss of the pH probe fluorescence, pattern N, corresponds to onset of H⁺ permeability, and loss of fura-2 fluorescence, pattern F, corresponds to onset of Ca²⁺ permeability. Localization of intracellular fura-2 fluorescence to the acrosomal compartment required extracellular Mn²⁺ to quench surface-bound fura-2 AM, the tetra-acetoxymethyl ester of fura-2 used to load the cells. Loss of acrosomal fura-2 fluorescence is due to quenching by tracer Mn²⁺ accompanying Ca²⁺. Onset of membrane permeability to both H⁺ and Ca²⁺, asseenby loss of patterns N and F, occurred in synchrony in populations of sperm bound to isolated, structurally intact zonae, with an overall time coursfe of 210 min postbinding. The loss of pattern N in individual sperm cells bound to zonae was rapid, with a half time of 2.1 min. Concomitant with this rapid loss of pattern N was a shift in the amplitude of flagellar motion from large to small. The lag times to pattern N loss in 50 individual cells ranged from 30 to 140 min. The variable lag times determine the population kinetics; the rate of the endpoinl reaction seen in the individual cells is rapid and constant. Dissipation of the H⁺ gradient with immediate loss of pattern N was readily achieved by addition of nigericin with no change in the time course of the onset of Ca²⁺ permeability of the membranes enclcsing the acrasome. Onset of Ca²⁺ permeability was always accompanied by onset of H⁺ permeability, but the alkalinization caused by H⁺ permeability induced by nigericin had no effect on Ca²⁺ permeability in intact sperm. This indicates that the permeabilization of the membranes marking the endpoint reaction of the B-to-S transition is most likely due to pore formation induced by punctate fusion of the plasma and outer acrasomal membranes, as would be expected for an exocytotic reaction.