This revised proposal addresses examines the role of a putative glycine receptor (GlyR) on the mammalian sperm surface. The acrosome reaction is an exocytotic event involving fusion of the plasma membrane with the underlying acrosomal membrane, followed by release of acrosomal contents. It is required for fertilization. The zona pellucida is believed to be a physiological inducer of the acrosome reaction, but the mechanisms that control exocytosis are not well understood. Ca2+ and Cl- fluxes are required for the induction of the acrosome reaction by the zona pellucida. Recent studies from the P.I.'s laboratory indicate that a GlyR/Cl- channel is involved in the zona-mediated pathway, although it is not certain whether such a receptor is present and what the nature of its role in the acrosome reaction might be. This application tests the hypothesis that GlyR activation produces Cl- efflux and that the resulting depolarization activates voltage-sensitive Ca2+ channels. The P.I. intends to characterize a sperm GlyR and to determine its role in the acrosome reaction. Studies will be carried out in both the human and pig systems.
In Aim 1 confocal microscopy will be used to localize BODIPY-strychnine, a probe for the putative GlyR, on live and on fixed human sperm.
Aim 2 will monitor Gly-dependent Cl- fluxes in human sperm using the MEQ fluorescent probe. The effects of certain GlyR agonists, antagonists, and potentiators on Cl- and acrosome reactions in human sperm will be determined.
Aim 4 will determine whether the GlyR-mediated Cl- efflux activates a Ca2+ influx through voltage-sensitive Ca2+ channels. Internal Ca2+ responses to Gly stimulation will be determined in single sperm by quantitative fluorescence microscopy, in collaboration with Nuccitelli. Additional studies will determine the effects of Ca2+ channel antagonists on this response.
Aims 5 -7 address the biochemical and functional characterization of the porcine GlyR.
In Aim 5, the P.I. will solubilze boar sperm membranes and isolate the receptor by affinity chromatography. Biochemical characterization of the isolated receptor will be performed.
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