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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD033368-04
Application #
6164907
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Tasca, Richard J
Project Start
1997-03-01
Project End
2002-11-30
Budget Start
2000-03-01
Budget End
2002-11-30
Support Year
4
Fiscal Year
2000
Total Cost
$227,537
Indirect Cost
Name
University of California Davis
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
094878337
City
Davis
State
CA
Country
United States
Zip Code
95618
Kumar, Priyadarsini; Meizel, Stanley (2008) Identification and spatial distribution of glycine receptor subunits in human sperm. Reproduction 136:387-90
Kumar, Priyadarsini; Ferns, Michael J; Meizel, Stanley (2006) Identification of agrinSN isoform and muscle-specific receptor tyrosine kinase (MuSK) [corrected] in sperm. Biochem Biophys Res Commun 342:522-8
Bray, Christopher; Son, Jung-Ho; Kumar, Priyadarsini et al. (2005) Mice deficient in CHRNA7, a subunit of the nicotinic acetylcholine receptor, produce sperm with impaired motility. Biol Reprod 73:807-14
Meizel, Stanley; Son, Jung-Ho (2005) Studies of sperm from mutant mice suggesting that two neurotransmitter receptors are important to the zona pellucida-initiated acrosome reaction. Mol Reprod Dev 72:250-8
Kumar, Priyadarsini; Meizel, Stanley (2005) Nicotinic acetylcholine receptor subunits and associated proteins in human sperm. J Biol Chem 280:25928-35
Bray, C; Son, J-H; Meizel, S (2005) Acetylcholine causes an increase of intracellular calcium in human sperm. Mol Hum Reprod 11:881-9
Meizel, Stanley (2004) The sperm, a neuron with a tail: 'neuronal' receptors in mammalian sperm. Biol Rev Camb Philos Soc 79:713-32
Son, Jung-Ho; Meizel, Stanley (2003) Evidence suggesting that the mouse sperm acrosome reaction initiated by the zona pellucida involves an alpha7 nicotinic acetylcholine receptor. Biol Reprod 68:1348-53
Bray, Christopher; Son, Jung-Ho; Meizel, Stanley (2002) A nicotinic acetylcholine receptor is involved in the arosome reaction of human sperm initiated by recombinant human ZP3. Biol Reprod 67:782-8
Bray, Christopher; Son, Jung-Ho; Kumar, Priyadarsini et al. (2002) A role for the human sperm glycine receptor/Cl(-) channel in the acrosome reaction initiated by recombinant ZP3. Biol Reprod 66:91-7

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