A role for the human sperm glycine receptor/Cl(-) channel in the acrosome reaction initiated by recombinant ZP3.
Previously, we have demonstrated an essential role for the neuronal glycine receptor (GlyR) in the acrosome reaction (AR) of mouse and porcine sperm initiated by the egg zona pellucida (ZP). In the present study, we have demonstrated presence of the GlyR in human sperm by immunoprecipitation and Western blot analysis, investigated the potential of a recombinant human ZP3 (rhZP3) preparation as an alternative research tool to solubilized human ZP, and shown that the human sperm GlyR is essential to the human AR initiated by rhZP3. Additionally, we have been able to demonstrate that rhZP3 possesses biological activity, because it is able to rapidly stimulate the AR in capacitated human sperm and its action is blocked by the addition of pertussis toxin. Moreover, spectrofluorometric studies using fura-2-loaded human sperm have shown that rhZP3 triggers a peak-and-plateau rise in intracellular Ca(2+) levels similar to that seen with solubilized mammalian ZP. These results suggest that the actions of rhZP3 and solubilized ZP are elicited via the same signal transduction pathways. Furthermore, incubation of human sperm with an antibody directed against the alpha1 subunit of the human spinal cord GlyR or with 50 nM strychnine caused significant inhibition in the rhZP3-initated AR. Finally, studies using fura-2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the Ca(2+) influx associated with addition of rhZP3. These results further support the view that rhZP3 and the ZP work through the same mechanisms, show that the GlyR is involved in rhZP3-initiated AR, and suggest that the GlyR may also play a role in the early signal transduction cascades associated with ZP-initiated AR in vivo.
Bray
Christopher
C
Department of Cell Biology and Human Anatomy, School of Medicine, University of California at Davis, Davis, California 95616-8643, USA.
Son
Jung-Ho
JH
Kumar
Priyadarsini
P
Harris
Jeffrey D
JD
Meizel
Stanley
S
D020101:Acrosome Reaction / Q000187:drug effects*; D000328:Adult; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D004527:Egg Proteins / Q000096:biosynthesis / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000494:pharmacology*; D037342:Pertussis Toxin; D011233:Precipitin Tests; D011956:Receptors, Cell Surface; D018009:Receptors, Glycine / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013075:Sperm Capacitation / Q000187:drug effects; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D013331:Strychnine / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000072216:Zona Pellucida Glycoproteins
D018118:Chloride Channels; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D018009:Receptors, Glycine; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse; D037342:Pertussis Toxin; D013331:Strychnine; D002118:Calcium