title: Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation.
abstract: The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.
authors.affiliation: The ARC Centre of Excellence in Biotechnology and Development, Reproductive Science Group, School of Environmental and Life Science, and Hunter Medical Research Institute, University of Newcastle, NSW, Australia.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Baker
authors.forename: Louise
authors.initials: L
authors.lastname: Hetherington
authors.forename: Heath
authors.initials: H
authors.lastname: Ecroyd
authors.forename: Shaun D
authors.initials: SD
authors.lastname: Roman
authors.forename: R John
authors.initials: RJ
authors.lastname: Aitken
mesh_terms: D000177:Acrosome / Q000502:physiology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003847:Deoxyglucose / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004822:Epididymis / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D051379:Mice; D008928:Mitochondria; D010766:Phosphorylation; D012402:Rotenone / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D014475:Uncoupling Agents; D012402:Rotenone; D014443:Tyrosine; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D000242:Cyclic AMP; D000251:Adenosine Triphosphatases; D005947:Glucose; D002118:Calcium