Calmodulin antagonists differentially affect capacitation-associated protein tyrosine phosphorylation of mouse sperm components.
Sperm capacitation in vitro is thought to be correlated with the increased protein tyrosine phosphorylation of a subset of sperm components. Our group recently used a pharmacological approach to demonstrate that calmodulin (CaM), a 17 kDa calcium sensor protein, has a role in sperm capacitation. In the present study, we have used several CaM antagonists in an attempt to characterize further the role of CaM in capacitation-associated protein tyrosine phosphorylation of sperm components. Our data demonstrate, first, that mouse spermatozoa incubated in a medium that favors capacitation undergo increased protein tyrosine phosphorylation in a time-dependent manner. Second, inclusion of six CaM antagonists individually in an in vitro incubation medium prevented sperm capacitation, as demonstrated by their diminished ability to undergo agonist-induced acrosome reaction. Third, half of the CaM antagonists (compound 48/80, W13 and CaM-binding domain) had no effect on protein tyrosine phosphorylation or sperm motility. Fourth, by contrast, three CaM antagonists (W7, ophiobolin A and calmidazolium) significantly inhibited protein tyrosine phosphorylation of sperm components (42, 56, 66, 82 and 95 kDa) and adversely affected their motility without altering viability as assessed by propidium iodine staining. Finally, inclusion of purified CaM in the capacitation medium significantly increased tyrosine phosphorylation of 82 kDa and 95 kDa components. Combined, these data suggest that CaM antagonists prevent capacitation by interfering with multiple regulatory pathways, and do so either with or without adverse effects on sperm motility and protein tyrosine phosphorylation.
Zeng
Hai-Tao
HT
Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Room D-3243 MCN, Nashville, TN 37232-2633, USA.
Tulsiani
Daulat R P
DR
D000177:Acrosome / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008954:Models, Biological; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D054830:Sesterterpenes; D013075:Sperm Capacitation; D013081:Sperm Motility; D013094:Spermatozoa / Q000378:metabolism* / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
D002147:Calmodulin; D004791:Enzyme Inhibitors; D007093:Imidazoles; D054830:Sesterterpenes; D013449:Sulfonamides; D013729:Terpenes; D014443:Tyrosine; C040764:ophiobolin A; C031938:calmidazolium; C017967:W 7