Na+/K+ATPase regulates sperm capacitation through a mechanism involving kinases and redistribution of its testis-specific isoform.
Incubation of bovine sperm with ouabain, an endogenous cardiac glycoside that inhibits both the ubiquitous (ATP1A1) and testis-specific alpha4 (ATP1A4) isoforms of Na(+)/K(+)ATPase, induces tyrosine phosphorylation and capacitation. The objectives of this study were to investigate: (1) fertilizing ability of bovine sperm capacitated by incubating with ouabain; (2) involvement of ATP1A4 in this process; and (3) signaling mechanisms involved in the regulation of sperm capacitation induced by inhibition of Na(+)/K(+)ATPase activity. Fresh sperm capacitated by incubating with ouabain (inhibits both ATP1A1 and ATP1A4) or with anti-ATP1A4 immunoserum fertilized bovine oocytes in vitro. Capacitation was associated with relocalization of ATP1A4 from the entire sperm head to the post-acrosomal region. To investigate signaling mechanisms involved in oubain-induced regulation of sperm capacitation, sperm preparations were pre-incubated with inhibitors of specific signaling molecules, followed by incubation with ouabain. The phosphotyrosine content of sperm preparations was determined by immunoblotting, and capacitation status of these sperm preparations were evaluated through an acrosome reaction assay. We inferred that Na(+)/K(+)ATPase was involved in the regulation of tyrosine phosphorylation in sperm proteins through receptor tyrosine kinase, nonreceptor type protein kinase, and protein kinases A and C. In conclusion, inhibition of Na(+)/K(+)ATPase induced tyrosine phosphorylation and capacitation through multiple signal transduction pathways, imparting fertilizing ability in bovine sperm. To our knowledge, this is the first report documenting both the involvement of ATP1A4 in the regulation of bovine sperm capacitation and that fresh bovine sperm capacitated by the inhibition of Na(+)/K(+)ATPase can fertilize oocytes in vitro.
Newton
Larissa D
LD
Faculty of Veterinary Medicine, Department of Production Animal Health, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada.
Krishnakumar
Sulochana
S
Menon
Ajitkumar Gopinadha
AG
Kastelic
John P
JP
van der Hoorn
Frans A
FA
Thundathil
Jacob C
JC
D000177:Acrosome / Q000201:enzymology*; D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity / Q000502:physiology; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology*; D013737:Testis / Q000378:metabolism
D002316:Cardiotonic Agents; D004791:Enzyme Inhibitors; D010042:Ouabain; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase