PARENT SESSION

(W672) NaK-ATPASE AS A SIGNALING MOLECULE DURING BOVINE SPERM CAPACITATION.

Thundathil, Jacob¹, Baines, Shaun¹, Hickey, Katie¹, Anzar, Muhammad¹, Buhr, Mary¹, ¹ University of Guelph, Guelph, ON, Canada

ABSTRACT- NaK-ATPase is a heteromeric, integral membrane protein composed of alpha and beta subunits, and found to be active in many cell types including testicular tissue and spermatozoa. Studies on somatic cells demonstrated that inhibition of NaK-ATPase with its specific inhibitor ouabain initiates responses, which are similar to signaling events associated with bovine sperm capacitation. The objectives of this study were to demonstrate the presence and investigate the role of NaK-ATPase in the regulation of motility, protein tyrosine phosphorylation, capacitation and calcium influx in bovine sperm. Semen samples collected from Holstein bulls were used for this study. A monoclonal antibody against beta 1 subunit of NaK-ATPase was used for immunoblotting and immunolabelling. Spermatozoa were incubated in capacitating conditions for 5h without or with ouabain to study the role of NaK-ATPase on sperm motility (using computer-assisted semen analyzer), tyrosine phosphorylation (by immunoblotting using antiphosphotyrosine antibody) and capacitation (by inducing acrosome reaction with LPC followed by staining the smears with FITC-PSA). Indo-1 AM loaded spermatozoa were incubated without or with ouabain in capacitating conditions and evaluated for calcium influx over a period of 4h using flowcytometry. Results demonstrated that NaK-ATPase is localized in the plasma membrane of spermatozoa. Ouabain inhibited progressive motility after 2h of incubation, suggesting the possible role of NaK-ATPase in the regulation of sperm motility. Our results indicated that inhibition of NaK-ATPase triggers signaling mechanisms leading to tyrosine phosphorylation in a cohort of sperm proteins and induces capacitation in spermatozoa. Since ouabain inhibition did not elicit an increase in intracellular calcium level in a mixed population of spermatozoa, the signaling role of NaK-ATPase during capacitation may be independent of its role as an ion transporter. To our knowledge, this is the first report on the signaling role of NaK-ATPase in mammalian sperm capacitation. Thanks to NSERC and L'Alliance Boviteq, Saint-Hyacinthe, QC, for financial support and Gencor Ltd. Guelph, ON, for providing semen samples for this study.

KEY WORDS: NaK-ATPase, Bull semen, Sperm capacitation, Tyrosine phosphorylation