ROLE OF NA/K-ATPase IN BULL SPERM CAPACITATION

Abstract

Sodium/potassium ATPase (Na/K-ATPase) is also a membrane signaling protein that induces capacitation in bull sperm when exposed to the highly-specific steroid hormone ouabain. Specific signalling roles of Na/K-ATPase subunit isoforms α1, α3, β1, β2 or β3, which have been identified in bull sperm head plasma membrane (HPM), are unknown. The first trial hypothesized that isoforms involved in capacitation would be concentrated in HPM rafts. For the first time, rafts were isolated from fresh, uncapacitated dairy bull sperm HPM as detergent-resistant membranes that contained the marker protein flotillin-1. The ATPase isoforms α1, α2, α3, β1, β2 and β3 were identified in matched raft, non-raft and HPM by SDS-PAGE and immunoblotting using specific monoclonal antibodies and quantified with Image Quant Software (GE Healthcare). The α2, identified in HPM for the first time, and the previously-known α1, 3 and β1, 2, 3 isoforms were not confined to, or enriched in, any fraction. The α3 and β1 were the major isoforms in the rafts, non-raft and HPM. Rafts had unique patterns and amounts of the bands of each isoform, which could indicate their signalling role. To examine induction of tyrosine phosphorylation (Tyr-p) and capacitation, sperm were incubated with 0-100 μM ouabain, progesterone (P4) or ouabain + P4 under capacitating conditions. Progesterone was hypothesized to act by mimicking ouabain to induce Tyr-p and/or microscopically-identified AR. Both P4 and ouabain stimulated Tyr-p of three sperm proteins, but ouabain also stimulated Tyr-p of four additional proteins and induced significantly more overall Tyr-p after 3 and 5 hours of incubation, and only ouabain induced the AR. Co-incubating sperm with ouabain plus P4 reduced ouabain-induced Tyr-p. Sperm from a subset of good fertility bulls were capacitated by ouabain and non-responsive to P4, while sperm from low-fertility bulls didn’t respond to ouabain until 5 hours of incubation, and were stimulated by P4 to produce significant Tyr-p. This suggests that P4 and ouabain may both interact with Na/K-ATPase, but ouabain significantly stimulated capacitation. The findings here provide a better understanding about the molecular mechanisms associated with capacitation and insights for developing methodology for predicting bull sperm fertility.