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Are isoforms of capacitating Na+K+‐ATPase localized to sperm head rafts?

Capacitation begins in the sperm head plasma membrane (HPM). Membrane rafts could house signaling molecules, but although these specialized microdomains have been microscopically visualized in sperm heads, rafts have been isolated for study only from homogenized whole sperm or tails, never purified HPM. Sodium/potassium ATPase (Na+K+‐ATPase) is a membrane‐bound signaling protein that induces capacitation in bull sperm in response to the steroid hormone ouabain, and its subunit isoforms α1, α3, β1, β2, and β3 are known in HPM. This study hypothesized that rafts exist in the HPM of bull sperm, with Na+K+‐ATPase subunit isoforms preferentially localized there. Western immunoblotting (WB) of HPM from fresh, uncapacitated bull sperm (n = 7 ejaculates), and detergent‐resistant membranes isolated by density gradient centrifugation from this HPM, contained the raft‐marker protein Flotillin‐1; the non‐raft fraction did not. HPM, raft, and non‐raft contained all known Na+K+‐ATPase isoforms including, for the first time, the previously unknown α2 isoform. Quantification (ImageQuant Software) found α3 and β1 were relatively dominant isoforms in the HPM raft. WB profiles of raft isoforms differed significantly from HPM and non‐raft profiles, with unique banding patterns and amounts, hinting that the capacitation signaling in the now‐identified HPM rafts may depend on unique sequences within the isoform structure.