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

Abstract

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.

Testis-Specific Isoform of Na+-K+ ATPase and Regulation of Bull Fertility

“…ATP1A4 is the predominant catalytic subunit of NKA, which accounts for two-thirds of the total sperm NKA activity [ 22 ]. However, the α2 isoform has recently been reported to be the predominant isoform on the raft fractions in bovine sperm head plasma membrane during ouabain-induced capacitation [ 72 ].…”

“…Various NKA isoforms involved in capacitation-associated signaling have been demonstrated in both raft and non-raft fractions of the plasma membrane of bull sperm. All of the NKA isoforms are present in the raft and non-raft fractions of head plasma membrane in bull sperm, and among various isoforms (α1, α2, α3, β1, β2, and β3) in the raft fraction, α3 and β1 were the most abundant isoforms [ 72 ]. The existence of α4 isoform has also been demonstrated in sperm raft and non-raft fractions, where its content increases during capacitation [ 20 ].…”