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, ß1, ß2, and ß3 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 ß1 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.

Interaction of ouabain and progesterone on induction of bull sperm capacitation.

The endogenous steroid hormone ouabain induces capacitation of bull sperm acting through its receptor Na+/K+-ATPase on the sperm plasma membrane. Progesterone (P4) is believed to act through the sperm membrane P4 receptor (mPR) to induce non-genomic signalling leading to capacitation and/or acrosome reaction (AR) in the sperm of some species, but the exact nature of this receptor molecule on bull sperm is not known. In amphibian oocytes, P4 acts through the low-affinity ouabain binding site on Na+/K+-ATPase to induce signalling highly reminiscent of ouabain's signalling that initiates capacitation. This study hypothesized that ouabain and P4 interact agonistically or antagonistically to induce bull sperm capacitation. Sperm were incubated with 0, 12.5, 25, 50 and 100 µM ouabain, P4 or ouabain + P4 (12.5, 25 and 50 µM each) under capacitating conditions, and capacitation was assessed microscopically looking at the acrosome status of sperm and as the amount of protein tyrosine phosphorylation (Tyr-P). Both steroids stimulated Tyr-P of certain sperm proteins, but ouabain caused tyrosine phosphorylation of more proteins than P4 and stimulated significantly more overall Tyr-P (P < 0.05). Ouabain also was the only steroid to stimulate significant microscopically-evident AR. When sperm were co-incubated with the two steroids, P4 partially inhibited ouabain-induced Tyr-P and AR. These results suggest that P4 and ouabain may both interact with Na+/K+-ATPase, but ouabain is the more effective hormone. Ouabain may, therefore, be the primary physiological inducer of bovine capacitation.