Na+, K+-ATPase in ram sperm - Its importance for kinematics, localisation and expression on the sperm surface

The goals of this study were to investigate (a) the localisation of Na+, K+-ATPase in ram sperm; (b) cryopreservation like-changes on median fluorescence intensity (MFI) and the expression of Na+, K+-ATPase on the sperm surface (labeled sperm); and (c) the influence of Na+, K+-ATPase inhibition on sperm kinematics. Exp. 1: pooled semen samples (n = 7) from four rams were diluted and frozen. The following sperm features were evaluated in fresh-diluted and post-thawed samples: MFI and expression of Na+, K+-ATPase (Bodipy (R) FL-Ouabain), plasma and acrosomal membrane integrity (IP; FITC-PNA), sperm viability and plasma membrane stability (Yo-Pro and Merocyanine), as well as sperm kinematics (CASA). Exp. 2: pooled semen samples (n = 5) from four rams were diluted and incubated in medium treated with or without Na+, K+-ATPase inhibitor (ouabain, 10(-4) M). Sperm kinematics (CASA) were determined prior to (0 h) and after incubation (3 h). In both fresh and thawed sperm, Na+, K+-ATPase was localised in the middle piece and cryopreservation did not influence the MFI in sperm with intact plasma membranes. However, cryopreservation reduced (P < 0.05) the overall percentage of sperm labeled with Bodipy (R) FL-Ouabain. Compared to the control, the inhibition of Na+, K+-ATPase reduced VAP at 0 h and both VAP and VSL at 3 h (P < 0.05). Thus, Na+, K+-ATPase is localised in the middle piece of fresh diluted and thawed sperm, and cryopreservation reduces the percentage of sperm expressing Na+, K+-ATPase in their surface, with no influence on the MFI of sperm with intact plasma membranes. Despite its localisation, Na+, K+-ATPase inhibition results in discrete kinematic modifications to ram sperm.