Pannexin channels increase propidium iodide permeability in frozen-thawed dog spermatozoa

Abstract

Pannexins (Panx) are proteins that form functional single membrane channels, but they have not yet been described in dogs. The aim of the present study was to detect Panx1, Panx2 and Panx3 in frozen–thawed dog spermatozoa using flow cytometry and immunofluorescence analyses, evaluating the relationship of these proteins with propidium iodide (PI) in frozen–thawed spermatozoa. Fresh and frozen–thawed dog spermatozoa from eight dogs were preincubated with 3 mM PI with or without 15 mM carbenoxolone (CBX) or 1 mM probenecid (PBD), two Panx channel inhibitors, and then incubated with rabbit anti-Panx1, anti-Panx2 and anti-Panx3 antibodies (1 : 200). Panx immunolocalisation was assessed by fluorescence microscopy. Flow cytometry data were evaluated by analysis of variance. All three Panx proteins were found in dog spermatozoa: Panx1 was mostly localised to the acrosomal and equatorial segment, Panx2 was found in the posterior region of the head and tail and Panx3 was localised to the equatorial and posterior head segment. The percentage of PI-positive cells determined by flow cytometry was reduced (P , 0.05) in the presence of Panx inhibitors. These results show that Panx proteins are present in dog spermatozoa and increase PI permeability in frozen–thawed dog sperm, suggesting that the percentage of PI-positive spermatozoa used as an indicator of non-viable cells may lead to overestimation of non-viable cells.