The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane

A flow cytometric procedure was used to follow the effect of bicarbonate, a key inducer of sperm capacitation in vitro, on the transbilayer behavior o f C6NBD-phospholipids in the plasma membrane of living acrosome-intact boar spermatozoa under physiological conditions. In the absence of bicarbonate, 97% of C6NBD-phosphatidylserine and 78% of C6NBD-phosphatidylethanolamine was rapidly translocated from the outer leaflet to the inner, whereas relat ively little C6NBD-phosphatidylcholine and C6NBD-sphingomyelin was transloc ated (15% and 5%, respectively). Inclusion of 15 mM bicarbonate/5%CO2 marke dly slowed down the rates of translocation of the aminophospholipids withou t altering their final distribution, whereas it increased the proportions o f C6NBD-phosphatidylcholine and C6NBD-sphingomyelin translocated (30% and 2 0%, respectively). Bicarbonate activated very markedly the outward transloc ation of all four phospholipid classes. The changes in C6NBD-phospholipid b ehavior were accompanied by increased membrane lipid disorder as detected b y merocyanine 540, and also by increased potential for phospholipase catabo lism of the C6NBD-phospholipid probes. All three changes were mediated via a cAMP-dependent protein phosphorylation pathway. We suspect that the chang es result from an activation of the nonspecific bidirectional translocase ( 'scramblase'), They have important implications with respect to sperm ferti lizing function.