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 of 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 relatively little C6NBD-phosphatidylcholine and C6NBD-sphingomyelin was translocated (15% and 5%, respectively). Inclusion of 15 mM bicarbonate/5%CO(2) markedly slowed down the rates of translocation of the aminophospholipids without altering their final distribution, whereas it increased the proportions of C6NBD-phosphatidylcholine and C6NBD-sphingomyelin translocated (30% and 20%, respectively). Bicarbonate activated very markedly the outward translocation of all four phospholipid classes. The changes in C6NBD-phospholipid behavior were accompanied by increased membrane lipid disorder as detected by merocyanine 540, and also by increased potential for phospholipase catabolism of the C6NBD-phospholipid probes. All three changes were mediated via a cAMP-dependent protein phosphorylation pathway. We suspect that the changes result from an activation of the non- specific bidirectional translocase ('scramblase'). They have important implications with respect to sperm fertilizing function.
Gadella
B M
BM
Institute of Biomembranes, Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. b.gadella@vet.uu.nl
Harrison
R A
RA
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000177:Acrosome / Q000378:metabolism; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008297:Male; D010069:Oxadiazoles / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D011744:Pyrimidinones / Q000378:metabolism; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism; D013552:Swine
C092351:1-palmitoyl-2-(6-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)caproyl)glycero-3-phosphoserine; D001639:Bicarbonates; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; C104907:N-6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminohexanoysphingomyelin; D010069:Oxadiazoles; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010743:Phospholipids; D011744:Pyrimidinones; D013109:Sphingomyelins; C052267:1,2-(palmitoyl-NBD-aminocaproyl)phosphatidylethanolamine; C003954:merocyanine dye; C044745:1-palmitoyl-2-(6-(N-(7-nitrobenz)-2-oxa-1,3-diazol-4-yl)aminocaproyl)phosphatidylcholine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D009327:4-Chloro-7-nitrobenzofurazan; D002118:Calcium