Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: Elimination of labeling artifacts

Reliable protocols were established for investigating asymmetric distributi ons of 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-caproyl (CGNBD)phospholip ids in the plasma membrane of boar sperm cells under physiological conditio ns. A method based on fluorescence resonance energy transfer was used to en sure that incorporation of the fluorescent phospholipids into the sperm pro ceeded via monomeric transfer. The total amount of incorporated phospholipi d fluorescence and the proportion of translocated phospholipid cytometric a nalysis before, and after, dithionite destruction of outer leaflet fluoresc ence. Catabolism of incorporated fluorescent phospholipids was blocked with phenylmethylsulfonyl fluoride. Membrane-damaged cells were detected with i mpermeant DNA stains, thereby enabling their exclusion from subsequent anal yses of the flow cytometric data, whence it could be demonstrated that the labeled phospholipids were incorporated only via the outer plasma membrane leaflet in living sperm cells. Phospholipid uptake and internalization was followed at 38 degrees C. After 1 hr of labeling, about 96% of the incorpor ated C6NBD-phosphatidylserine, 80% of C6NBD-phosphatidylethanolamine,18% of C6NBD-phosphatidylcholine, and 4% of C6NBD-sphingomyelin were found to hav e moved across the plasma membrane bilayer to the interior of the spermatoz oa. These inward movements of fluorescent phospholipids were ATP-dependent and could be blocked with sulfhydryl reagents. Movements from the inner to the outer leaflet of the sperm plasma membrane were minimal for intact fluo rescent phospholipids, but were rapid and ATP-independent for fluorescent l ipid metabolites. The described method enables, for the first time, assessm ent of changes in lipid asymmetry under fertilizing conditions. (C) 1999 Wi ley-Liss, Inc.