Stability of transbilayer phospholipid asymmetry in viable ram sperm cells after cryotreatment

The transbilayer dynamics of lipids in the plasma membrane of mammalian sperm cells is crucial for the fertilization process. Here, the transbilayer movement and distribution of phospholipids in the plasma membrane of fresh, ejaculated and cryopreserved ram spermatozoa was studied by labeling cells with fluorescent analogues of phosphatidylserine and phosphatidylcholine. By co-labeling cells with the DNA-binding dye propidiumiodide as well as by employing fluorescence microscopy and flow cytometry we were able to determine the transbilayer redistribution of fluorescent phospholipid analogues in intact (propidiumiodide-negative) and in impaired (propidiumiodide-positive) spermatozoa. The transbilayer distribution of the fluorescent phosphatidylserine and phosphatidylcholine analogues was not perturbed in intact sperm cells after cryopreservation. In those cells, the phosphatidylserine analogue became rapidly enriched on the cytoplasmic leaflet by the activity of a putative aminophospholipid translocase similar to intact cells of fresh, ejaculated samples. However, upon cryopreservation the activity of the putative aminophospholipid translocase was significantly reduced in intact cells. Employing annexin V-FITC, we found that even after cryopreservation the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet is maintained in intact cells, but not in impaired cells. The phosphatidylcholine analogue redistributed very slowly remaining essentially confined to the exoplasmic leaflet of the plasma membrane of intact cells from both fresh, ejaculated and cryopreserved samples. The physiological consequences of a perturbed transbilayer asymmetry in sperm plasma membranes is discussed.