Abstract
Head plasma membranes were isolated from fresh or cryopreserved ejaculated boar spermatozoa and the lipids were extracted for determination of lipid fluidity (n = 6 for fresh and cryopreserved) and for compositional analysis (n = 5 for fresh, 6 for cryopreserved). Composition of the egg yolk extender was also determined. For fluidity determination, the mixed lipids were allowed to form natural liposomes. Bilayer fluidity of these liposomes was analyzed in the presence or the absence of 1 mM Ca2+ with the probes tPNA. which preferentially locates into gel-phase areas, and cPNA, which enters fluid and gel-phase areas equally and thus assesses bulk lipids. Fluidity of liposomes declined significantly during controlled-rate cooling for all samples. Compared to lipids from fresh membranes, gel lipids from cryopreserved cells lost fluidity at a significantly more rapid rate, as did bulk lipids in the presence of Ca2+ (P < 0.0001). Fluidity increased during subsequent rewarming 15 to 50°C), again at a slower rate for lipids from fresh cells, with the cryopreservation effect being significant for all probe/ Ca2+ combinations (P ⩽ 0.05). Calcium altered the fluidity characteristics of membrane lipids from fresh but not cryopreserved sperm when analyzed during cooling with cPNA (P < 0.01) and during rewarming with cPNA (P < 0.0001) and tPNA (P < 0.05). Lipids from cryopreserved cells contained significantly less sphingomyelin (14.6 ± 1.1 vs 22.4 ± 1.6 mol%) and more phosphatidylcholine (51.5 ± 2.0 vs 40.5 ± 2.4%). The octadecanoate (18:0) content in both phosphatidylserine and phosphatidylethanolamine decreased after cryopreservation (P < 0.05). The polyunsaturated falty acids docosatetraenoate (22:4) and/or arachidonate (20:4) increased in these phospholipids and in sphingomyelin and phosphatidylinositol (P > 0.05). The alterations in the molecular interactions, composition, and Ca2+ sensitivity of membrane lipids may interfere with the normal membrane events of fertilization.
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