Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant

Abstract

One effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. We hypothesized two modes of sublethal cryodamage: one is peroxidation-related involving plasma membrane damage due to lipid peroxidation; the other is membrane stress-related involving membrane embrittlement during phase transitions occurring during freeze-thaw. If the peroxidation-related mode contributed substantially to sublethal cryodamage, the hypothesis predicts that lipid peroxidation inhibitors should reduce this damage. To test this prediction, we examined the effect of the lipid peroxidation inhibitors, hypotaurine, bovine serum albumin (BSA), and alpha-tocopherol (Vit. E) on the time to loss of motility (TLM), taken as a measure of cell viability over time, for sperm samples cryopreserved in glycerol plus egg yolk medium. These agents had no effect on TLM of these samples, indicating that this mode contributes little to sublethal cryodamage. If the membrane stress-related mode contributed, the hypothesis predicts rapid recovery of motility in the presence of egg yolk plus glycerol, but slow recovery in the presence of glycerol alone. It also predicts that an appropriate polyol may be both necessary and sufficient for cryopreservation. In the presence of egg yolk plus glycerol, motility recovery was complete within 5 minutes, but the percent motile cells then decreased linearly with time. With glycerol alone in the range 3-12%, at 5 minutes post-thaw the percent motile cells was 5-10%, but by 40 minutes post-thaw had risen to 60-80%, approaching that in the fresh sample, and was maintained up to 4 hours. In the absence of glycerol, the percentage of motile cells post-thaw was nil and remained nil up to 4 hours. The polyols, erythritol, ribitol, and sorbitol had similar effects to that of glycerol, but the recovery of motility was not as complete. These results indicate that the membrane stress-related mode contributes substantially to sublethal cryodamage. They also indicate that glycerol and other polyols can function alone as cryoprotectants, but that recovery of motility is slow in these systems.