EVIDENCE THAT MEMBRANE STRESS CONTRIBUTES MORE THAN LIPID-PEROXIDATION TO SUBLETHAL CRYODAMAGE IN CRYOPRESERVED HUMAN SPERM - GLYCEROL AND OTHER POLYOLS AS SOLE CRYOPROTECTANT

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 cryodamag e: one is peroxidation-related involving plasma membrane damage due to lipid peroxidation; the other is membrane stress-related involving me mbrane embrittlement during phase transitions occurring during freeze- thaw. If the peroxidation-related mode contributed substantially to su blethal cryodamage, the hypothesis predicts that lipid peroxidation in hibitors should reduce this damage. To test this prediction, we examin ed the effect of the lipid peroxidation inhibitors, hypotaurine, bovin e serum albumin (BSA), and alpha-tocopherol (Vit. E) on the time to lo ss of motility (TLM), taken as a measure of cell viability over time, for sperm samples cryopreserved in glycerol plus egg yolk medium. Thes e agents had no effect on TLM of these samples, indicating that this m ode contributes little to sublethal cryodamage. If the membrane stress -related mode contributed, the hypothesis predicts rapid recovery of m otility in the presence of egg yolk plus glycerol, but slow recovery i n 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 linearl y 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 mai ntained up to 4 hours. In the absence of glycerol, the percentage of m otile cells post-thaw was nil and remained nil up to 4 hours. The poly ols, erythritol, ribitol, and sorbitol had similar effects to that of glycerol, but the recovery of motility was not as complete. These resu lts indicate that the membrane stress-related mode contributes substan tially to sublethal cryodamage. They also indicate that glycerol and o ther polyols can function alone as cryoprotectants, but that recovery of motility is slow in these systems.