Alvarez J G, Storey B T
Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia 19104-6080.
J Androl. 1993 May-Jun;14(3):199-209.
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.
冷冻保存对人类精子的一个影响是亚致死性冷冻损伤，即解冻后细胞活力在后期比新鲜细胞更快丧失。我们假设了亚致死性冷冻损伤的两种模式：一种是与过氧化相关的，涉及由于脂质过氧化导致的质膜损伤；另一种是与膜应激相关的，涉及冻融过程中相变期间的膜脆化。如果与过氧化相关的模式对亚致死性冷冻损伤有很大贡献，那么该假设预测脂质过氧化抑制剂应能减少这种损伤。为了验证这一预测，我们研究了脂质过氧化抑制剂次牛磺酸、牛血清白蛋白（BSA）和α-生育酚（维生素E）对在甘油加蛋黄培养基中冷冻保存的精子样本活力丧失时间（TLM）的影响，TLM被用作细胞活力随时间变化的指标。这些试剂对这些样本的TLM没有影响，表明这种模式对亚致死性冷冻损伤的贡献很小。如果与膜应激相关的模式起作用，该假设预测在有蛋黄加甘油的情况下活力会快速恢复，但在仅有甘油的情况下恢复缓慢。它还预测一种合适的多元醇对于冷冻保存可能既是必要的也是充分的。在有蛋黄加甘油的情况下，活力恢复在5分钟内完成，但有活力细胞的百分比随后随时间呈线性下降。在3 - 12%的范围内仅使用甘油时，解冻后5分钟有活力细胞的百分比为5 - 10%，但解冻后40分钟已上升至60 - 80%，接近新鲜样本中的水平，并维持长达4小时。在没有甘油的情况下，解冻后有活力细胞百分比为零且在长达4小时内保持为零。多元醇赤藓醇、核糖醇和山梨醇与甘油有相似的效果，但活力恢复不那么完全。这些结果表明与膜应激相关的模式对亚致死性冷冻损伤有很大贡献。它们还表明甘油和其他多元醇可以单独作为冷冻保护剂起作用，但在这些系统中活力恢复较慢。
