Sztein J M, Noble K, Farley J S, Mobraaten L E
The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA.
Cryobiology. 2001 Feb;42(1):28-39. doi: 10.1006/cryo.2001.2300.
Mouse sperm has proven to be more difficult to cryopreserve than sperm of other mammalian species. Published reports show that only three cryoprotectant agents (CPAs), alone or combined, have been studied: glycerol and dimethyl sulfoxide (DMSO), as permeating agents, and raffinose, as a nonpermeating agent. To date, the most consistent results for mouse sperm cryopreservation have been achieved by use of raffinose/skim milk as cryoprotectant with rapid cooling at 20 degrees C per minute. In this study, we compared the cryoprotection provided by permeating (glycerol, formamide, propanediol, DMSO, adonitol) or nonpermeating (lactose, raffinose, sucrose, trehalose, d-mannitol) compounds for freezing mouse sperm. Different solutions were made using 3% skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a concentration of 0.3 M, with a final osmolality of approx. 400 mOsm. Sperm samples from CB6F1 (hybrid) and C57BL/6J (inbred) mice collected directly into each CPA were frozen/thawed under identical conditions. After thawing and CPA elimination (centrifugation) raffinose (59%), trehalose (61%), and sucrose (61%) sustained the best motility (P = < 0.1) of the nonpermeating agents, whereas the best of the permeating agents was DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (prefreeze) to sugars was less harmful than the exposure to glycols. Coincidentally, sperm frozen in trehalose (41%), raffinose (40.5%), and sucrose (37.5%) were the samples less injured among all different postthawed CPA tested. The in vitro fertilization results demonstrated that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%, trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%).
事实证明，小鼠精子比其他哺乳动物的精子更难冷冻保存。已发表的报告显示，仅对三种冷冻保护剂单独或联合使用进行了研究：作为渗透剂的甘油和二甲基亚砜（DMSO），以及作为非渗透剂的棉子糖。迄今为止，小鼠精子冷冻保存最一致的结果是通过使用棉子糖/脱脂乳作为冷冻保护剂并以每分钟20摄氏度的速度快速冷却来实现的。在本研究中，我们比较了渗透型（甘油、甲酰胺、丙二醇、DMSO、阿东糖醇）或非渗透型（乳糖、棉子糖、蔗糖、海藻糖、D-甘露醇）化合物对小鼠精子冷冻的保护作用。使用3%脱脂乳溶液作为缓冲液或稀释剂制备不同溶液，所有不同的冷冻保护剂均以0.3 M的浓度溶解于其中，最终渗透压约为400 mOsm。将直接收集到每种冷冻保护剂中的CB6F1（杂交）和C57BL/6J（近交）小鼠的精子样本在相同条件下冷冻/解冻。解冻并去除冷冻保护剂（离心）后，棉子糖（59%）、海藻糖（61%）和蔗糖（61%）在非渗透剂中保持了最佳活力（P = < 0.1），而最佳的渗透剂是DMSO（42%）。对膜完整性进行分析，结果表明，精子简单暴露（预冻）于糖类的危害小于暴露于二醇类。巧合的是，在所有测试的解冻后冷冻保护剂中，冷冻于海藻糖（41%）、棉子糖（40.5%）和蔗糖（37.5%）中的精子样本受损最小。体外受精结果表明，用糖类冷冻的杂交小鼠精子（乳糖产生双细胞胚胎的比例为80%，棉子糖为80%，海藻糖为79%）比用二醇类冷冻的精子（甘油为11%）更具生育能力。
