Sieme Harald, Oldenhof Harriëtte, Wolkers Willem F
Clinic for Horses-Unit for Reproductive Medicine, University of Veterinary Medicine, Hannover, Germany.
Clinic for Horses-Unit for Reproductive Medicine, University of Veterinary Medicine, Hannover, Germany.
Anim Reprod Sci. 2016 Jun;169:2-5. doi: 10.1016/j.anireprosci.2016.02.004. Epub 2016 Feb 3.
Sperm cryopreservation facilitates storage and transport for use in artificial reproduction technologies. Cryopreservation processing, however, exposes cells to stress resulting in cellular damage compromising sperm function. Cryoprotective agents are needed to minimize cryopreservation injury, but at higher concentration they are toxic to cells. In this review, we describe cryoinjury mechanisms, and modes of action of different types of cryoprotective agents. Furthermore, measures are discussed how to minimize toxic effects caused by adding and removing cryoprotective agents. Cryoprotective agents can be divided into permeating and non-permeating agents. Permeating agents such as glycerol can move across cellular membranes and modulate the rate and extent of cellular dehydration during freezing-induced membrane phase transitions. Permeating protectants provide intracellular protection because they are preferentially excluded from the surface of biomolecules thereby stabilizing the native state. Non-permeating agents can be divided into osmotically active smaller molecules and osmotically inactive macromolecules. Both, permeating and non-permeating protectants form a protective glassy state during freezing preserving biomolecular and cellular structures. Freezing extenders for sperm contain salts, buffer compounds, sugars, proteins and lipids, and typically contain glycerol as the main permeating cryoprotective agent providing intracellular protection. Non-permeating protectants including sugars and proteins are used as bulking agents and to increase the glass transition temperature of the freezing extender. Ultra-heat-treated milk and egg yolk are frequently added as membrane modifying agents to enhance the inherent sperm cryostability. The protocol how to use and add cryoprotectants is a compromise between their beneficial and potentially detrimental effects.
精子冷冻保存便于在人工生殖技术中进行储存和运输。然而，冷冻保存过程会使细胞受到应激，导致细胞损伤，从而损害精子功能。需要使用冷冻保护剂来尽量减少冷冻保存损伤，但浓度较高时它们对细胞有毒性。在本综述中，我们描述了冷冻损伤机制以及不同类型冷冻保护剂的作用方式。此外，还讨论了如何将添加和去除冷冻保护剂所造成的毒性影响降至最低的措施。冷冻保护剂可分为渗透性和非渗透性两类。甘油等渗透性保护剂能够穿过细胞膜，并在冷冻诱导的膜相变过程中调节细胞脱水的速率和程度。渗透性保护剂提供细胞内保护，因为它们优先被排除在生物分子表面之外，从而稳定天然状态。非渗透性保护剂可分为具有渗透活性的小分子和无渗透活性的大分子。渗透性和非渗透性保护剂在冷冻过程中都会形成保护性玻璃态，从而保存生物分子和细胞结构。精子冷冻稀释液含有盐、缓冲化合物、糖、蛋白质和脂质，通常含有甘油作为主要的渗透性冷冻保护剂，提供细胞内保护。包括糖和蛋白质在内的非渗透性保护剂用作填充剂，并提高冷冻稀释液的玻璃化转变温度。经常添加超高温处理的牛奶和蛋黄作为膜修饰剂，以增强精子固有的冷冻稳定性。如何使用和添加冷冻保护剂的方案是在其有益效果和潜在有害影响之间进行权衡的结果。
