FUNDAMENTAL CRYOBIOLOGY AND PRESERVATION OF SPERM

一项国际努力正在进行中，以确定相关基因的特征， 与人类胚胎发育和疾病有关， 这是此类研究中的一个重要模型。一个特别强大 一种方法是创造转基因动物，其中已知的DNA片段 序列插入宿主DNA中。其结果是创造了 突变株的数量迅速增加，以至于 在标准育种群体中保持这些品系正成为一种 棘手的问题。一个更具成本效益的方法是 精子的低温生物学保存事实上，这种方法应该 比其他选择更具成本效益， 冷冻保存胚胎，只要功能存活 冷冻解冻精子的比例相当高然而，直到两年前， 试图生存的努力都失败了。最近，中度存活的 冷冻保存的精子已经报道使用经验方法，但 成功和失败的程序令人不安 在不同的实验室里是矛盾的。我们研究的目的是 来研究小鼠精子对潜在的基本 决定冷冻后存活或死亡的低温生物学因素， 解冻，并使用这些信息来制定程序， 这些细胞的保存具有高和一致的功能存活。 冷冻和解冻受试者细胞及其细胞器， 渗透条件以及细胞的物理和生物反应 这主要取决于它们对水的渗透性和低温保护性 溶质，以及这些渗透性如何影响的浓度 可以耐受的冷冻保护溶质以及它们如何影响 冰在哪里形成。因此，拟议研究的一个主要目标是 将通过各种生物物理学方法来确定这些相互关系， 接近。第一步是测定渗透压 精子的行为，它们对水的渗透性和对冷冻保护的作用 溶质，以及这些渗透性的温度系数。这 信息将用于最大限度地提高耐受浓度， 冷冻保护剂第二步是利用水渗透率数据， 预测精子在不冷冻的情况下冷却的最大速率 并将这些预测与实验进行比较。第三 第一步将是研究存活率，冷却速率， 升温速率、冷冻保护剂类型和浓度，以确定最佳 最大限度的生存条件。造成损伤的一个因素可能是 来自分子氧的自由基对膜的损伤。这 将通过持续将精子维持在低温度下来测试假设。 通过使用细菌膜制剂来测定氧张力。

An international effort is under way to characterize the genes associated with embryonic development and disease in humans, and the mouse is considered an important model in such studies. An especially powerful approach is to create transgenic animals in which DNA segments of known sequence are inserted into the host DNA. The result has been the creation of a rapidly increasing number of mutant lines, so much so that the maintenance of such lines in standard breeding colonies is becoming a formidable problem. A far more cost-effective method would be the cryobiological preservation of their sperm. Indeed, this approach should be substantially more cost-effective than the alternative of cryobiologically preserving embryos, provided that the functional survival of frozen-thawed sperm is reasonably high. However, until two years ago, attempts to achieve survival had failed. Recently, moderate survivals of cryopreserved sperm have been reported using empirical approaches, but the procedures that succeeded and those that failed are disturbingly contradictory in different labs. The purpose of our proposed research is to study the response of mouse sperm to the underlying fundamental cryobiological factors that determine survival or death after freezing and thawing, and to use this information to develop procedures for the preservation of these cells with high and consistent functional survival. Freezing and thawing subject cells and their organelles to highly abnormal osmotic conditions, and the cells' physical and biological responses depend importantly on their permeability to water and cryoprotective solutes, and on how these permeabilities influence the concentrations of cryoprotective solutes that can be tolerated and how they influence when and where ice forms. Accordingly, a major aim of the proposed research will be to determine these interrelations by a variety of biophysical approaches. The first step will be the determination of the osmotic behavior of sperm, their permeability to water and to cryoprotective solutes, and the temperature coefficients of these permeabilities. This information will be used to maximize the tolerated concentration of cryoprotectants. The second step will be to use water permeability data to predict the maximum rate at which sperm can be cooled without freezing internally and to compare those predictions with experiment. The third step will be to investigate the relation between survival, cooling rate, warming rate, cryoprotectant type and concentration to determine optimal conditions for maximum survival. A contributing factor to injury may be membrane damage from free radicals derived from molecular oxygen. This hypothesis will be tested by continuously maintaining sperm under low oxygen tensions by the use of a bacterial membrane preparation.