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.
