AQUAPORINS, ICE FORMATION IN CELLS,/CRYOPRESERVATION

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DESCRIPTION (provided by applicant): Cryobiologically preserved cells are playing an increasingly important direct and indirect role in health-related areas. The direct roles include assisted reproduction and the use of cryopreserved cells and tissues in clinical medicine. The indirect role includes the maintenance of an accelerating number of mutant lines of embryos and sperm of model animals like the mouse. These mutant lines are vital to uncovering the genetic basis of diseases. Although many of these cell types can be successfully frozen, a sizeable number currently cannot. Furthermore, only a few tissues and fewer organs can be cryopreserved as of now. A major cause of injury in cells during freezing is the formation of intracellular ice, and high survivals demand that it be avoided or minimized. One problem is that the usual steps taken to avoid it may introduce injury from other causes. One theory of intracellular ice formation is that external ice makes contact with the cell surface and grows through pre-existing pores in the cell membrane to nucleate the cell interior. One important class of pre-existing pores in many cells are those formed by aquaporins. These are transmembrane proteins discovered about a decade ago that form Angstrom-size pores. The pores in some of the aquaporins (e.g. AQP-1) allow the passage of water only; others (e.g. AQP-3) allow the passage of water and small non-electrolytes like glycerol. A major specific aim of this proposal is to determine whether these pores constitute a route of entry for external ice, and consequent (lethal) ice formation in the cell interior. This will be tested by comparing the ice-nucleation temperatures of normal oocytes from mouse and the frog Xenopus with those of oocytes in which aquaporins have been expressed. In order for external ice to grow through the plasma membrane, it must first make contact with that membrane. The investigators will test this supposition by determining the effects of varying the fraction of ice in the external medium and, thus varying the probability of contact between cell and ice both in the presence and absence of aquaporins. Another factor may be the crystalline form of the external ice. Antifreeze proteins affect that form and are known to influence ice nucleation temperatures. The investigators will test their effect in combination with the other two (presence and absence of aquaporins and the magnitude of the frozen fraction). Finally, the investigators will use the above information to determine whether intracellular freezing can be minimized in ways that do not introduce other deleterious factors, thus providing novel approaches to enhancing the survival of difficult types of cells and tissues.