Submitted March 24, 2008
Returned for revision April 18, 2008
Accepted June 11, 2008
Reproductive Technology
Effect of Warming Rate on the Survival of Vitrified Mouse Oocytes and on the Recrystallization of Intracellular Ice
Shinsuke Seki
and
Peter Mazur *
* To whom correspondence should be addressed. E-mail: pmazur{at}utk.edu.
Abstract
Successful cryopreservation demands there be little or no intracellular ice. One procedure is classical slow equilibrium freezing, and it has been successful in many cases. However, for some important cell types, including some mammalian oocytes, it has not. For the latter, there are increasing attempts to cryopreserve them by vitrification. However, even if intracellular ice formation (IIF) is prevented during cooling, it can still occur during the warming of a vitrified sample. Here, we examine two aspects of this occurrence in mouse oocytes. One takes place in oocytes that are partly dehydrated by an initial hold for 12 min at -25°C. They are then cooled rapidly to -70°C and warmed slowly or they are warmed rapidly to intermediate temperatures and held. These oocytes undergo no IIF during cooling but blacken from IIF during warming. The blackening rate increases about 5-fold for each 5-degree rise in temperature. Upon thawing, they are dead. The second aspect involved oocytes that had been vitrified by cooling to -196°C while suspended in a concentrated solution of cryoprotectants, and warmed at rates ranging from 140 to 3300°C/min. Survivals after warming at 140 and 250°C/min were low (<30%). Survivals after warming at 
2200°C/min were high (80%). When warmed slowly, they are killed apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing are discussed.
Key words:
Assisted Reproductive Technology •
Ovum •
cryopreservation •
vitrification
