Vitrification is the most sought after route to the cryopreservation of animal embryos and oocytes and other cells of medical, genetic, and agricultural importance. Current thinking is that successful vitrification requires that cells be suspended in and permeated by high concentrations of protective solutes and that they be cooled at very high rates to below − 100°C. We report here that neither of these beliefs holds for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultra high rates (10,000,000°C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only 1/3rd of standard.

A hypothesis that accounts for most of the available literature on insulin-stimulated GLUT4 translocation is that insulin action controls the access of GLUT4 vesicles to a constitutively active plasma-membrane fusion process. However, using an in vitro fusion assay, we show here that fusion is not constitutively active. Instead, the rate of fusion activity is stimulated 8-fold by insulin. Both the magnitude and time course of stimulated in vitro fusion recapitulate the cellular insulin response. Fusion is cell cytoplasm and SNARE dependent but does not require cell cytoskeleton. Furthermore, insulin activation of the plasma-membrane fraction of the fusion reaction is the essential step in regulation. Akt from the cytoplasm fraction is required for fusion. However, the participation of Akt in the stimulation of in vitro fusion is dependent on its in vitro recruitment onto the insulin-activated plasma membrane.

The permeability of cells is important for cryopreservation. Previously, we showed in mice that the permeability to water and cryoprotectants of oocytes and embryos at early cleavage stages (early embryos) is low because these molecules move across the plasma membrane predominantly by simple diffusion through the lipid bilayer, whereas permeability of morulae and blastocysts is high because of a water channel, aquaporin 3 (AQP3). In this study, we examined the pathways for the movement of water and cryoprotectants in bovine oocytes/embryos and the role of AQP3 in the movement by determining permeability, first in intact bovine oocytes/embryos, then in bovine morulae with suppressed AQP3 expression, and finally in mouse oocytes expressing bovine AQP3. Results suggest that water moves through bovine oocytes and early embryos slowly by simple diffusion, as is the case in mice, although channel processes are also involved in the movement. On the other hand, water appears to move through morulae and blastocysts predominantly by facilitated diffusion via channels, as in mice. Like water, cryoprotectants appear to move through bovine oocytes/early embryos mostly by simple diffusion, but channel processes could also be involved in the movement of glycerol and ethylene glycol, unlike that in mice. In bovine morulae, although glycerol and ethylene glycol would move predominantly by facilitated diffusion, mostly through AQP3, as in mice, dimethylsulfoxide appears to move predominantly by simple diffusion, unlike in mice. These results indicate that permeability-related properties of bovine oocytes/embryos are similar to those of mouse oocytes/embryos, but species-specific differences do exist.

Vitrification is now the main route to the cryopreservation of human and animal oocytes and preimplantation embryos. A central belief is that for success, the cells must be placed in very high concentrations of cryoprotective solutes and must be cooled extremely rapidly. We have shown recently that these beliefs are incorrect. Over 90% of mouse oocytes and embryos survive being cooled relatively slowly even in solutions containing only 1/3rd the normal solute concentrations, provided that they are warmed ultra-rapidly at 107°C/min by a laser pulse. Nearly all vitrification solutions contain both permeating and non-permeating solutes, and an important question is whether the former protect because they permeate the cells and promote intracellular vitrification (as is almost universally believed), or because they osmotically withdraw a large fraction of intracellular water prior to cooling. The answer for the mouse system is clearly the latter. When oocytes or embryos are placed in 1 molal concentrations of the impermeable solute sucrose, they osmotically lose ~85% of their cellular water in less than 2 minutes. If the cells are then cooled rapidly to −196°C, nearly 90% remain viable after warming, again provided that the warming is ultra rapid.

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