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Intracellular ice is generally lethal. One way to avoid it is to vitrify cells; that is, to convert cell water to a glass rather than to ice. The belief has been that this requires both the cooling rate and the concentration of glass-inducing solutes be very high. But high solute concentrations can themselves be damaging. However, the findings we report here on the vitrification of mouse oocytes are not in accord with the first belief that cooling needs to be extremely rapid. The important requirement is that the warming rate be extremely high. We subjected mouse oocytes in the vitrification solution EAFS 10/10 to vitrification procedures using a broad range of cooling and warming rates. Morphological survivals exceeded 80% when they were warmed at the highest rate (117,000°C/min) even when the prior cooling rate was as low as 880°C/min. Functional survival was >81% and 54% with the highest warming rate after cooling at 69,000 and 880°C/min, respectively. Our findings are also contrary to the second belief. We show that a high percentage of mouse oocytes survive vitrification in media that contain only half the usual concentration of solutes, provided they are warmed extremely rapidly; that is, >100,000°C/min. Again, the cooling rate is of less consequence. [ABSTRACT FROM AUTHOR]

The permeability to water and cryoprotectants of the plasma membrane affects the suitable condition for cryopreservation of the cell. Previously, we showed that water, glycerol, ethylene glycol, Me2SO, and acetamide move through mouse morulae by facilitated diffusion. We also showed that the facilitated diffusion of water, glycerol, and ethylene glycol relies on aquaporin 3, an aquaglyceroporin (Edashige et al., 2007), and suggested that AQP9, another aquaglyceroporin, can transport Me2SO, acetamide, and other cryoprotectants when AQP9 was exogenously expressed in mouse oocytes (Edashige et al., in Cryo 2011). In the present study, we examined the transporting-specificity of AQP9 endogenously expressed in mouse morulae. In addition, we examined whether AQP9 expressed in morulae was involved in the tolerance to cryopreservation. As shown in previous studies, the suppression of the endogenous expression of AQP9 in morulae decreased the permeability to Me2SO and acetamide but not that to other cryoprotectants. And the exogenous expression of AQP9 in mouse oocytes, in which water/cryoprotectant channels are not expressed at the functional level, significantly increased not only the permeability to Me2SO and acetamide but also to glycerol and ethylene glycol. Cystic fibrosis transmembrane conductance regulator (CFTR), which has been reported to modulate the transporting-property of AQP9, was markedly expressed in oocytes but not in morulae. The suppression of the endogenous expression of CFTR in oocytes decreased the permeability to glycerol and ethylene glycol of oocytes enhanced by exogenous expression of AQP9 but not that to Me2SO and acetamide. These results indicate that the transporting-property of AQP9 is modulated by CFTR, and suggest that AQP9 endogenously expressed in morulae transports only Me2SO and acetamide because CFTR is not expressed in morulae. When mouse morulae were vitrified with DFS20, a Me2SO -based solution, by a one-step method, most of intact morulae survived and had the ability to develop into blastocysts after warming, whereas only 40% of AQP9-suppressed morulae survived and no embryos had the ability to develop, suggesting that AQP9 plays an important role in the tolerance to vitrification. These results will provide important information for understanding the movement of cryoprotectants in mouse embryos. Source of funding: The Japan Society for the Promotion of Science. Conflict of interest: None declared. keisuke@cc.kochi-u.ac.jp [Copyright &y& Elsevier]

The germplasm of mutant mice is stored as frozen oocytes/embryos in many facilities worldwide. Their transport to and from such facilities should be easy and inexpensive with dry ice at -79 °C. The purpose of our study was to determine the stability of mouse oocytes with time at that temperature. The metaphase II oocytes were cryopreserved with a vitrification solution (EAFS10/10) developed by M Kasai and colleagues. Two procedures were followed. In one, the samples were cooled at 187 °C/min to -196 °C, warmed to -80 °C, held at -80 °C for 1 h to 3 months, and warmed to 25 °C at one of three rates. With the highest warming rate (2950 °C/min), survival remained at 75% for the first month, but then slowly declined to 40% over the next 2 months. With the slowest warming (139 °C/min), survival was only ∼ 5% even at 0 time at -80 °C. In the second procedure, the samples were cooled at 294 °C/min to -80 °C (without cooling to -196 °C) and held for up to 3 months before warming at 2950 °C/min. Survival was ∼ 90% after 7 days and dropped slowly to 35% after 3 months. We believe that small non-lethal quantities of intracellular ice formed during the cooling and that the intracellular crystals increased to a damaging size by recrystallization during the 3 month's storage at -80 °C. From the practical point of view, this protocol yields sufficient stability to make it feasible to ship oocytes worldwide in dry ice.

Movement of water and cryoprotectants through the plasma membrane needs to be accelerated for successful cryopreservation of zebrafish oocytes/embryos, which are much larger than their mammalian counterparts. Aquaporin-3 is a water/solute channel that can transport not only water but also various cryoprotectants. In this study, we attempted to increase the permeability of immature zebrafish oocytes at stage III to water and cryoprotectants by exogenous expression of rat aquaporin-3. Immature zebrafish oocytes were injected with rat aquaporin-3 cRNA and cultured for 5-12 h. Permeability to water and cryoprotectants was then determined based on changes in the volumes of the oocytes in a hypertonic sucrose solution and various cryoprotectant solutions at 25 C. The permeability to water of the aquaporin-3 cRNA-injected oocytes was three times higher than that of intact and water-injected oocytes. The permeability of the aquaporin-3 cRNA-injected oocytes to ethylene glycol, glycerol, propylene glycol, and DMSO was also 2-4 times higher than that of intact oocytes. Thus, the permeability of immature zebrafish oocytes to water and cryoprotectants was enhanced by exogenous expression of aquaporin-3. Cryopreservation of teleost oocytes may be realized through a further increase in permeability.