Permeability of Mouse Oocytes and Embryos at Various Developmental Stages to Five Cryoprotectants

Abstract

Abstract.To assess the permeability of mouse oocytes and embryos, matured oocytes and embryos at various stages of development were placed in five cryoprotectant solutions at 25 C for 25 min. From the cross-sectional areas of the oocytes/embryos, the relative change in volume was analyzed. In oocytes, shrinkage was least extensive and recovery was quickest in the propylene glycol solution, showing that propylene glycol permeates the oocytes most rapidly. Dimethyl sulfoxide, acetamide, and ethylene glycol permeated the oocytes slightly more slowly than propylene glycol. The oocytes in glycerol shrunk extensively and then expanded marginally, indicating slow permeation. The volume changes of 1-cell and 2-cell embryos were similar to those of oocytes, showing little change in permeability. In 8-cell embryos, the volume recovered much faster than in the earlier stages especially in glycerol and acetamide. In morulae, the volume recovery was much faster in glycerol and in ethylene glycol; in ethylene glycol, the extent of shrinkage was small and the recovery was fast, indicating an extremely rapid permeation. Although the permeability of oocytes/embryos generally increased as embryo development proceeded, the degree of increase varied greatly among the cryoprotectants. Interestingly, the volume change in propylene glycol was virtually unaffected by the stage of development. Such information will be valuable for determining a suitable protocol for the cryopreservation of oocytes/embryos at different stages of development. Key words: Mouse, Embryo, Oocyte, Permeability, Cryoprotectant (J. Reprod. Dev. 51: 235-246 , 2005) he survival of mammalian oocytes and embryos after cryopreservation varies with the stage of maturation and development. In general, oocytes/embryos at earlier stages appear to be more sensitive to cryopreservation. In the mouse, the rate of survival after cryopreservation is lower for unfertilized oocytes than embryos and increases as development proceeds up to the 8-cell or morula stage, although it may decrease as the blastocoel enlarges Cryoprotectants for mammalian oocytes and embryos are virtually limited to these five agents both in slow freezing and in vitrification [8].Although the protective action of cryoprotectants is considered colligative [9], each agent has its own specific properties. Among them, the permeating p r o p e r t y i s o f g r e a t i m p o r t a n c e , b e c a u s e permeation of the cell with a cryoprotectant is critical for the successful cryopreservation of

Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method

“…Vitrified 8-cell stage embryos had the highest blastocyst formation and hatching rates, which implied that this stage is the most appropriate for vitrification. This could be due to higher permeability to cryoprotectants at the 8-cell stage, as compared with earlier developmental stages [37]. Our results correlate with previous successful reports on the vitrification of human cleavage-stage embryos [8][9][10]31].…”

Developmental Ability of Vitrified Mouse Oocytes Expressing Water Channels

“…The water-permeability and glycerol-permeability of the oocytes/embryos were measured from the change in volume of oocytes, zygotes and a blastomere of 2-cell and 4-cell embryos during exposure to glycerol/PB1 at 25 C by the method described elsewhere [4,8]. The osmolality of 10% (v/v) glycerol in water was 1.59 Osm/kg (calculated from published data about the colligative properties of glycerol in aqueous solutions) [14], and that of PB1 medium was 0.30 Osm/kg (measured with an osmometer; OM801, Vogel, Giessen, Germany).…”

“…We also showed in the mouse that permeability to water and cryoprotectants was low in oocytes and early embryos, whereas permeability to water, glycerol and ethylene glycol was high in morulae [4], and that aquaporin 3 (AQP3), an aquaglyceroporin, plays an important role in the high permeability [5,6]. Therefore, the high tolerance of morulae to cryopreservation would be attributable to their high permeability via water/cryoprotectant channels.…”

Preservation of Embryonic Stem Cells

“…The unusually high (as high as 7 M) concentration of CPA required, however, can result in significant metabolic and osmotic injury to cells (Chen et al 2000;Chen et al 2001a;Fahy et al 2004a;Fowler and Toner 2005;Heng et al 2005;Hunt et al 2006). Consequently, it is necessary to use multiple steps of CPA loading/dilution and maintain a short exposure time (within a few minutes) to high concentration CPA in each step to minimize injury (Reubinoff et al 2001), which makes the procedure complicated, stressful, and particularly, difficult to control in that the time for the diffusion of CPAs into the cells to reach equilibrium usually takes at least 5-10 minutes (He et al 2008b;Heng et al 2005;Jain and Paulson 2006;Pedro et al 2005). In addition, a cocktail of various CPAs rather than one CPA has been commonly used to reduce the CPA toxicity.…”