122 INTRACELLULAR ICE FORMATION IN VITRIFIED MOUSE MORULAE DURING WARMING

Vitrification is an ice-free cryopreservation method. However, ice can form during warming if the concentration of cryoprotectant is insufficient. For the best use of vitrified embryos, it is important to know the condition in which intracellular ice (ICI) is formed, since ICI is fatal to the cell. So, we tried to estimate the conditions in which ICI forms in vitrified embryos during warming. Compacted morulae were obtained from ICR mice. Vitrification solutions were prepared by diluting ethylene glycol (EG) with FS solution (PB1 medium containing 30% Ficoll and 0.5 M sucrose) at 10, 20, 30, 40, and 50% (EFS10, EFS20, EFS30, EFS40, and EFS50, respectively). Morulae were treated with the solutions for 30, 60, or 120 s at 25�C, loaded in a straw, and vitrified using liquid nitrogen. The sample was warmed by various methods. The survival of embryos was assessed by their ability to develop to expanded blastocysts in culture. The difference in survival was analyzed by the chi-square test. In addition, the morphology of damaged embryos was examined to estimate the type of injury. It was confirmed that 20% or more EG was necessary to prevent the formation of extracellular ice during cooling without embryos. In the rapid warming protocol (control), in which vitrified samples were directly immersed in 25�C water, high embryonic survival was obtained with EFS30 (79–88%) and EFS40 (96–99%), but not with EFS20 (12–45%) and EFS50 (0–14%). This suggests that embryos vitrified with EFS20 were injured by ICI, whereas those vitrified with EFS50 were injured by the chemical toxicity of the solution. When embryos vitrified with EFS30 and EFS40 were warmed slowly by holding the straw in air at 25�C for 120 s, the survival rate decreased significantly (44–64%), probably because the cytoplasm of the embryos that had vitrified in a state of supercooling devitrified during slow warming. To estimate the condition in which ICI forms, embryos vitrified with EFS30 and EFS40 were kept at intermediate temperatures for various times before being warmed in 25�C water. Compared with control, survival rates decreased significantly by keeping samples for 3 min at -80�C (27–65%), -60�C (16–39%), -40�C (6–26%), or -20�C (33–46%). These results suggest that ICI forms at a wide range of temperatures between -80�C and -20�C, but more likely forms at temperatures between -60�C and -40�C. When the holding time was prolonged to 1 h and 24 h, the survival decreased further (0–58% and 0–14%, respectively). This shows that ICI forms not only quickly but also slowly at an intermediate temperature. From the morphological examination, embryos that should be injured by ICI swelled at recovery in PB1 medium and collapsed in culture, whereas those that should be injured by the toxicity of solution underwent decompaction after culture. This observation reinforces our supposition that slowly warmed embryos are injured by ICI.