ABSTRACT
Physical and chemical alterations caused by the freezing and thawing and their effects on survivals/developments in vitro were investigated. Of a total of 452 two-cell mouse embryos, the overall survival rate of the frozen-thawed embryos was 76.1% (344/452). The blastocyst formation of the frozen-thawed embryos was 32.6% (44/136) compared to 74.5% (117/157) in the fresh embryos (P<0.05). The total number of cells in a blastocyst also decreased from 96.0 +/- 19.0 (n=26) in the fresh embryos to 42.0 +/- 11 .34 (n=30) in the frozen-thawed embryos (P<0.05). Fluorescence recovery after photobleaching (FRAP) measurement revealed about 5-fold decrease in the cell membrane fluidity with a characteristic time constant (tau) of 1.46 +/- 0.13 sec (n=5) in the frozen-thawed embryos as opposed to 0.28 +/- 0.04 sec (n=5) in the fresh embryos (P<0.05). The relative amount of H(2)O(2) in an embryo as quantified by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) showed 62.8 +/- 23.5 (n=24) and 34.2 +/- 14.5 (n=20) in the frozen-thawed embryos and in the fresh embryos, respectively (P<0.05). The distribution of actin filaments in the frozen-thawed embryos revealed an uneven distribution, particularly discontinuities at the "actin band," which contrasted to an even distribution shown in the fresh embryos. Mitochondrial staining by Rhodamine 123 showed that there was no significant difference between the two treatments in the number and in the distribution of viable mitochondria, but a marked aggregation was seen in the arrested embryos. No Annexin V binding was detected in two-cell or four-cell embryos while the binding was positive in the arrested embryos. The mitochondrial membrane potential measured by a membrane potential-sensitive fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol- carbocyanine iodide (JC-1) revealed a marked depolarization in the frozen-thawed embryos. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was employed to quantify the DNA fragmentation. In 75.0% cells of blastocysts (n=24) in the frozen-thawed embryos, the DNA fragmentation was detected as opposed to 37.0% in the fresh embryos (n=20) (P<0.05). Taken together, it is proposed that during the cryopreservation, two-cell mouse embryos are subjected to physical and chemical alterations, including destruction of the cell membrane integrity, redistribution of actin fibers, mitochondrial depolarizations, and increased reactive oxygen species (ROS) productions, which then may trigger the apoptotic cascade leading to a decrease in the survival rate and in the developmental rate of the embryos.