However, 13% of embryos in stages two- through eight-cell flashed sequentially; i.e., an observable time (and temperature) interval elapsed between the freezing of one blastomere and that of a neighboring one. The mean time interval was 13.5 sec (Table 5). The mean temperature difference was —4.8°C. The mean temperature of the first freezing event was —24.5, —24.0, and —24.3°C (excluding one that froze at the EIF temperature of —7°C) for two-cell, four-cell, and eight-cell embryos respectively. That is 14°C higher than the mean flash temperature of —38.2°C for simultaneous flashing. The mean flash temperature for the second flash in the sequence was —29.1°C. That is 5°C lower than the first flash, but still 9°C higher than the mean for simultaneous flashing. read
We can only speculate as to the cause of the 14°C difference between the flash temperature of the 87% of the two- to eightcell embryos in which the blastomeres flashed simultaneously and the 14°C-higher temperature of the 13% in which they flashed sequentially. Because Th should be constant, the latter cells must be freezing heterogeneously. It is possible, but seems highly unlikely, that these 13% contained a weak nucleator whereas the other 87% did not. These stages contain no aquaporins that might permit the passage of ice from the medium and no gap junctions that would permit blastomere-to-blastomere propagation of ice. More likely, we think, is that a defect develops in the plasma membrane during the cooling of the 13%, a defect that allows external ice to pass through the membrane.
Perhaps this defect is a result of forces generated by EIF. Pedro et al. have reported that the ability of mouse oocytes/embryos to survive the osmotic swelling resulting from exposure to hypotonic saline solutions depends significantly on their developmental stage. One-cell embryos (zygotes) were the most resistant; eight-cell embryos and early morulae were the least resistant. Perhaps the putative defect mentioned in the previous sentence is a manifestation of that fact.
IIF in Morulae Flash temperatures in the 38 morulae examined fell into three groups. In the first group, three morulae flashed simultaneously at —40°C and one at —34°C. Group 2 is comprised of 24 morulae that underwent simultaneous flashing between —14 and —26°C. The third group consisted of two morulae that flashed simultaneously at —8 and — 10°C and eight morulae that flashed sequentially at —6 and —8°C.