Type of Intracellular Ice Formation: RESULTS 2

In contrast, the mean IIF flash temperature of morulae (— 23.1 °C) was substantially higher than those of any other stage. The data in Figure 2 and Table 3 apply to the situation in which all blastomeres flashed simultaneously. The ensuing sections pertain to the situation in which flashing in individual blastomeres or small groups of blastomeres was sequential. The percentages that flashed simultaneously were 91.1%, 82.9%, and 86.0% in two-cell, four-cell, and eight-cell embryos, respectively. With morulae, the percentage was lower— namely, 78.9%—but not significantly so.

Propagation of IIF in Two- to Eight-Cell Embryos and in Early Morulae from a Flashed Blastomere to Neighboring Blastomeres
Table 4 shows that the percentages of embryos in which the blastomeres flashed sequentially were 8.9%, 17.1%, and 14.0% in two-cell, four-cell, and eight-cell embryos. The percentage was 21.1% in morulae. Figure 3 shows photographs of sequential flashing of blastomeres in an eight-cell embryo (Fig. 3-1) and in an early morula (Fig. 3-2). The times shown are the seconds that elapsed between the flashing of one blastomere and that of a next (usually adjoining) one. Because the cells were being cooled at 10 or 20°C/min, the time interval also represents a temperature interval. Two conclusions are evident. One is that the time interval between flashing in the morulae was considerably shorter (Fig. 3-2) than that in the eight-cell embryos (Fig. 3-1). The second point is that this sequential flashing in both stages is occurring at a much higher temperature (—7 to — 16°C ) than that observed for simultaneous flashing. The temperature of —7°C is essentially the same as the temperature at which EIF occurred. Link

FIG. 3. Photographs illustrating sequential flashing in an eight-cell embryo and a morula embryo. Photographs 1A and 2A depict the embryos in 1.0 M EG just prior to EIF;photos 1B and 2B, just after EIF. They then underwent sequential flashing of the blastomeres (1-I, 1-II, 1-III, 1-IV for an eight-cell embryo and 2-I, 2-II, 2-III, 2-IV for a morula embryo) at the temperatures depicted. We also show the time for IIF to spread from one flashed blastomere(s) to an adjoining blastomere(s). The magnification is the same as in Figure 1.
TABLE 4. Ratio of simultaneous flashing and sequential flashing in 2- to 8-cell embryos and in early morulae.

FIG. 4. Frequency distribution of the flash or IIF temperatures of morulae exhibiting sequential flashing. The black bars show the temperatures at which the first blastomere or small group of blastomeres in a morula exhibited flashing. The open bars show the temperatures at which the last blastomere or small group of blastomeres flashed in a given morula.
Figure 4 is a plot of the frequency distribution of the temperatures at which the first blastomere or small group flashed (black bars) and the corresponding temperature at which the last group flashed (white bars) in each of the multicellular developmental stages studied. (Note that —6 and —8°C on the far right of the abscissa are repeated two or three times. The reason is that these temperatures are traversed in ramps 2, 3, 4, and 5, as can be seen from inspection of Table 2.)

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