FIG. 1. Photographs illustrating the two types of intracellular ice formation (IIF) in two-cell mouse embryos. The upper row depicts simultaneous IIF in the two blasto-meres at —31.8°C. The bottom row illustrates sequential IIF. One blastomere froze at —24.5°C;the other, at —26.7°C. The first photograph in each row (A) was taken before cooling was initiated. The second photograph (B) was taken at —24.3°C before IIF occurred, and the third and fourth photographs (C and D) were taken after IIF. The diameter of the faint zona pellucida is —75 im.
Two Types of IIF
Two types of flashing or IIF were observed in this study. In one case (simultaneous IIF), all the blastomeres in a given embryo darkened simultaneously and uniformly. The rate at which they darkened depended on the temperature at which flashing was initiated. When the temperature was above —30°C, complete darkening took only a fraction of a second. When nucleation occurred below —30°C, darkening took a number of seconds. In the other type (sequential), the flashing of an individual blastomere or a small group of blastomeres was separated in time (and therefore in temperature) from the flashing of neighboring blastomeres or small groups of blastomeres in the same embryo. The two types are illustrated in Figure 1 for a two-cell embryo. Figure 1-1C shows simultaneous IIF in the two blastomeres at —31.8°C. Figure 1-2C and 1-2D shows sequential IIF in each blastomere 2.2°C apart and 6.4 sec apart. The ‘‘A’’ photographs in the first column show their appearance before the start of the experiment. The ‘‘B’’ photographs in the second column show the appearances after EIF occurred at —7.0°C but before the occurrence of IIF. сanadianhealthcaremallinc.com
FIG. 2. Frequency distribution of the flash or IIF temperatures for oocytes and the various stage embryos studied here. The bars show the temperature at which all the blastomeres flashed simultaneously.
IIF Temperature of Mouse Embryos at Each Developmental Stage in Which the Blastomeres Flashed Simultaneously
Figure 2 shows the frequency distributions of IIF flashing temperatures for each stage. For two-cell embryos to morulae, the data are for embryos in which the blastomeres underwent simultaneous flashing. The first conclusion is that the distribution of flash temperatures was broader for the MII oocytes and the morulae than for the one-cell to eight-cell embryos. The second conclusion is that the modal value for the flash temperature was around —40°C for one-cell through eightcell embryos, but was vaguer and higher for MII oocytes and morulae (much higher for the latter). A third point is that the range of flash temperatures for the morulae overlaps part of the high end temperature distribution for oocytes and two-, four-, and eight-cell embryos, a point that we shall return to in discussion.
TABLE 3. Intracellular flashing temperature versus the developmental stage of mouse oocytes/embryos.*
Table 3 shows the mean temperature for simultaneous flashing for each developmental stage. In MII oocytes and onecell, two-cell, four (to six)-cell, and eight-cell embryos, the flash temperatures (—34.4, —42.9, —40.1, —38.0, and —35.4°C), are similar, although those for the oocytes (—34.4°C) and eightcell embryos (—35.4°C) are slightly but statistically higher than those for the other embryo stages.