FIG. 5. The time that elapsed between the flashing of the first blastomere in a two- to eight-cell embryo or morula and the flashing of the next blastomere or small group of blastomeres in that embryo.
Figure 5 shows the average number of seconds that elapsed between the flashing of the first blastomere in two-, four (six)-, and eight-cell embryos and early morulae and that of the second, usually adjoining, blastomere. (Strictly speaking, in the four-cell through morula stages, we usually could not observe flashing events in each individual blastomere, but rather separate flashing in small groups of blastomeres. Thus, in four- to six-cell embryos, eight-cell embryos, and morulae, we observed 2-3, 2-4, and 4-11 separate freezing events, respectively. However, for economy of writing, we will sometimes refer to the flashing of a small group of blastomeres as equivalent to the flashing of a single blastomere.) Figures 4 and 5 both show striking differences between the morulae and the earlier stages. With respect to temperature (Fig. 4), the initial flashing in the eight morulae occurred at —6 or —8°C, followed at nearly the identical temperature by the flashing of the last group in that morula. In contrast, in the two-cell to eight-cell stages, the first flashing (black bars) occurred at much lower temperatures (with one exception) and over a broad range (—12 to —42°C). And the last component to flash in a given embryo (white bars) did so at a still lower temperature. These latter temperatures are somewhat higher than those observed for simultaneous flashing (Fig. 2).
With respect to the time interval between the first flashing event and the second (Fig. 5), the interval in the early morulae was 0.6 6 0.1 sec, a much shorter time than the intervals of 10 to 18 sec in the three earlier stages. The time differences in the latter group were not significant. The temperature and time abscissas in Figures 4 and 5 are interrelated by the fact the cooling rate was 10°C/min between —3.2 and —7°C in ramp 4 and was 20°C/min below —7°C in ramp 5 (Table 2). Thus in ramp 4, the temperature dropped 1.7°C over a 10-sec. interval. In ramp 5, it dropped 3.3°C in 10 sec.
The compound GA blocks the ability of gap junctions to readily transmit arginosuccinate between fibroblast cells, presumably by at least partly closing the pores in the junctions. Irimia and Karlsson used it for that purpose to test the ability of ice to propagate from one tissue culture cell to an apposed neighbor. When we applied it to mouse morulae, it produced a 6-fold increase in the time required for ice to propagate from an initial frozen blastomere (or small group of blastomeres) to a neighboring blastomere (or small group) from 0.6 6 0.1 to 3.6 6 0.7 sec—a highly significant increase. The 3.6-sec propagation delay, however, was still shorter than the 10-sec delay observed in eight-cell embryos. We used a 30 iM concentration of GA, the same as that used in. Davidson et al. report that GA is effective and nontoxic over a broad concentration range (2-100 iM). Permeation through gap junctions is also reported to be reduced by eliminating Ca+ from the medium; however, in our hands, the ice propagation delay was essentially the same in PBS lacking Ca++ as in PBS containing it. Moreover, the substantial ice propagation delay induced by GA was essentially the same when Ca++ was present (3.8 6 0.7 sec) as when it was absent (3.2 6 0.8 sec).