The four morulae in group 1 show that morulae are capable of cooling to Th before nucleating. Therefore, these four possessed intact membranes during cooling and possessed no internal heterogeneous nucleators. Because the 24 morulae in the second group flash at much higher temperatures, they must be nucleating heterogeneously. We suggest the following interpretation of their behavior. One blastomere undergoes IIF at, say, —20°C, perhaps because its membrane is damaged or perhaps for stochastic reasons. (Note that the range of—14°C to —26°C for morula flashing overlaps the higher end of the temperature range of flashing in the earlier stages.) At —20°C, the Kelvin effect is strong enough to permit ice in that blastomere to easily pass through the gap junctions into neighboring blastomeres to initiate IIF in them.
The third and final group includes the morulae that flashed between —6°C and —8°C, 80% of which flashed sequentially. It would seem more than a coincidence that these flashing events occurred right at the temperature at which extracellular freezing occurred (mean of —7.3°C). Some flashed almost immediately after EIF in ramp 2; some flashed during the warming segment of ramp 3; and some flashed during the subsequent cooling of ramp 4. If this is more than a coincidence, it suggests that external ice crystals exert a force on the morula that produces defects in the plasma membrane of one or more of the blastomeres that lead to IIF in it or them. comments
In group 2 morulae, the temperature is low enough to permit that internal ice to propagate swiftly through gap junctions to nucleate neighboring blastomeres. But in these group 3 morulae, the temperature is —6 to —8°C, and that may be marginal with respect to the existence of any ice crystal with a radius of curvature small enough to pass through the pore in a gap junction. The conclusion that the ice is propagating from one cell in a morula to others through the pores in gap junctions is supported by the finding that GA, which reportedly closes the pores, increases the propagation time 6-fold.
NOTE ADDED IN PROOF
During the production of this paper, a paper by Seki and Mazur showing that the temperatures of intracellular ice formation of oocytes and embryos subjected to a prior vitrification are the same as those of freshly collected oocytes and embryos was accepted for publication.
We appreciate the assistance of the following undergraduate and graduate students of Drs. Edashige and Kasai in collecting and vitrifying the oocytes/embryos sent to Knoxville: Dr. B. Jin, M. Tanaka, S. Ohta, K. Matsuo, T. Kuwano, M. Fuchiwaki, T. Kouya, T. Hara, and S. Takahashi.