Category Archives: Intracellular Ice Formation

Type of Intracellular Ice Formation: Conclusion 2

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

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Type of Intracellular Ice Formation: Conclusion

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

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Type of Intracellular Ice Formation: Membrane Changes Associated with Development 2

The amount by which the melting point of a curved ice crystal in pure water is suppressed below that of a planar crystal is given by the Kelvin equation, namely,
where v, Tm, rSL, 9, and Lf are the molar volume of water, the melting point of the planar crystal, the interfacial tension between ice and water, the contact angle between ice and the inner wall of the pore (values can be 0-90°), and the latent heat of fusion of ice, respectively. The symbols a and r are the radius of the pore and the radius of curvature of the ice crystal (Fig. 6). Values for all except 9 are known or have been estimated. buy birth control online

Acker et al. state that v should be the molar volume of ice, not water, and that a correction term needs to be added to the equation to correct for situations where the liquid is a solution, not pure water, and they have calculated AT for various pore radii and contact angles using both the original and corrected equations. Table 5 shows the calculations for pore radii of 7.5 and 12 A, and for 9 = 50° and 75°. There is only about a 3°C difference in AT with the two equations, mostly due to substituting the molar volume of ice for the molar volume of liquid water. We observed a mean flash temperature of —23.1°C for the simultaneous freezing of the morulae (Table 3), which is consistent with the AT values calculated for a gap junction pore radius of 7.5 A in Table 5. Additional support for the role of gap junctions in cell-to-cell ice propagation is our finding that the propagation rate is slowed 6-fold by the presence of the gap junction inhibitor GA.
FIG. 6. Configuration of an ice-water interface in a hypothetical cylindrical pore in a cell membrane. See Membrane Changes Associated with Development for a definition of the symbols. (Reprinted in slightly modified form from Mazur with permission from Elsevier.)
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Type of Intracellular Ice Formation: Membrane Changes Associated with Development

Two important structures in the membranes of many cells are aquaporins on cell surfaces and gap junctions between tightly apposed cells in multicellular systems. As shown in Table 1, neither is present at any stage between the MII oocytes and four- to six-cell embryos. The early eight-cell embryo also shows no evidence of the presence of gap junctions or AQP 3; however, its membranes may possibly contain AQP 9. At this stage, the surfaces of the eight blastomeres are stacked on each other but not structurally joined. Later in the eight-cell stage, the membranes that form the boundary between two blastomeres now form tight junctions, and gap junctions appear. Simultaneously, AQP 3 now becomes detectable in the membranes facing the outside medium.

Gap junctions almost universally form across the dual bilayer constituting the tight junctions between cells. Most gap junctions are formed by a family of connexin proteins, and in the center of these junctions is a transmembrane pore that varies from 4 to 12 A in radius depending on the connexin that forms them. According to Saez et al., the pores are hourglass in shape. The outer surfaces have radii of 12-20 A that narrow to 7.5 A in the center. Reading here

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Type of Intracellular Ice Formation: DISCUSSION 2

The observed temperatures of IIF (flashing temperatures) fell essentially into two groups with respect to developmental stage and into two groups with respect to the type of nucleation. With respect to developmental stage, IIF in oocytes and one-cell embryos and simultaneous IIF in the blastomeres of two- to eight-cell embryos occurred between —35 and —43°C. That range overlaps the calculated Th, a fact that leads us to conclude that their flashing is a consequence of homogeneous nucleation. In theory, one might expect a slight lowering of Th with an increase in the number of blastomeres per stage. Because embryo volume remains nearly constant with development, the volume of individual blastomeres has to decrease. However, as indicated above, the dependence of Th on water volume is weak, and our failure to see a volume effect may simply be a consequence of the noise in the system. further

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Type of Intracellular Ice Formation: DISCUSSION

The purpose of this study was to determine whether the temperature at which intracellular ice is formed in mouse preimplantation embryos is influenced by the developmental stage. Knowledge of those temperatures is essential for computing the likelihood of IIF as a function of cooling rate. At the cooling rate used here (predominantly 20°C/min), modeling shows that mouse oocytes and embryos lose only — 12% of their water osmotically during cooling to —70°C [16, Fig. 1.9]. That means that the chemical potential of water inside the cells will become increasingly higher than that of the ice and solution outside the cell. Put differently, it means that the cell water becomes increasingly supercooled. At some temperature, the supercooling can no longer be maintained.

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Type of Intracellular Ice Formation: Effect of GA

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).

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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.
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Type of Intracellular Ice Formation: RESULTS

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. с

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Type of Intracellular Ice Formation: The Linkam Cryostage, Freezing Protocols, and Ramps

Using LN2 vapor for cooling and electrical resistors for heating, the Linkam cryostage with its associated control hardware and Pax-it software allows samples to be subjected to sequential ramps in which cooling rate, limiting temperature, holding time, and warming rate can be specified. The ramps used here are shown in Table 2. The procedure was as follows: the oocytes/embryos were cooled rapidly to —5.0°C, and cooled slowly to —8.0°C (ramps 1 and 2).

TABLE 2. Cooling and warming rates programmed into the Linkam cryostage for oocytes and embryos frozen in 1.0 M ethylene glycol/PBS.

External ice formation (EIF) occurred at a mean of —7.2 6 0.06°C. The sample was then warmed (ramp 3) to —3.2°C, which is just at the melting point of the medium. Most, but not all, of the external ice melted. Recooling was then initiated in ramp 4 after a 10-sec hold at the end of ramp 3. The purpose of ramp 3 was to provide time for the external liquid medium, the external ice, and the supercooled water in the cell to come to near equilibrium before recooling began. If ramp 3 was omitted, the observed temperatures of IIF were about 20°C higher. Most cases of IIF occurred during the rapid cool (20°C/min) in ramp 5. IIF was manifested by abrupt blackening of the cell. Above —30°C, cells completely blackened in a fraction of a second. Below that temperature, blackening could take a number of seconds. The nucleation or IIF temperature was taken to be the temperature at which the blackening first became evident. comments

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