The temperature and type of intracellular ice formation in preimplantation mouse embryos as a function of the developmental stage

Abstract

Our studies the past 5 yr have concentrated on intracellular ice formation (IIF) in mature mouse oocytes at the metaphase stage of meiosis II. Here we report an analogous investigation of the temperature of intracellular ice nucleation in preimplantation embryo stages from one-cell to early morula suspended in 1 M ethylene glycol/PBS and cooled at 20 degrees C/min to -70 degrees C. Physical modeling indicates that oocytes and preimplantation embryos undergo very little osmotic shrinkage at that cooling rate. As a consequence, their interior becomes increasingly supercooled until the supercooling is abruptly terminated by IIF. Four categories of IIF were observed. The first two were 1) those undergoing IIF at temperatures well below the temperature of external ice formation (EIF; -7.2 degrees C) vs. 2) those undergoing IIF within 1 degrees C of the EIF temperature. The other two categories were those multicellular stages in which 3) all the blastomeres underwent IIF simultaneously vs. 4) those in which blastomeres underwent IIF sequentially. Embryos in categories 1 and 3 constituted the majority (80-90%), and for them, the mean IIF temperatures of one-cell, two-cell, four- to six-cell, and early eight-cell ranged from -37 degrees C to -43 degrees C, temperatures that indicate that IIF is a consequence of homogeneous nucleation. However, the IIF nucleation temperature of early morulae in categories 1 and 3 was markedly higher; namely, -23.1 +/- 1.5 degrees C. This marked rise in nucleation temperature coincides with the appearance of aquaporin 3 and gap junctions in early morulae (compacted eight-cell), and is presumably causally related.

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 blastomeres 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 μm.

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.

FIG. 3.

Photographs illustrating sequential flashing in an eight-cell embryo (1) and a morula embryo (2). 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.

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.

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.

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 [21] with permission from Elsevier.)