**2.2 Cryoprotectant permeability of mammalian embryos**

Prevention of ice formation by cryoprotectants is the key principle of protecting embryos from damage at cryogenic temperatures. To prevent intracellular ice formation, cryoprotectant molecules must penetrate the cells to exert intracellular cryoprotecting function. By measuring the changes in the volume of embryos immersed in cryoprotectant solutions (Emiliani et al., 2000), permeability of different cryoprotectant molecules at different developmental stages of preimplantation mouse embryos were compared (Table 1).

Embryos of different developmental stages show a differential permeating response to glycerol. The glycerol permeates compact morulae effectively and pre-compact 8-cell morulae moderately. One-cell embryos, 2-cell embryos and oocytes are virtually impermeable to glycerol. The highest permeability of embryos to cryoprotectant among all combinations is on compact morula to ethylene glycol. Acetamide also permeates at a relatively high degree in mouse 8-cell morulae but its permeability decreases dramatically in embryos at earlier developmental stages (Pedro et al., 2005). In sheep morula, ethylene glycol permeates faster than propylene glycol, dimethyl sulfoxide (DMSO) and glycerol (Songsasen et al., 1995). DMSO, on the other hand, shows moderate permeability on embryos at different preimplantation stages, with little difference. Being the most permeating cryoprotectants on 1-cell embryos and 2-cell embryos, propylene glycol permeates better than ethylene glycol. In general, there is a trend of increased permeability of the membrane towards various cryoprotectants when embryos develop (Mazur & Schneider, 1986; Pedro et al., 2005).

cellular part of a mouse zygote is about 85m (Zernicka-Goetz et al., 1997). In the presence of the perivitelline space, the cellular component is still in close contact with the zona pellucida, and is subjected to the immediate influence by the external medium due to the high permeability of the zona pellucida. The mouse embryo reaches two-cell stage and eight-cell stage at day 2 (dpc 1.5) and day 3 (dpc 2.5), respectively, after fertilization. Compaction usually occurs at day 3, causing a tight cell-cell association between the eight blastomeres to form a compact morula. At day 4 (dpc3.5), a blastocoel is evidenced as a cavity accumulated with fluid to form the blastocyst. The blastocoel expansion is limited by the non-growing zona pellucida. The blastocyst is composed of an embryonic inner cell mass and an extraembryonic trophectoderm which immediately surrounds the expanding blastocoel. At day 4, the blastocyst hatches from a breach in the zona pellucida and attaches

Fig. 3. Embryos and larva of model organisms to be cryopreserved. A, L1 stage larva of *C. elegans*; B, stage 15 (11-13hours) embryo of *Drosophila* (Weigmann et al., 2003); C; zebrafish at

Prevention of ice formation by cryoprotectants is the key principle of protecting embryos from damage at cryogenic temperatures. To prevent intracellular ice formation, cryoprotectant molecules must penetrate the cells to exert intracellular cryoprotecting function. By measuring the changes in the volume of embryos immersed in cryoprotectant solutions (Emiliani et al., 2000), permeability of different cryoprotectant molecules at different developmental stages of

Embryos of different developmental stages show a differential permeating response to glycerol. The glycerol permeates compact morulae effectively and pre-compact 8-cell morulae moderately. One-cell embryos, 2-cell embryos and oocytes are virtually impermeable to glycerol. The highest permeability of embryos to cryoprotectant among all combinations is on compact morula to ethylene glycol. Acetamide also permeates at a relatively high degree in mouse 8-cell morulae but its permeability decreases dramatically in embryos at earlier developmental stages (Pedro et al., 2005). In sheep morula, ethylene glycol permeates faster than propylene glycol, dimethyl sulfoxide (DMSO) and glycerol (Songsasen et al., 1995). DMSO, on the other hand, shows moderate permeability on embryos at different preimplantation stages, with little difference. Being the most permeating cryoprotectants on 1-cell embryos and 2-cell embryos, propylene glycol permeates better than ethylene glycol. In general, there is a trend of increased permeability of the membrane towards various cryoprotectants when embryos develop (Mazur &

onto the endometrium for further development.

50% epipoly; D, mouse embryo at zygote stage.

Schneider, 1986; Pedro et al., 2005).

**2.2 Cryoprotectant permeability of mammalian embryos** 

preimplantation mouse embryos were compared (Table 1).

The findings indicate a dynamic change in permeability of cell membrane to different cryoprotectants during development. This permeability change does not correlate with the molecular size of the cryoprotectants. In addition, the dynamic changes in cryoprotectant permeability do not seem to be caused by the increase in the total surface area of the embryos. The mouse 8-cell embryos undergo compaction at late day 4, thus decreasing the total surface area drastically, but it is best penetrated at least by ethylene glycol, compared with 1-, 2- and pre-compacted 8-cell embryos (Pedro et al., 2005), which have a higher surface area to volume ratios. Altogether, these findings support the notion that permeability is a dynamic physiological change related to the cellular differentiation state, not a simple passive mechanism dictated by the physical size and surface area. Understanding the changes of permeability of embryos to cryoprotectants at the molecular level may help further develop the use of cryopreservation technologies on mammalian embryos and, more importantly, on other organisms that cannot be cryopreserved yet.


Table 1. Permeability of various common cryoprotectants to mouse preimplantation embryos (Information derived from Pedro et al., 2005)
