**2.1 Mammalian pre-implantation embryos at different stages**

The mammalian zygote (Figure 3D) is formed by fertilization of the oocyte by spermatozoa, normally in the oviduct. With advancement of the embryo culture technology, fertilization can be initiated outside the body (i.e. *in vitro* fertilization). The embryonic development continues up to hatched blastocysts, i.e. maximum of 4 days in mouse and 6 days in human, without compromising the development of the embryos after they are transferred into the recipient's uterus/oviduct. The preimplantation stag eembryo is composed of a single cell or multiple blastomeres surrounded by an outer membrane called the zona pellucida, glycoprotein layer of a thickness of about 6m in mouse and 8m in human. The embryos have an outer diameter of about 0.10 mm in mouse and 0.12 mm in human. The volume enclosed by the mouse zona pellucida is limited to about 200 ρl and the diameter of the

Cryopreservation of Embryos from Model Animals and Human 265

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

**Cryoprotectants Permeability to different mouse embryo** 

**property 1-cell 2-cell Pre-compact** 

MW=59 Moderate Moderate High High

MW=78.1 High High High High

MW=92 Impermeable Impermeable Moderate High

Table 1. Permeability of various common cryoprotectants to mouse preimplantation

The higher the concentration of cryoprotecting agent is in a solution, the lower is the likelihood water crystals would be formed in the solution in a rapid-cooling process. However, most cryoprotecting molecules are toxic to embryos with toxicity positively correlating to their concentrations and the exposure time. When choosing the appropriate cryoprotectants, toxicity must be considered. Among five common permeating cryoprotecting agents to be tested, toxicity was determined to be dimethylformamide> erythritol > DMSO > glycerol and ethylene glycol on mouse morulae (Kasai et al., 1981). By electron probe microanalysis, Pogorelov et al. (2006 & 2007a) detected a dramatic decrease in intracellular potassium and sodium content in two-cell embryos treated with procedures mimicking vitrification in ethylene glycol, demonstrating a potential stress exerted on the

Moderate Moderate High High

High High High High

**morula (8-cell)**

**Compact morula (8-cell)** 

cryopreserved yet.

Acetamide Amide,

DMSO Organosulfur,

Glycerol Polyol,

**Molecular** 

Polyhydric alcohol, MW=62.1

Polyhydric alcohol, MW=76.1

embryos (Information derived from Pedro et al., 2005)

**2.3 Cryoprotectant toxicity to mammalian embryos** 

**Common name** 

Ethylene glycol

Propylene glycol

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 onto the endometrium for further development.

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