N OCIETY' - the Society for Reproductive Biology

COMPARISON OF NORMAL AND METHOXYLATED ETHYLENE GLYCOL FOR THECRYOPRESERVATION OF 2-CELL AND BLASTOCYST STAGES MOUSE EMBRYOSM. Pangestu1.2 and J.M. Shawl1)lnstitute ofReproduction and Development, Monash University, Clayton 3168 Australia,2)Faculty ofAnimal Husbandry, Jenderal Soedirman University, Purwokerto 53122 IndonesiaIntroductionAlthough there are a number of highly effectivecryopreservation (freezing) protocols for mouse oocytes andembryos, it remains almost impossible to freeze oocytes andearly embryos for most domestic species. The problemappears to be common to all species with "dark" (supposedlylipid rich) cytoplasm. It has recently been shown thatmethoxylation Le. replacing one or more OH group(s) byOCH3 group(s) of the cryoprotectant ethylene glycol (EG)increases its permeability, reduces its viscosity and increasesits capacity to bind lipids. As methoxylated ethylene glycol(s)have been successfully used for cryopreservation of kidney(1), it was possible that they would suit lipid rich embryosbetter than conventional EG, even though these compoundsare more toxic than EG. This study evaluated the efficacyand toxicity of cryoprotectants containing EG ormethoxylated EG's on early (2-cell) and late (blastocyst)stage mouse embryos.Materials and MethodsMouse embryos were generated by superovulating C57xCBAF1 females with PMSG (1OIU/ml), and hCG (1OIU/ml) 48 hlater followed by mating to Fl males. Two cell embryoswere collected 48 h after the hCG injection. Blastocysts forthe vitrification studies were derived from in vitro cultured 2­cell embryos. The baseline cryoprotectant solution contained20% dimethyl sulfoxide (DMSO), 20% ethylene glycol (EG)and 0.6 M sucrose (2). Part of the EG component (1/4 or1/2) was substituted for Ethylene Glycol Dimethyl Ether("DE", 1,2-Di methoxyethane, Sigma Cat. E 1129) orEthylene' Glycol Monomethyl Ether ("ME", 2­Methoxyethanol, Sigma Cat. E 5378) (Table 1).All embryos were cryopreserved and thawed inopen pulled straws (2). After thawing and 2 stepdilution embryos were washed and cultured inMTF medium and incubated at 37°C, in 5% CO 2 inair. Two cell embryos were cultured for 72 h todetermine the proportion developing intoblastocysts. Meanwhile the embryos vitrified at theblastocyst stage were in vitro cultured for 24 h todetermine their viability (re-expanding andhatching).ResultsThe development of 2-cell and blastocyst stageembryos vitrified in the baseline vitrificationsolution was comparable to that of controls andembryos vitrified in solutions containing 5% DE orME (Table 1). At a concentration of 10% both DEor ME reduced the proportion of 2-cell embryosdeveloping into blastocysts.ConclusionUp to 10% of the EO can be replaced by either ofthese methoxylated compounds, but some toxicitywas evident at the highest concentration (10%).This indicates that further studies on lipid richembryos should be performed.Table 1. Composition ofthe vitrification solutions and in vitro development of vitrified embryosVitrification solution compositionStage vitrified2 cell embryos BlastocystsVitrification Methoxylated EthyleneBlastocyst(%)(%)DMSO%Hatchingsolution compoundNglycol %N ViableNon-treatedBaselineDE5DEIOME5ME1005%10%5%10%20%15%10%15%10%2020202020222424262425100888869*9276485049505049100100100100100100444649444651*Statistically different compared to controls =P