I15 Should Cryoprotectants be Formulated According to the Mouse Genome?

Mike Legge, Mat Byers and Stephen Bird. Molecular Embryology Group, Department of Biochemistry, University of Otago, PO BOX 56 Dunedin, New Zealand

The use of penetrating cryporotectants such as dimethyl sulphoxide and I,2-propanediol is well established for mouse embryo cryopreservation. However, the sensitivity of both oocytes and embryos to adverse effects of both cryoprotectants and the freezing process is well documented (Duliquost et al. 1999). For mouse genome cryopreservation this is especially important as there is a correlation between mouse embryo genotype and cryopreservation success. (Schmidt et al. 1985). With the increasing necessity to archive mouse genomes by embryo and gamete cryopreservation the improvement in cryoprotectant formulations is becoming increasingly important. Recently we identified the non-enzymatic formation of formaldehyde in cryoprotectant solutions and that at the micro-molar concentrations present oocyte zona pellucida hardening was established and that this effect could be overcome by the use of reduced thiol compounds (Karren and Legge, 1996). Aldehydes are known to be detrimental to biological systems especially by modifying energy substrate metabolism and protein cross-linking. The aim of the current research is to further characterise the relationship of the aldehydes in cyoprotectants, to determine whether the product resulting from the scavenging reaction was potentially toxic to embryos and to initiate investigations into cryprotectant effects on enzymes involved into embryo energy metabolism. Using dimethyl sulphoxide as a model system we have characterised the aldehyde profile by high pressure liquid chromatography using 2,4 dinitrophenyl hydrazine aldehyde derivatives and a C-18 column (Nilsson-Thorell et al. 1993). Six aldehydes have been identified of which the two major components are formaldehyde and acetylaldehyde. Addition of cysteine as a free thiol scavenger significantly reduced the aldehyde content of the cryoprotectant. Using NMR spectroscopy we have established that the formaldehyde-cysteine scavenger product is 4-thiazolidine-4-carboxylic acid, a non-toxic compound. In-vitro kinetic analysis of lactate dehydrogenase exposed to 0.5, 1.0, 1.5 and 2.0 M dimethyl sulphoxide demonstrates a progressive loss of enzyme activity to 75% of the control activity after 30 minutes exposure to the cryoprotectant. In summary we believe that the ability of embryos and gametes to under go successful cryopreservation and thawing is dependent upon the non-enzymaticaly formed compounds in the cryoprotectants and the biochemical interaction with these compounds on embryo metabolism. This in turn may well be reflected in the embryo genotype and we propose that a pharmacogenomics approach should be considered for resolving the interaction with mouse embryo genomes and cryprotective agent.

Funding Source: Lottery Health Research, NZ. University of Otago Research Bequest.


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Karren, G and Legge, M (1996). The non-enzymatic formation of formaldehyde in mouse oocyte freezing mixtures. Human Reproduction, 11; 101-106.

Nilsson-Thorell,CB., Muscala, N et al.(1993) Heat sterilization of fluids for peritoneal dialysis gives rise to aldehydes. Peritoneal Dialysis. International. 15; 348-352.

Schmidt, PM., Hansen, CT and Wilde, DE. (1985) Viability of frozen-thawed mouse embryos is affevted by genotype. Biology of Reproduction. 32; 507-514.