Cryopreservation of mammalian oocytes by using sugars: Intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates

Accumulation of intra- and extracellular sugars such as trehalose, glucose, and raffinose is central to survival strategies of a variety of organisms coping with extreme conditions including freezing and almost complete drying. The objective of the present study was to investigate the potential application of intra- and extracellular raffinose in combination with low concentrations of dimethylsulfoxide (Me₂SO) to mammalian oocyte cryopreservation. To this end, the fertilization and embryonic development of cryopreserved metaphase II (M II) mouse oocytes were studied in comparison to unfrozen controls. For cryopreservation, M II oocytes were microinjected with 0.1M raffinose, and then cooled to -196°C in the presence of either 0.3M raffinose and 0.5M Me₂SO (cryopreservation group 1) or 0.3M raffinose and 1.0M Me₂SO (cryopreservation group 2). The control groups included untreated oocytes (untreated control) and oocytes microinjected with raffinose, but not frozen (injection control). The post-thaw survival rates were 83.9% and 80.6% for the cryopreservation group 1 and 2, respectively. The fertilization and blastocyst rates in the cryopreservation group 1 (90.0% and 77.8%, respectively) and 2 (94.6% and 72.5%, respectively) were also high and similar to the ones of the injection controls (97.8% and 78.5%, respectively) and untreated controls (98.8% and 83.6%, respectively). These results are consistent with the findings of our earlier studies and support the use of sugars as intra- and extracellular cryoprotectants. Furthermore, the results of the present study indicate that the presence of intra- and extracellular sugars alleviates high concentrations of conventional penetrating cryoprotectants, and thus minimizes their toxicity.