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

Abstract

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.