Cryoprotectant loading from the perspective of oocyte cytoskeleton

Abstract

Conventional cryoprotectants (CPAs) are toxic at their typical freezing concentrations and induce significant injuries to the oocyte cytoskeleton. The objective of this study was to find CPA combinations and experimental conditions that are more compatible with the oocyte cytoskeleton. In vitro study. Metaphase II (MII) mouse oocytes were exposed to different experimental conditions, and then triple stained for microfilaments, microtubules, and DNA. Experiments were repeated at least three times. A total of 227 MII oocytes were analyzed. To assess adverse effect of conventional CPAs, MII oocytes were exposed to 1.5M dimethylsulfoxide (DMSO) at 37°C and room temperature (RT) for 10 min, subsequently fixed and stained. In parallel, MII oocytes were exposed to 0.1 M intra- and extracellular trehalose at 37°C for >1 hour to evaluate the effect of sugars on the cytoskeleton. Untreated MII oocytes served as controls. A short exposure of 10 min to 1.5 M DMSO at 37°C disrupted microfilament network (67%) and the spindle (94%) of MII oocytes causing dispersion of chromosomes (61%). Similar changes in microfilament organization (46%), spindle morphology (88%), and chromosome dispersion (42%) were observed after a 10-min exposure to 1.5 M DMSO at RT. In contrast, oocytes exposed to 0.1 M intra- and extracellular trehalose displayed normal microfilament organization (100%), spindle morphology (98%), and chromosome alignment (97%) similar to untreated controls (100%, 95%, and 95%, respectively). Next, 1/3 (0.5 M) of typical DMSO concentration was tested to reduce the toxicity. Indeed, exposure to 0.5 M DMSO at 37°C for 10 min resulted in normal microfilament organization, spindle morphology, and chromosome alignment in vast majority of oocytes (100%, 85%, and 92%, respectively). Although 0.5 M DMSO is insufficient to provide adequate cryoprotection alone, combining it with low concentrations of intra- and extracellular trehalose results in excellent cryosurvival rates over 90%. When exposed to 0.1 M intra- and extracellular trehalose and 0.5 M DMSO together at 37°C for 10 min, the proportion of oocytes showing normal microfilament organization (100%), spindle morphology (92%), and chromosome alignment (85%) was similar to controls. Our results suggest that sugars such as trehalose are non-deleterious to the cytoskeleton and can be used with low concentrations of CPAs to reduce the CPA toxicity while maximizing the overall cryoprotection.