Transcript analysis of heat shock protein 72 in vitrified 2-cell mouse embryos and subsequent in vitro development

The aim of the study was to compare the effects of two different concentrations of cryoprotectants by cryotopvitrification on survival, developmental capacity and Heat shock protein 72 (Hsp72) expression of two-cell mouse embryos.

In this experimental study, transcript analysis of Hsp72 gene was performed on non-vitrified and vitrified 2-cell mouse embryos via a nested quantitative polymerase chain reaction (nqPCR) subsequent to normalization with Hprt1 as the reference gene. The different cryoprotectant combinations were 15% (vit1:7.5% of each ethylene glycol (EG) and dimethyl sulfoxide (DMSO), 30% (vit2:15% EG + 15% DMSO) and control group with no cryoprotectants. Vitrified and fresh 2-cell embryos were cultured to obtain cleavage and blastocyst formation rates. The results were analyzed via one-way analysis of variance and the mean values were compared with least significant difference (LSD) (p< 0.05).

The relative expression of Hsp72 in vit2 (30% v/v) was significantly higher than vit1 (15% v/v). Survival rates were the same for both vitrification treatments and significantly lower than the control group. Cleavage and blastocyst rates in vit1 were significantly higher than vit2 while those in two vitrified groups were significantly lower than the control group.

Our developmental data demonstrated that vit1 treatment (7.5% EG and 7.5% DMSO) was more efficient than vit2 (15% EG and 15% DMSO) in mouse embryos. The cryotopvitrification with two concentrations of cryoprotectants caused the relative changes of Hsp72 transcript level, but the stability of the gene in vit1 was significantly higher than vit2 and closer to the fresh 2-cell embryos.