Effect of vitrification on the expression of genes in porcine blastocysts derived from in vitro matured oocytes

Abstract

This study aimed to examine the effect of vitrification on the expression of genes that are crucial for porcine early embryo development; cathepsin B (CTSB), growth differentiation factor 9 (GDF9), caudal type homeobox 2 (CDX2), and OCT-4, which play an important role in the maintenance of embryonic cell pluripotency. Their gene expression was investigated in expanded blastocysts (day 6–7) derived from in vitro matured oocytes. The quantitative real-time PCR method was used to assess the amount of relative specific transcripts in 20 vitrified (treatment group) and 32 fresh non-vitrified (control group) blastocysts. Vitrification was performed using 7.5% dimethyl sulfoxide (DMSO) plus 7.5% ethylene glycol (EG), and in the final step, 15% DMSO plus 15% EG and a 0.5 M sucrose solution and cryotop as a vitrification device. The blastocysts were warmed in 1 M, 0.5 M, and 0.25 M sucrose solution and kept in a culture medium for six hours before their fixation and further qPCR analysis. A significant upregulation in the targeted genes CTSB (p<.006), GDF9 (p<.04), and CDX2 (p<.003) was observed in the vitrified embryos compared to the fresh control group. Interestingly, the OCT-4 mRNA expression level was not affected by vitrification and remained comparable to that of the fresh non-vitrified embryos. In summary, the results of this pilot study showed, that vitrification induced substantial alteration in the expression of CTSB, GDF9, and CDX2 genes but did not influence the expression of OCT-4 gene in porcine in vitro derived blastocysts. Our data on the expression of developmentally important genes in vitrified porcine blastocyst may facilitate: (1) future improvements in culture conditions and/or cryopreservation protocol and (2) understanding the mechanism(s) of cryoinjuries inducing compromised post-thaw embryo development followed by the poor pregnancy outcome after blastocyst transfer.