Gene expression and development of mouse zygotes following droplet vitrification

The concept of ultra-rapid vitrification has emerged in recent years; the accelerated cooling rate reduced injury attributed to cryopreservation and improved post-freezing developmental competence of vitrified oocytes and embryos. The objectives of the present study were to develop a simple and effective ultra-rapid vitrification method (droplet vitrification) and evaluate its effects on post-thaw development and apoptosis-related gene expression in mouse zygotes. Presumptive zygotes were equilibrated for 3 min in equilibration medium and washed 3 times in vitrification solution. A drop (5 μL) of vitrification solution containing 10–12 embryos was placed directly onto surface of liquid nitrogen, with additional liquid nitrogen poured over the drop. For thawing and cryoprotectant removal, vitrified drops were put into dilution medium for 3 min, followed by M2 medium for 5 min. Although cleavage rate did not differ significantly among the control (90.8 ± 2.8%; mean ± S.E.M.), toxicity control (83.5 ± 3.2%), and vitrified (86.2 ± 3.1%) zygotes, rates of blastocyst and hatched blastocyst formation were lower (P < 0.01) in vitrified zygotes (49.7 ± 4.7% and 36.0 ± 4.7%) and toxicity controls (47.3 ± 4.6% and 40.3 ± 4.6%) compared with controls (65.5 ± 4.1% and 54.2 ± 4.3%). Exposure of zygotes to vitrification solution, as well as the vitrification process, down-regulated the expression of Bax, Bcl2, and p53 genes in blastocysts. Although droplet vitrification was efficient and easy, it altered the transcriptional activities of Bax, Bcl2, and p53 genes in vitrified embryos, indicating a strong relationship between reduced developmental competence and the altered transcriptional activities of these genes.