title: Amelioration of mitochondrial dysfunction and apoptosis of two-cell mouse embryos after freezing and thawing by the high frequency liquid nitrogen infusion.
abstract: Liquid nitrogen (LN2) infusions are currently used in a slow controlled-rate freezing during cryopreservation. The effects of two different LN2 infusion frequencies (conventional, slow 50 infusions/min and high 120 infusions/min) were studied with frozen-thawed two-cell mouse embryos and their subsequent development to blastocysts. The embryos that were subjected to the high frequency LN2 infusion (HFLI) showed a significantly higher survival rate over the low frequency LN2 infusion (LFLI) (50.7 vs. 34.6%, P < 0.05). The blastocyst formation was also higher in HFLI (76.7%) than LFLI (44.0%, P < 0.05) with respective to the number of cells in a blastocyst of 71.6 8.0 (n = 20) and 62.5 +/- 4.7 (n = 20) (P < 0.05). The relative amount of H2O2 in an embryo that was assessed by a fluorescence intensity of 2',7'-dichlorofluorecein (DCF) showed a difference between the procedures with 16.6 +/- 1.6 (n = 21) and 23.4 +/- 1.8 (n = 24) for HFLI and LFLI, respectively (P < 0.05). Mitochondrial staining by Rhodamine 123 showed that the number and distribution of viable mitochondria were similar in both procedures, but fewer mitochondria were observed with a marked aggregation in the arrested embryos, indicating a mitochondrial disintegration. The mitochondrial membrane potential was visualized by a membrane potential-sensitive fluorescent probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). There was a decrease in the number of mitochondria that had a high membrane potential, and they showed a peripheral redistribution along the cell membrane in LFLI. A fluorescent staining of the actin filaments revealed a discontinuity that was noticeably at the peripheral "actin band" in LFLI. The DNA fragmentation was assessed by the dUTP nick end-labeling (TUNEL). The results showed a higher DNA fragmentation of blastocyst nuclei in LFLI compared to HFLI (65.6 vs. 36.0%, P < 0.05). Based on these observations, it was concluded that HFLI was better than LFLI in the case of freezing the mouse 2-cell embryos for preserving cytoskeletons and mitochondrial integrities. This could subsequently lead to a higher survival and developmental rate of the cryopreserved mouse embryos.
authors.affiliation: Department of Biology, Kyonggi University, Suwon, Korea.
authors.forename: In Pyo
authors.initials: IP
authors.lastname: Sohn
authors.forename: Hak Joon
authors.initials: HJ
authors.lastname: Ahn
authors.forename: Dong Wook
authors.initials: DW
authors.lastname: Park
authors.forename: Myung Chan
authors.initials: MC
authors.lastname: Gye
authors.forename: Do Hyun
authors.initials: DH
authors.lastname: Jo
authors.forename: Suk Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Churl Ki
authors.initials: CK
authors.lastname: Min
authors.forename: Hyuck Chan
authors.initials: HC
authors.lastname: Kwon
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003599:Cytoskeleton / Q000378:metabolism; D053938:DNA Fragmentation; D004622:Embryo, Mammalian / Q000033:anatomy & histology / Q000502:physiology*; D005260:Female; D006861:Hydrogen Peroxide / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000502:physiology*; D009584:Nitrogen / Q000737:chemistry*; D016877:Oxidants / Q000378:metabolism
chemical_list: D000199:Actins; D016877:Oxidants; D006861:Hydrogen Peroxide; D009584:Nitrogen