Amelioration of mitochondrial dysfunction and apoptosis of two-cell mouse embryos after freezing and thawing by the high frequency liquid nitrogen infusion.
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.
Sohn
In Pyo
IP
Department of Biology, Kyonggi University, Suwon, Korea.
Ahn
Hak Joon
HJ
Park
Dong Wook
DW
Gye
Myung Chan
MC
Jo
Do Hyun
DH
Kim
Suk Young
SY
Min
Churl Ki
CK
Kwon
Hyuck Chan
HC
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
D000199:Actins; D016877:Oxidants; D006861:Hydrogen Peroxide; D009584:Nitrogen