PARENT SESSION
Cryopreservation
528
THE IMPACT OF CRYOPRESERVATION ON BIOPHYSICAL AND BIOCHEMICAL PROPERTIES AND DEVELOPMENTAL RATE OF MOUSE EMBRYO.
Ahn, Hak Jun1,4, Son, In Pyo2,4, Kwon, Hyuck Chan3, Gye, Myung Chan2, Cho, Do Hyun1, Min, Chul Ki1, 1 4 2 3
ABSTRACT- The aim of this study were to assess biophysical and biochemical changes of the ultrastructure including apoptosis, and to evaluate functional changes such as production of ROS and embryo development after freezing and thawing. To assess alteration of the plasma membrane fluidity, fluorescent recovery velocity was measured by LASER after staining with fluorecein labeled WGA. Intracellular location and membrane potential of the mitochondria were evaluated by confocal microscope after staining with JC-1. The free calcium was tagged with Fluo-3. Relative amount of H2O2 production was measured by H2DCFDA. Cell apoptosis was assessed by TUNEL method. Total cell number in the blastocyst was counted by fluorescent microscope after Acrydine Orange staining. Fluorescent recovery velocity was 1.46±0.13 sec and 0.28±0.04 sec for study and control group respectively (p<0.05), suggesting alteration of the plasma membrane. The distribution pattern of the mitochondria was similar in both groups. The membrane potentials (Ym) were represented as color intensity after JC-1 staining. Relative intensity of red color measured at 590 nm (high membrane potential) versus relative intensity of green color measured at 510 nm (low membrane potential) were 17.2±3.8 vs. 14.4±0.9 and 13.2±2.0 vs. 10.8±1.2 for control and study group respectively (p<0.05).The study group showed free calcium release into cytoplasm. The relative amount of produced H2O2 was higher for study group (62.8±23.5 vs. 43.2±14.5; p<0.05). The expression pattern of Annexin V staining was not significantly different between two groups. However, DNA fragmentation rate was higher for study group (74.7 % vs. 32.3%; p<0.05). 74.7 % and 33.7 % of embryos developed into blastocyst for control and study group (p<0.05). Total cell number in the blastocyst was 95.87±19.14 and 42.00±11.34 for control and study groups respectively (p<0.05). The process of freezing and/or thawing negatively affected the integrity of cell membrane, function of mitochondria and rate of ROS production. Hampered development of embryo after cryopreservation may be related with these ultrastructural and functional alterations. These results may rationalize the use of antioxidants and anti-apoptotic substances in process of cryopreservation.
KEY WORDS: Cryopreservation, Mouse embryo, Mitochondria, Apoptosis