생쥐모델을 이용한 동결보존제의 독성조사 = Toxic Effect of Cryoprotectants on Embryo Development in a Murine Model

다국어 초록 (Multilingual Abstract)

Objectives: The aim of this study was to assess toxicities of cryoprotectants. Methods: Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and 1, 2-propanediol (PROH), were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or PROH. Embryo development was evaluated up to the blastocyst stage. Blastocysts were stained with bis-benzimide to evaluate the cell count and with terminal deoxynucleotidyl transferase mediated dUTP nick labeling (TUNEL) to assess apoptosis. Results: The total cell count of blastocysts that were treated with DMSO at the 2-cell stage was significantly lower than that were treated with PROH ($75.9{\pm}27.0$) or the control ($99.0{\pm}18.3$) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the PROH treated group (p<0.05). Both DMSO ($14.2{\pm}1.5$) and PROH ($11.2{\pm}1.4$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.2{\pm}0.9$, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the PROH treated group (p<0.001). Conclusions: The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or PROH at room temperature. When comparing two cryoprotective agents, PROH appeared to be less toxic than DMSO at least in a murine embryo model.

참고문헌 (Reference)

1 "gical characterization of blastocyst hatching mechanisms by use of a mouse antihatching model" 288-294, 1995

2 "Sutton RL" 87 : 101-05, 1991

3 "Survival rate of frozen-thawed bovine IVF embryos in relation to exposure time using various cryoprotectants" 306-12, cryobiology1993

4 "Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation." 43 : 21-31, 2001

5 "Prevention of freezing damage to living cells by dimethyl sulphoxide" 1394-5, nature1959

6 "Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation" 13 : 376-380, 1998

7 "Osmotically inactive volume, hydraulic conductivity, and permeability to dimethyl sulphoxide of human mature oocyte" 117 : 27-33, 1999

8 "Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols" 75 : 754-762, 2001

9 "Embryo culture systems. In "Handbook of in vitro Fertilization"." 2nd ed, CRC Press 205-264, 2000

10 "Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries" 110 : 11-19, 1997

11 "Deleterious effect of equilibration temperature on the toxicity of propanediol during cryopreservation of mouse zygotes" 14 : 51-54, 1997

12 "Crystallization of ice in aqueous solutions of glycerol and dimethly sulphoxide Physiological characterization of blastocyst hatching mechanisms by use of a mouse antihatching model" 205 288-16 94, cryobiology1996fertilsteril1995

13 "Crystallization of ice in aqueous solutions of glycerol and dimethly sulphoxide" 33 : 205 -216, 1996

14 "A more accurate determination of the quantity of ice crystallized at low cooling rates in the glycerol and 1 Comparison with equilibrium" 2-, cryobiology1984183-91