Development of Effective Cryopreservation Method for Mouse Oocytes

Choi, Su Jin; Kim, Soo Kyung; Kim, Ji Sun; Cho, Jae Won; Jun, Jin Hyun; Byun, Hye Kyung

Abstract

OBJECTIVE
The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte.
METHODS
Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1,2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-beta-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent).
RESULTS
Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different.
CONCLUSION
For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.