Effects of Osmotic Stress and Cryoprotectant Toxicity on Mouse Oocyte Fertilization and Subsequent Embryonic Development In Vitro

Abstract

Supplementation of cryoprotectant agent (CPA) into freezing solution causes osmotic change and this change together with toxicity of CPA may damage the oocytes before freezing. The objectives of the present study were to evaluate the tolerance of mouse oocytes to osmotic changes and to compare the toxicity of the permeable CPAs. Denuded mouse oocytes were exposed to (1) 0.5 M, 1.0 M, or 2.0 M sucrose in HEPES-buffered modified human tubal fluid (mHTF), respectively, for 3 min at the room temperature; exposed to (2) either 1.0 M or 2.0 M ethylene glycol (EG), 1,2-propanediol (PROH), dimethyl sulfoxide (Me2SO) and glycerol in HEPES-buffered mHTF for 3 min at room temperature, respectively. Following exposure, the oocytes were inseminated by in vitro fertilization (IVF), and the zygotes were further cultured to assess embryonic development in vitro. Additional analyses included morphologic evaluation of meiotic spindle organization and chromosome alignment as well as aneuploidy. Exposure to 2.0 M sucrose significantly impaired fertilization and blastocyst formation rates (p < 0.01). Although the fertilization and blastocyst formation rates were not affected by exposure to 1.0 M CPAs, the percentages of fertilization and blastocyst formation were reduced significantly (p < 0.05) when the oocytes were exposed to 2.0 M EG, PROH, Me2SO and glycerol. Analysis of meiotic spindle and chromosome alignment showed higher (p < 0.05) percentages of abnormalities in the oocytes that were exposed to 2.0 M PROH, glycerol or sucrose compared to Me2SO- or EG-treated oocytes. Ploidy analysis also showed a lower incidence of aneuploidy in EG group (p < 0.05) compared to other CPAs, indicating that EG as CPA has less cytotoxicity.