Lowering intracellular and extracellular calcium contents prevents cytotoxic effects of ethylene glycol‐based vitrification solution in unfertilized mouse oocytes

We investigated the characteristics of the changes in intracellular calcium (Ca 2+ ) concentration ([Ca 2+ ] i ) and the viability of the unfertilized mouse oocytes exposed to various concentrations of ethylene glycol (EG)‐containing solutions or vitrification solutions. Oocytes exposed to EG (1, 5, 10, 20, and 40% (v/v)) exhibited a rapid and dose‐dependent increase in [Ca 2+ ] i . The survival rate was 100% when oocytes were exposed to the EG concentration up to 5% through 5 min, while all oocytes were dead within 3 min when exposed to 10, 20, or 40% EG. When extracellular Ca 2+ was removed, increase in [Ca 2+ ] i at 10 and 20% EG was less than that at the same concentrations of EG with extracellular Ca 2+ . The survival rates of the oocytes exposed to 10, 20, and 40% EG at 3 min were 100, 97, and 0%, respectively. In the presence of 20 μM 1,2‐bis( o ‐aminopheoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid tetra acetoxymethyl ester (BAPTA‐AM), a Ca 2+ chelator, a small increase in [Ca 2+ ] i exposed to 10, 20, and 40% EG was observed until 4 min. Subsequently prolonged elevation of the [Ca 2+ ] i was observed in the oocytes exposed to 40% EG but not with 10 and 20% EG. The survival rate of the oocytes, in the presence of 20 μM BAPTA‐AM, exposed to 10 and 20% EG was 100% throughout 5 min, while the oocytes exposed to 40% EG were alive only for 3 min. Treatment by the vitrification solution with various concentrations of EG (10, 20, and 40%) caused a smaller increase in [Ca 2+ ] i , while the survival rates were higher compared to those without vitrification solution at the same concentrations of EG. These data suggested that the sustained [Ca 2+ ] i rises by EG in unfertilized mouse oocytes resulted in cell death. Therefore, the lowering of [Ca 2+ ] i in the oocytes exposed to the cryoprotectant may improve the viability of cryopreserved unfertilized oocytes. Mol. Reprod. Dev. 68: 250–258, 2004. © 2004 Wiley‐Liss, Inc.