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 (Ca2+) concentration ([Ca2+](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 [Ca2+](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 Ca2+ was removed, increase in [Ca2+](i) at 10 and 20% EG was less than that at the same concentrations of EG with extracellular Ca2+. 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 microM 1,2-bis(o-aminopheoxy)ethane-N,N,N',N'-tetraacetic acid tetra acetoxymethyl ester (BAPTA-AM), a Ca2+ chelator, a small increase in [Ca2+](i) exposed to 10, 20, and 40% EG was observed until 4 min. Subsequently prolonged elevation of the [Ca2+](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 microM 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 [Ca2+](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 [Ca2+](i) rises by EG in unfertilized mouse oocytes resulted in cell death. Therefore, the lowering of [Ca2+](i) in the oocytes exposed to the cryoprotectant may improve the viability of cryopreserved unfertilized oocytes.
Takahashi
Toshifumi
T
Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata 990-9585, Japan. totakaha@med.id.yamagata-u.ac.jp
Igarashi
Hideki
H
Doshida
Masakazu
M
Takahashi
Kazuhiro
K
Nakahara
Kenji
K
Tezuka
Naohiro
N
Kurachi
Hirohisa
H
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D015925:Cryopreservation; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D051379:Mice; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*
D002614:Chelating Agents; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019855:Ethylene Glycol; D002118:Calcium