Cold‐induced calcium elevation triggers DNA fragmentation in immature pig oocytes

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Abstract
Fluo-4 loaded immature oocytes were cooled from 30°C to various lower temperatures between 20 and 10°C and changes in intracellular calcium (Ca2+) levels were measured. Pig oocytes cooled to 14°C exhibited a clear biphasic Ca2+ rise. Lower temperatures produced similar responses, while higher temperatures did not exert any effect. The Ca2+ response appeared to rely on ryanodine dependent stores as removal of extracellular Ca2+ and intracytoplasmic injection of heparin did not modify cold-induced Ca2+ elevation, while procaine or ruthenium red virtually eliminated the response. Confocal analysis of subcellular Ca2+ distribution during cooling revealed that the ion rises sharply within the nucleus. As Ca2+ imbalance may activate nuclear endonucleases, DNA integrity of cooled pig oocytes was evaluated by TUNEL and comet assays. Most cooled oocytes showed clear signs of DNA fragmentation. Oocytes injected with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid tetrapotassium salt (BAPTA), a Ca2+ chelator, maintained their DNA integrity thus confirming that intracellular Ca2+ is involved in triggering DNA fragmentation. The protective effect exerted by ruthenium red and/or procaine further confirmed this hypothesis. These data show that a moderate and transient cooling is sufficient to cause an intracellular Ca2+ rise that leads to DNA damage. The addition of inhibitors of ryanodine dependent Ca2+ stores may represent a valuable protective treatment to reduce chilling injuries. Mol. Reprod. Dev. 65: 289–297, 2003.