Chilling injury mechanism in the immature oocytes of zebrafish (Danio rerio)

Abstract

Immature zebrafish oocytes are sensitive to chilling, and their survival is markedly reduced by exposure to 0°C. In the present study, we investigated the involvement of cold-sensitive channels and lipid mediators in chilling injury in immature zebrafish oocytes. The oocytes were injected with inhibitors of a cold-sensitive channel (TRPA1), cytosolic phospholipase A₂α (cPLA₂α), cyclooxygenases (COXs), arachidonate 5-lipoxygenase (ALOX5), and lysophosphatidylcholine acyltransferase 2 (LPCAT2). The cells were then chilled at 0–12°C for 5–30 min, incubated at 25°C for 2 h, and stained with propidium iodide. Oocytes were damaged when exposed to temperatures below 12°C. When oocytes were chilled at 0°C for 15 min, the survival rate was very low (9%). However, when the oocytes were injected with a TRPA1-specific inhibitor, their survival markedly improved (70%). This strongly suggests that activation of the cold-sensitive TRPA1 channel triggers chilling injury in oocytes. When a cPLA₂α-specific inhibitor was injected, the survival of chilled oocytes markedly improved (60%). This strongly suggests that lipid mediators are involved in chilling injury in oocytes. When oocytes were injected with specific inhibitors of COXs, ALOX5, and LPCAT2, the survival of chilled oocytes significantly improved by 47%, 28%, and 43%, respectively. These results strongly suggest that eicosanoids and platelet-activating factor are involved in the chilling injury in oocytes. The results of this study may facilitate advancements in successful cryopreservation of fish oocytes.