Cold Stress Induces Apoptosis in Silver Pomfret via DUSP-JNK Pathway

We cultured silver pomfret for 20 days, decreasing water temperature from 18 to 8 degrees C, and sampled muscle every 5 days. Muscle fiber degeneration and apoptosis began to increase at 13 degrees C detected by HE and TUNEL staining. Further analysis of transcriptome revealed that several apoptosis-related pathways were highly enriched by differentially expressed genes (DEGs). We analyzed 10 DEGs from these pathways by RT-qPCR during the temperature-decreasing process. JNK1, PIDD, CytC, Casp 3, and GADD45 were up-regulated after 15 and 20 days, while DUSP3, JNK2, and PARP genes were down-regulated after 15 and 20 days. DUSP5 was up-regulated from 10 to 20 days, and C-JUN was up-regulated after 20 days. We analyzed apoptosis in PaM cells under different temperatures (26 degrees C, 23 degrees C, 20 degrees C, 17 degrees C, and 14 degrees C). The cell viability significantly declined from 14 to 20 degrees C; the TUNEL and IHC results showed that the apoptosis signal increased with the temperature dropping, especially in 17 degrees C and 14 degrees C; DUSP5, JNK1, CytC, C-JUN, Casp 3, and GADD45 were up-regulated at 17 degrees C and 14 degrees C, and PIDD was up-regulated at 20 degrees C, 17 degrees C, and 14 degrees C. DUSP3 was up-regulated at 20 degrees C but down-regulated at 17 degrees C and 14 degrees C, and PARP was down-regulated at 17 degrees C and 14 degrees C. JNK2 was up-regulated at 20 degrees C but down-regulated at 17 degrees C and 14 degrees C. Our results suggest that DUSP could help inhibit apoptosis in the initial stage of cold stress, but low temperature could down-regulate it and up-regulate JNK-C-JUN, inducing apoptosis in a later stage. These data provide a basis for the study of the response mechanism of fish to cold.

Automated summary

This study found that muscle fiber degeneration and apoptosis in silver pomfret increased when the water temperature dropped to 13 degrees C. Transcriptome analysis revealed the enrichment of several apoptosis-related pathways. RT-qPCR analysis of differentially expressed genes showed that some genes were up-regulated or down-regulated during the temperature-decreasing process. Cell experiments at different temperatures also showed that cell viability declined and apoptosis signal increased with lower temperatures, especially at 17 degrees C and 14 degrees C. The results suggest that DUSP may inhibit apoptosis in the initial stage of cold stress, but low temperature down-regulates DUSP and up-regulates JNK-C-JUN, leading to apoptosis.