Stress-induced apoptosis in larval embryos of Japanese flounder

Abstract

Stress-induced apoptosis was characterized in larval embryos of Japanese flounder using a caspase-3-like activity assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining of whole mounts. Severe stress conditions, such as heat shock by temperature shift from 16°C to 28°C or 29°C for 1 h, and ultraviolet (UV) irradiation at 20-100 mJ/cm², induced extensive apoptosis in embryos. Following the induction of stress, many TUNEL-positive apoptotic cells were found in the yolk sac, heart, and larval fin. Caspase-3-like activity against a fluorogenic substrate, acetyl-Asp-Glu-Val-Asp-α-4-methylcoumaryl-7-amide, increased in a dose-dependent manner during stress-induced apoptosis. In addition, C2-ceramide treatment (5-10 μM) induced a similar apoptosis pattern to stress treatment. These findings indicate that a pro-apoptotic pathway, which is caspase-3 mediated and acts through ceramide signaling, plays a major role in induction of apoptosis under stress conditions.