Copper inhibits hatching of fish embryos via inducing reactive oxygen species and down-regulating Wnt signaling

The copper ion (Cu2+) has been reported to suppress the hatching of fish. However, little is known about the underlying mechanism. In this study, copper nanoparticles (CuNPs) and Cu2+ were shown to significantly suppress hatching of zebrafish in a dosage-dependent manner, and reactive oxygen species (ROS) scavengers NAC (Nacetylcysteine) & GSH (reduced glutathione)) and signaling agonist Bio (6bromoindirubin3'oxine) significantly alleviated the suppressing effects of Cu2+ and CuNPs on egg hatching. Mechanistically, NAC, GSH, and BIO recovered the egg hatching in copper-treated group via increasing the embryonic motility rather than stimulating the expression and secretion of hatching enzymes before hatching. Additionally, no significant difference in egg hatching was observed between the control and Cu2+-treated group at 72 hpf (hours post fertilization) in cox17 mutant embryos, in which little ROS was producd after copper stimulation. This may be the first report that Cu2+ and CuNPs suppress embryonic motility and the subsequent hatching via inducing ROS and at the same time down-regulating Wnt signaling in fish embryos.