Parental copper stress induces offspring developmental defects by altering DNA methylation in zebrafish [WGBS]

Here, we describe environmental Cu2+ in promoting zebrafish (Danio rerio) developmental abnormalities in subsequent generation. This study for the first time demonstrated that: 1. Cu2+ induced alterations in sperm methylome could be passed down to offspring and cause developmental defects in the nervous and digestive system, even the Cu2+ concentration is at 10 μg/L, the National Quality Standards of fishery water (GB11607-89); 2. DNA methylomes of F0 sperms and F1 offspring were altered in a loci-specific manner and were correlated with transcriptional expression of genes which function importantly in the development of CNS, retina, and digestive system development during F1 embryogenesis; 3. Loci-specific hypoDMRs in the pmpcb promoter and loci-specific hyperDMRs in the crebl2 and tab2 promoters might show adaptive and harmful responses to Cu2+ stresses in next generational inheritance, respectively. Those data revealed that Cu2+ induced alterations in sperm methylome may be passed down to offspring and cause the embryonic developmental defects and the resulted inferior seeding, suggesting that environmental Cu2+ might pose a dramatic and long-lasting threat to the sustainability of fish population and even to humans with imbalanced copper homeostasis. Adult zebrafish (F0 fish) at 3 months of age were divided into three groups: exposure separately to normal water environment (control), 0.078 μM (5 μg/L), and 0.156 μM (10 μg/L) Cu for over 21 days, with three biological repetitions for each group. Their spawning eggs were collected separately and cultured in normal water environment (no Cu added).The sperms of 10-12 adult zebrafish (F0) for each of the 3 groups (control males; 0.078 μM Cu stressed males; 0.156 μM Cu stressed males) and 50-60 larvae chosen biasedly based on their phenotypes (F1 offspring) at 96 hpf in each of 3 groups (control; 0.078 μM group; 0.156 μM group) were collected for genomic DNA extraction using the ammonium acetate method.