High temperature induces reproductive dysfunction in male zebrafish via DNA methylation-mediated tssk6 downregulation

High temperature (HT) poses a significant threat to aquatic organisms, yet the molecular mechanisms underlying HT-induced reproductive impairment in fish remain unclear. In this study, we demonstrated that both short-term (< 21 days) and long-term (> 120 days) exposure to HT (33℃) induced reproductive dysfunction in male zebrafish, including reduced fertilization rates, impaired sperm quality, inhibited testicular development, and increased offspring mortality. Transcriptome analyses showed that short-term HT exposure primarily disrupted testicular energy metabolism, whereas long-term exposure impaired sperm-egg recognition and binding. By analyzing differentially expressed genes involve in reproductive processes, we detected a significant downregulation of testis-specific serine kinase 6 (tssk6) in zebrafish testis under HT conditions. Notably, injection of tssk6-mRNA partially restored the HT-induced reduction in sperm quality. Further investigation revealed that long-term HT increased DNA methylation in the tssk6 promoter region, thereby inhibiting its expression, suggesting tssk6 as a potential biomarker for reproductive health. These findings establish an epigenetic mechanistic link between environmental HT and reproductive function in aquatic organisms.