Abstract

Hypoxia is an environmental stressor in aquaculture that adversely affects fish reproduction. However, the underlying mechanisms remain poorly understood. This study investigated the role of autophagy in hypoxia-induced testicular dysfunction in male yellow catfish. Chronic hypoxia significantly reduced gonadosomatic index (GSI) and decreased spermatozoa abundance. These changes were accompanied by reduced levels of 11-ketotestosterone (11-KT) and testosterone (T), as well as downregulation of key genes involved in steroidogenesis, including star, cyp11a1 , and hsd17b1 . Notably, hypoxia inhibited testicular autophagy, as evidenced by decreased LC3II/LC3I ratio and Beclin 1 expression, along with increased P62 accumulation. To further elucidate the role of autophagy, pharmacological interventions were performed using rapamycin and chloroquine. Activation of autophagy by rapamycin partially restored testicular structure, androgen levels, and steroidogenic gene expression under hypoxic conditions. In contrast, inhibition of autophagic flux by chloroquine under normoxia mimicked the effects of hypoxia, leading to impaired spermatogenesis and reduced androgen production. Additionally, hypoxia and chloroquine both decreased testicular cholesterol levels, whereas rapamycin reversed these effects. Autophagy may regulate steroidogenesis through lipophagy-mediated cholesterol mobilization, thereby linking metabolic homeostasis to reproductive function. Collectively, these findings demonstrate that hypoxia impairs spermatogenesis by inhibiting autophagy and disrupting cholesterol-dependent steroidogenesis. This study provides new insights into the regulatory role of autophagy in fish reproduction under environmental stress.