Comparative Phosphoproteomics Reveals a Role for AMPK in Hypoxia Signaling in Testes of Oriental River Prawn (Macrobrachium nipponense)

Hypoxia is one of the major stresses in aquaculture animals. Recently, we reported that hypoxia disrupts the endocrine system and inhibits testicular function of oriental river prawns (Macrobrachium nipponense), but the molecular mechanism of testes responded to hypoxia remains largely unknown. In the present study, we aimed to integrate whole phosphoproteomic profiles of hypoxia-treated testes of the oriental river prawn (Macrobrachium nipponense). We successfully isolated sperm cells and evaluated the mitochondrial morphology and function using laser confocal microscopy, flow cytometry, and biochemical analyses. Quantitative proteomics identified 117 differentially abundant phosphorylated proteins, and these proteins are mainly involved in the pathways related to cellular processes, including autophagy, apoptosis, and the FoxO signaling pathway. Protein-protein interaction analysis clustered these phosphoproteins into three groups, many of which have been suggested to impact carbohydrate metabolism, autophagy, and signal regulation in testes. Western blotting confirmed that phosphorylated proteins including AMPK, ULK1, and TP53 (of the AMPK pathway) may contribute to testicular dysfunction caused by hypoxia. Further, we investigated the potential roles of AMP-activated protein kinase (AMPK)'s in testes mitochondrial autophagy and apoptosis in M. nipponense as induced by hypoxia. Simultaneous knockdown of AMPK alpha in sperm cells led to a decrease in FOXO3a phosphorylation at Ser413, upregulation of caspase-3 and caspase-9 activities, and an increased apoptosis rate. These results improve our understanding of hypoxia-induced energy metabolism disorders in the testes of M. nipponense.