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 AMPKα 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.