SUMOylation of PGAM2 regulates mitochondrial metabolism and cuproptosis-related damage during boar sperm cryoinjury.

Boar spermatozoa are highly susceptible to oxidative stress during cryopreservation, resulting in impaired protein homeostasis, energy metabolism, and mitochondrial function. This study investigated the role of SUMO1 modification of phosphoglycerate mutase 2 (PGAM2) in regulating mitochondrial metabolism and cuproptosis-related damage in boar spermatozoa. Comparative proteomic analysis of fresh and frozen-thawed spermatozoa identified 875 proteins, of which 761 were differentially expressed and 26 SUMOylation-related proteins were downregulated after freezing and thawing. Structural prediction and co-immunoprecipitation supported PGAM2 as a SUMO1-modified protein. Freezing-thawing and H2O2 treatment enhanced the association between SUMO1 and PGAM2 while reducing PGAM2 abundance (P < 0.01). Inhibition of SUMOylation with ML-792 further decreased PGAM2 and pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) levels (P < 0.05) and aggravated the loss of sperm motility, DNA fragmentation, mitochondrial reactive oxygen species accumulation, mitochondrial membrane depolarization, and ATP depletion (P < 0.05). ML-792 also promoted intracellular Cu+ accumulation and lipid peroxidation and reduced Oligo-DLAT levels (P < 0.05). SENP2 knockdown enhanced the SUMO1-PGAM2 association and exacerbated H2O2-induced metabolic and mitochondrial dysfunction, as indicated by increased lactate dehydrogenase activity and malondialdehyde levels, a reduced NAD+/NADH ratio, ATP depletion, and mitochondrial membrane depolarization (P < 0.05). These changes were accompanied by increased PDHA1 and FDX1 expression and decreased Oligo-DLAT levels (P < 0.05). Collectively, the dynamic balance between SUMOylation and deSUMOylation is essential for maintaining PGAM2-associated metabolic homeostasis. Disruption of this balance aggravates oxidative stress-induced mitochondrial dysfunction and cuproptosis-related metabolic injury, providing a potential molecular target for improving boar semen cryopreservation.