Methods
GSNOR dsRNA was employed to knock down the expression of GSNOR, and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), a pan-NOS inhibitor, was used to prevent protein S-nitrosylation. Mitochondrial amount and function in embryo development were assessed by performing immunofluorescence staining, Western blot, fluorescent probe and real-time reverse transcription PCR.
Results
GSNOR knock-down significantly impaired blastocyst formation and quality and markedly induced the increase in protein S-nitrosylation. Notably, GSNOR knock-down-induced overproduction of S-nitrosylation caused mitochondrial dysfunction, including mitochondrial membrane potential depolarization, mitochondria-derived reactive oxygen species (ROS) increase and ATP deficiency. Interestingly, GSNOR knock-down-induced total mitochondrial amount increase, but the ratio of active mitochondria reduction, suggesting that the damaged mitochondria were accumulated and mitochondrial clearance was inhibited. In addition, damaged mitochondria produced more ROS, and caused DNA damage and apoptosis. Importantly, supplementation with L-NAME reverses the increase in S-nitrosylation, accumulation of damaged mitochondria, and oxidative stress-induced cell death. Interestingly, autophagy was downregulated after GSNOR knock-down, but reversed by L-NAME treatment. Thus, GSNOR maintains mitochondrial homeostasis by promoting autophagy and the clearing of damaged mitochondria in porcine preimplantation embryos.