S-nitrosoglutathione reductase maintains mitochondrial homeostasis by promoting clearance of damaged mitochondria in porcine preimplantation embryos

Objectives S-nitrosoglutathione reductase (GSNOR), a protein denitrosylase, protects the mitochondria from mitochondrial nitrosative stress. Mammalian preimplantation embryos are mitochondria-rich, but the effects of GSNOR on mitochondrial function in preimplantation embryos are not well-studied. In the present study, we investigate whether GSNOR plays a role in mitochondrial regulation during porcine preimplantation embryo development. Materials and Methods GSNOR dsRNA was employed to knock down the expression of GSNOR, and N omega-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.

Automated summary

The study found that knocking down GSNOR significantly impaired blastocyst formation and quality, while markedly increasing protein S-nitrosylation. The overproduction of S-nitrosylation caused by GSNOR knock-down led to mitochondrial dysfunction, including mitochondrial membrane potential depolarization, increased production of mitochondria-derived reactive oxygen species (ROS), and ATP deficiency. Additionally, damaged mitochondria accumulated and mitochondrial clearance was inhibited, leading to more ROS production, DNA damage, and apoptosis. Supplementing with L-NAME reversed the effects of increased S-nitrosylation, damaged mitochondria accumulation, and oxidative stress-induced cell death. Autophagy was downregulated after GSNOR knock-down, but was reversed by L-NAME treatment, suggesting that GSNOR maintains mitochondrial homeostasis by promoting autophagy and clearance of damaged mitochondria in porcine preimplantation embryos.