Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, China.
Department of Animal Science, Chungbuk National University, Cheongju, South Korea.
Cell Prolif. 2021 Mar;54(3):e12990. doi: 10.1111/cpr.12990. Epub 2021 Jan 17.
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.
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.
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.
S-亚硝基谷胱甘肽还原酶（GSNOR）是一种蛋白质去硝基酶，可保护线粒体免受线粒体硝化应激。哺乳动物的着床前胚胎富含线粒体，但 GSNOR 对着床前胚胎中线粒体功能的影响尚未得到充分研究。本研究旨在探讨 GSNOR 在猪着床前胚胎发育过程中线粒体调控中的作用。
采用 GSNOR dsRNA 敲低 GSNOR 的表达，并用 Nω-硝基-L-精氨酸甲酯盐酸盐（L-NAME）抑制所有 NOS，以防止蛋白质 S-亚硝基化。通过免疫荧光染色、Western blot、荧光探针和实时反转录 PCR 评估胚胎发育过程中线粒体的数量和功能。
GSNOR 敲低显著损害囊胚形成和质量，并显著诱导蛋白质 S-亚硝基化增加。值得注意的是，GSNOR 敲低诱导的 S-亚硝基化过度产生导致线粒体功能障碍，包括线粒体膜电位去极化、线粒体来源的活性氧（ROS）增加和 ATP 缺乏。有趣的是，GSNOR 敲低诱导的总线粒体数量增加，但活性线粒体比例减少，提示受损线粒体被积累且线粒体清除被抑制。此外，受损线粒体产生更多的 ROS，并导致 DNA 损伤和细胞凋亡。重要的是，L-NAME 的补充可逆转 S-亚硝基化、受损线粒体的积累以及氧化应激诱导的细胞死亡增加。有趣的是，GSNOR 敲低后自噬下调，但 L-NAME 处理可逆转。因此，GSNOR 通过促进自噬和清除猪着床前胚胎中的受损线粒体来维持线粒体的动态平衡。
