Guo Yi-Xuan, Zhang Guo-Min, Yao Xiao-Lei, Tong Ran, Cheng Chun-Yu, Zhang Ting-Ting, Wang Shu-Ting, Yang Hua, Wang Feng
Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, 210095, China.
Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, 210095, China.
Theriogenology. 2019 Mar 1;126:55-62. doi: 10.1016/j.theriogenology.2018.12.007. Epub 2018 Dec 3.
The aim of this study was to investigate effects of nitric oxide (NO) on steroidogenesis and apoptosis in goat luteinized granulosa cells (LGCs). We cultured goat LGCs from healthy follicles in culture medium supplemented with the NO donor sodium nitroprusside (SNP) or the NO synthase inhibitor N-Nitro-l-arginine methyl ester hydrochloride (l-NAME), then examined steroid synthesis, oxidative stress and apoptosis in vitro. The results showed that SNP treatment significantly increased the cGMP concentration in the LGCs (P < 0.05), whereas the l-NAME treatment significantly decreased cGMP concentration (P < 0.05). Then Inhibition of NO production significantly inhibited the expression of CYP19A1, a key gene that is involved in sex steroid hormones synthesis and is responsible for the decrease of E. Inhibition of NO production resulted in an increased percentage of apoptosis, which was accompanied by upregulating expression levels of apoptosis-related markers BAX, CASP3 and CASP9. These data indicate that NO is required for goat LGCs steroidogenesis and cell survival. Furthermore, Inhibition of NO production decreased the expression of mitochondrial biogenesis related genes and proteins (PPARGC1A, NRF-1 and TFAM) and the mtDNA copy number. Simultaneously, inhibition of NO production suppressed the transcription and translation of SOD, GPX1, and CAT, and decreased the glutathione level and increased the 8-OHdG level. However, SNP treatment increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the anti-oxidant stress system and steroid synthesis. Together, our results indicate that NO may up-regulate the expression of PPARGC1A and its downstream factors through the cGMP pathway, thereby decreasing granulosa cell apoptosis, and may participate in the regulation of granulocyte steroid production through the mitochondrial-dependent pathway.
本研究旨在探讨一氧化氮（NO）对山羊黄体化颗粒细胞（LGCs）类固醇生成和细胞凋亡的影响。我们在添加了NO供体硝普钠（SNP）或NO合酶抑制剂盐酸N-硝基-L-精氨酸甲酯（L-NAME）的培养基中培养来自健康卵泡的山羊LGCs，然后在体外检测类固醇合成、氧化应激和细胞凋亡。结果表明，SNP处理显著增加了LGCs中的cGMP浓度（P<0.05），而L-NAME处理显著降低了cGMP浓度（P<0.05）。抑制NO生成显著抑制了CYP19A1的表达，CYP19A1是参与性甾体激素合成并导致雌激素减少的关键基因。抑制NO生成导致细胞凋亡百分比增加，同时凋亡相关标志物BAX、CASP3和CASP9的表达水平上调。这些数据表明，NO是山羊LGCs类固醇生成和细胞存活所必需的。此外，抑制NO生成降低了线粒体生物发生相关基因和蛋白质（PPARGC1A、NRF-1和TFAM）的表达以及mtDNA拷贝数。同时，抑制NO生成抑制了SOD、GPX1和CAT的转录和翻译，降低了谷胱甘肽水平并增加了8-OHdG水平。然而，SNP处理增加了参与线粒体功能和生物发生的基因表达，提高了抗氧化应激系统和类固醇合成。总之，我们的结果表明，NO可能通过cGMP途径上调PPARGC1A及其下游因子的表达，从而减少颗粒细胞凋亡，并可能通过线粒体依赖性途径参与粒细胞类固醇生成的调节。
