Oxidative stress disrupts the intracellular redox balance that modulate many signaling pathways, including nuclear factor erythroid 2-related factor 2 (Nrf2)/Keap1 signaling. However, the antioxidant roles of Nrf2 in the testis before adulthood have not been reported. Accordingly, in this study, we aimed to investigate the effects of the Nrf2 antioxidant system on protection of testicular cells against oxidative stress at different stages of development in the testis of mice before adulthood. Male mice (1, 2, 4, and 8 weeks old) were used, and their relative testes weights were calculated. Malondialdehyde (MDA) contents and superoxide dismutase (SOD) activity were detected to evaluate the antioxidant capacity in the testes. Additionally, Nrf2 signaling pathway and mitochondrial apoptotic pathway proteins were evaluated by western blotting, and the localizations of Nrf2, protein gene product (PGP) 9.5, and activated-caspase 3 in testicular cells were examined using immunohistochemistry. The results showed that the activities of caspase-8 and caspase-3 and the number of activated-caspase 3-positive testicular cells per tubule were increased after 1 week of age. Moreover, MDA contents were increased and SOD activity was decreased with age in mouse testes before adulthood. The expression of PGP9.5 was increased, as well as the number of positive testicular cells per tubule. In addition, Nrf2 translocation to the nuclei of testicular cells also increased, accompanied by activation of the Nrf2/Keap1 signaling pathway. Moreover, nuclear factor-κB was inhibited, and the mitochondrial apoptotic pathway was activated in mouse testes before adulthood. Overall, our findings demonstrated that oxidative stress increased with age in mouse testes before adulthood and that oxidative stress could induce apoptosis in testicular cells. However, testicular cells are still in a rapid proliferative state owing to the antioxidant protection of Nrf2. Thus, our study provided new insights into oxidative stress-mediated impairment of spermatogenesis with age in mouse testes before adulthood and evidence for the protective role of Nrf2 in male fertility.

中文翻译：

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核因子红细胞2相关因子对成年前小鼠睾丸氧化应激和细胞凋亡的保护作用

氧化应激会破坏调节许多信号通路的细胞内氧化还原平衡，包括核因子红细胞 2 相关因子 2 (Nrf2)/Keap1 信号通路。然而，尚未报道 Nrf2 在成年前睾丸中的抗氧化作用。因此，在本研究中，我们旨在研究 Nrf2 抗氧化系统对成年前小鼠睾丸发育不同阶段的睾丸细胞免受氧化应激的影响。使用雄性小鼠（1、2、4 和 8 周龄），并计算它们的相对睾丸重量。检测丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性以评估睾丸的抗氧化能力。此外，通过蛋白质印迹评估 Nrf2 信号通路和线粒体凋亡通路蛋白，使用免疫组织化学检查睾丸细胞中 Nrf2、蛋白质基因产物 (PGP) 9.5 和活化半胱天冬酶 3 的定位。结果表明，caspase-8 和 caspase-3 的活性以及每个小管中激活的 caspase 3 阳性睾丸细胞数在 1 周龄后增加。此外，成年前小鼠睾丸的 MDA 含量增加，SOD 活性随着年龄的增长而降低。PGP9.5 的表达增加，每个小管的阳性睾丸细胞数量也增加。此外，Nrf2 向睾丸细胞核的易位也增加，伴随着 Nrf2/Keap1 信号通路的激活。此外，在成年前的小鼠睾丸中，核因子-κB 被抑制，线粒体凋亡途径被激活。全面的，我们的研究结果表明，成年前小鼠睾丸的氧化应激随着年龄的增长而增加，并且氧化应激可诱导睾丸细胞凋亡。然而，由于 Nrf2 的抗氧化保护，睾丸细胞仍处于快速增殖状态。因此，我们的研究为成年前小鼠睾丸中氧化应激介导的精子发生损伤提供了新的见解，并提供了 Nrf2 在男性生育能力中的保护作用的证据。