Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Oxid Med Cell Longev. 2020 Dec 31;2020:5954635. doi: 10.1155/2020/5954635. eCollection 2020.
Spermatogonial stem cells (SSCs) are the only adult stem cells that pass genes to the next generation and can be used in assisted reproductive technology and stem cell therapy. SSC cryopreservation is an important method for the preservation of immature male fertility. However, freezing increases the production of intracellular reactive oxygen species (ROS) and causes oxidative damage to SSCs. The aim of this study was to investigate the effect of melatonin on goat SSCs during cryopreservation and to explore its protective mechanism. We obtained SSCs from dairy goat testes by two-step enzymatic digestion and differential plating. The SSCs were cryopreserved with freezing media containing different melatonin concentrations. The results showed that 10 M of melatonin increased significantly the viability, total antioxidant capacity (T-AOC), and mitochondrial membrane potential of frozen-thawed SSCs, while it reduced significantly the ROS level and malondialdehyde (MDA) content ( < 0.05). Further analysis was performed by western blotting, flow cytometry, and transmission electron microscopy (TEM). Melatonin improved significantly the enzyme activity and protein expression of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) ( < 0.05), thereby activating the antioxidant defense system of SSCs. Furthermore, melatonin inhibited significantly the expression of proapoptotic protein (Bax) and increased the expression of antiapoptotic proteins (Bcl-2 and Bcl-XL) ( < 0.05). The mitochondrial apoptosis pathway analysis showed that the addition of melatonin reduced significantly the mitochondrial swelling and vacuolation, and inhibited the release of cytochrome C from mitochondria into the cytoplasm, thereby preventing the activation of caspase-3 ( < 0.05) and inhibiting SSC apoptosis. In addition, melatonin reduced significantly the autophagosome formation and regulated the expression of autophagy-related proteins (LC3-I, LC3-II, P62, Beclin1, and ATG7) ( < 0.05), thereby reversing the freeze-induced excessive autophagy. In summary, melatonin protected goat SSCs during cryopreservation via antioxidant, antiapoptotic, and autophagic regulation.
精原干细胞（SSC）是唯一能将基因传递给下一代的成体干细胞，可用于辅助生殖技术和干细胞治疗。SSC 冷冻保存是保存未成熟雄性生育力的重要方法。然而，冷冻会增加细胞内活性氧（ROS）的产生，并导致 SSC 的氧化损伤。本研究旨在探讨褪黑素在山羊 SSC 冷冻保存过程中的作用及其保护机制。我们通过两步酶消化和差速贴壁法从奶山羊睾丸中获得 SSC。用含有不同褪黑素浓度的冷冻培养基对 SSC 进行冷冻保存。结果表明，10μM 褪黑素显著提高了冻融 SSC 的活力、总抗氧化能力（T-AOC）和线粒体膜电位，而显著降低了 ROS 水平和丙二醛（MDA）含量（<0.05）。进一步通过 Western blot、流式细胞术和透射电镜（TEM）分析。褪黑素显著提高了超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的酶活性和蛋白表达（<0.05），从而激活了 SSC 的抗氧化防御系统。此外，褪黑素显著抑制了促凋亡蛋白（Bax）的表达，增加了抗凋亡蛋白（Bcl-2 和 Bcl-XL）的表达（<0.05）。线粒体凋亡途径分析表明，添加褪黑素可显著减少线粒体肿胀和空泡化，并抑制细胞色素 C 从线粒体释放到细胞质，从而阻止 caspase-3 的激活（<0.05），抑制 SSC 凋亡。此外，褪黑素可显著减少自噬体的形成，并调节自噬相关蛋白（LC3-I、LC3-II、P62、Beclin1 和 ATG7）的表达（<0.05），从而逆转冻融引起的过度自噬。综上所述，褪黑素通过抗氧化、抗凋亡和自噬调节来保护山羊 SSC 冷冻保存过程中的活力。
