Vitamin C modulates function and gene expression of mouse testis TM4 Sertoli cells. Vitamin C modulates function and gene expression of mouse testis TM4 Sertoli cells

Vitamin C (ascorbic acid, AA), as essential micro-nutrient and antioxidant, can affect multiple cellular processes. Previously, we showed that Vitamin C supplement could promote the proliferation and suppress apoptosis of porcine Sertoli cells, via modulating the global nucleic acid methylation levels to alter transcriptome and proteomics. Due to the differences between pig and rodent species, here we investigated the effects of Vitamin C on mouse TM4 Sertoli cells. Vitamin C (250µM) treatment of mouse TM4 Sertoli cells for 36h could significantly increase cell viability, promote cell proliferation and decrease the cell percentage with early apoptosis. Vitamin C (250µM, 36h) also significantly decreased ROS level, elevated mitochondrial function, altered the levels of nucleic acid N6-methylation (m6A) and modified the levels of histone H3 trimethylation (H3K4me3, H3K9Me3 and H3K36me3) of mouse TM4 Sertoli cells. Vitamin C (250µM, 36h) promoted the secretion of anti-müllerian hormone and Estrodiol, but decreased lactate metabolite in mouse TM4 Sertoli cells. RNA-seq identified 112 differentially expressed genes (DEGs) (74 up- and 38 down-regulated) (P≤0.05, |log2Fold Change|≥1) as induced by Vitamin C in mouse TM4 Sertoli cells. Gene enrichment analysis found multiple significantly enriched biological terms or pathways, including GO terms of regulation of response to biotic stimulus, homeostasis of number of cells, positive regulation of tumor necrosis factor production, GTPase activity, calmodulin binding etc., and KEGG signal pathways of calcium signal pathway, cGMP-PKG signaling pathway, steroid biosynthesis, arginine biosynthesis etc. 9 DEGs were selected for RT-qPCR validation and 8 of them showed the change trend consistent to RNA-seq. Taken together, Vitamin C (250µM, 36h) could modulate the functions of mouse TM4 Sertoli cells, through modifying the transcriptome to affect multiple signal pathways. Overall design: To explore the effect of valine on testis of male mouse

维生素C（抗坏血酸，AA）作为必需微量营养素与抗氧化剂，可调控多种细胞进程。本团队此前的研究显示，维生素C补充剂可通过调控全局核酸甲基化水平，改变转录组与蛋白质组，进而促进猪睾丸支持细胞（porcine Sertoli cells）增殖并抑制其凋亡。鉴于猪与啮齿类动物存在物种差异，本研究针对小鼠TM4睾丸支持细胞（mouse TM4 Sertoli cells）展开了维生素C作用效果的探究。以250μM浓度的维生素C处理小鼠TM4睾丸支持细胞36小时，可显著提升细胞活力、促进细胞增殖，并降低早期凋亡细胞比例。该处理还可显著降低细胞内活性氧（Reactive Oxygen Species, ROS）水平、提升线粒体功能，改变核酸N6-甲基化（m6A）水平，并调控小鼠TM4睾丸支持细胞的组蛋白H3三甲基化修饰水平（包括H3K4me3、H3K9Me3与H3K36me3）。250μM、36小时的维生素C处理可促进小鼠TM4睾丸支持细胞分泌抗缪勒管激素（Anti-Müllerian Hormone, AMH）与雌二醇（Estradiol, E2），同时降低细胞内乳酸代谢产物水平。RNA测序（RNA Sequencing, RNA-seq）鉴定出112个由维生素C诱导的差异表达基因（Differentially Expressed Genes, DEGs），其中74个基因上调、38个基因下调（P≤0.05，|log₂折叠变化|≥1）。基因富集分析显示，存在多个显著富集的生物学条目与通路，涵盖基因本体（Gene Ontology, GO）中的调控生物刺激应答、细胞数量稳态、肿瘤坏死因子产生正调控、GTP酶活性、钙调蛋白结合等术语，以及京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）中的钙信号通路、cGMP-PKG信号通路、类固醇生物合成、精氨酸生物合成等通路。选取9个差异表达基因进行实时定量聚合酶链反应（Real-time Quantitative Polymerase Chain Reaction, RT-qPCR）验证，其中8个基因的表达变化趋势与RNA测序结果一致。综上，250μM、36小时的维生素C处理可通过调控转录组、影响多条信号通路，进而修饰小鼠TM4睾丸支持细胞的功能。实验整体设计：本研究旨在探究缬氨酸对雄性小鼠睾丸的影响。