Vitamin C Modulates Function and Gene Expression of Mouse TM4 Sertoli Cells

doi:10.13304/j.nykjdb.2024.0583

Abstract

Abstract:

To investigate the effects of vitamin C on the function and gene expression of mouse TM4 Sertoli cells， the cells were treated with 0， 100， 250 and 500 μmol·L^-1 of L-ascorbic acid 2-phosphate magnesium hydrate （AA2P） for different time length. Cell viability under different treatments was used to select the optimal conditions for AA2P treatment of TM4 cells. Further， different methods were used to detect cell proliferation， apoptosis， reactive oxygen species （ROS） level， mitochondrial function， RNA methylation （m⁶A）， histone H3 trimethylation levels （H3K4me3， H3K9me3 and H3K36me3）， anti-Müllerian hormone （AMH）， estradiol， lactate， as well as gene expression alteration. The results showed that treatment of TM4 cells with 250 μmol·L^-1 AA2P for 36 h significantly increased cell viability， promoted cell proliferation， reduced early apoptosis， decreased ROS level， enhanced mitochondrial function， altered m⁶A level and histone H3 trimethylation （H3K4me3， H3K9me3 and H3K36me3） levels， promoted the secretion of AMH and estradiol， and reduced lactate level. RNA-seq results revealed that AA2P treatment （250 μmol·L^-1， 36 h） induced significant differential expression of 112 genes， which were involving in multiple GO （gene ontology） items and KEGG （kyoto encyclopedia of genes and genomes） pathways. To sum up， vitamin C could regulate the function and gene expression of mouse TM4 Sertoli cells.

Key words: mouse, TM4 cells, vitamin C, transcriptome, signal pathways

摘要：

为探究维生素C对小鼠TM4支持细胞的功能和基因表达影响，采用0、100、250和500 μmol·L^-1的L-抗坏血酸2-磷酸倍半镁水合盐（L-ascorbic acid 2-phosphate magnesium，AA2P）处理小鼠TM4支持细胞不同时间，分析不同AA2P处理水平和处理时间下的细胞活力；进一步采用多种研究方法检测细胞的增殖、凋亡、活性氧（reactive oxygen species，ROS）水平、线粒体功能、RNA甲基化（m⁶A）和组蛋白H3三甲基化（H3K4me3、H3K9me3和H3K36me3）水平、抗苗勒氏管激素（anti-Müllerian hormone，AMH）、雌二醇、乳酸以及基因表达的变化。结果表明，采用250 μmol·L^-1 AA2P处理TM4细胞36 h能显著提高细胞活力，促进细胞增殖，降低细胞早期凋亡和活性氧水平，增强线粒体功能，改变m⁶A水平和组蛋白H3三甲基化（H3K4me3、H3K9me3和H3K36me3）水平，促进AMH和雌二醇的分泌，降低乳酸水平。RNA-seq检测结果显示，250 μmol·L^-1 AA2P处理TM4细胞36 h诱导112个基因表达出现显著差异。GO（gene ontology）和KEGG（Kyoto encyclopedia of genes and genomes）富集到多条通路。综上表明，维生素C可调节小鼠TM4支持细胞的功能和基因表达。

关键词: 小鼠, TM4细胞, 维生素C, 转录组学, 信号通路

CLC Number:

S865.1+3

Xinxin WANG, Ziwei WU, Ting FANG, Qiao MOU, Fang WANG, Han ZHAO, Zhiqiang DU, Caixia YANG. Vitamin C Modulates Function and Gene Expression of Mouse TM4 Sertoli Cells[J]. Journal of Agricultural Science and Technology, 2025, 27(5): 81-89.

王昕昕, 吴子薇, 方婷, 牟巧, 王芳, 赵晗, 杜志强, 杨彩侠. 维生素C调节小鼠TM4支持细胞的功能和基因表达[J]. 中国农业科技导报, 2025, 27(5): 81-89.

Figures/Tables 7

Table 1 Primer sequences for RT-qPCR

基因名称

Gene name

引物序列

Prime sequence （5’-3’）

Gapdh

F： AGGTCGGTGTGAACGGATTTG

R： TGTAGACCATGTAGTTGAGGTCA

Adra1d

F： AGGTCGGTGTGAACGGATTTG

R： TGTAGACCATGTAGTTGAGGTCA

Gbp2

F： CTGCTAAACTTCGGGAACAGGA

R： AGAGAGCTTGGCAGAGAGGT

Inca1

F： CCAATTCTGAGGGCCACTGAT

R： TAACTCGGGCTGGAATGCTT

Mapkapk5

F： GCTGATGATGGATAAGGCGGT

R： TACCGTCCTTTGGCTTGGTG

Nos3

F： CCACTGAGCAGCTATTGGGT

R： TGCAGTCCCGAGCATCAAAT

Npas4

F： ACCAGATCAACGCCGAGATT

R： CACCATAGAGTGGCCCAGAT

Scand1

F： CGGGTCGAGACGCAGG

R： GTAGGGCGCCGTGTTCAAA

Tmem30c

F： AACTGTACCGCCGACTGAAG

R： CCCCCGTCACTGTGTAAGTC

Trim34a

F： GGAGGTGAGGGTCCAAAGATTC

R： GAGGCCATTATGTTGCCCCT

Fig. 1 Effects of AA2P on the viability， proliferation and apoptosis of mouse TM4 cellsA： Representative plots of TM4 cells treated with different AA2P treatments for different time with scale bar 100 μm； B： Cell viability； C： Representative images of EdU assay with scale bar 20 and 50 μm； D： Representative images of Annexin V assay with scale bar 50 μm； E： Proliferation rate of cell； F： Apoptosis rate of cell. *， ** and *** indicate significant differences at P<0.05， P<0.01 and P<0.001 levels， respectively

Fig. 2 ROS level and mitochondrial features of mouse TM4 cells treated by AA2PA~C： Representative images of ROS， MitoTracker and RH123 staining with scale bar 50 μm； D~F： Relative fluorescence intensities of ROS， MitoTracker and RH123 staining. * and ** indicate significant differences at P<0.05 and P<0.01 levels， respectively

Fig. 3 Global levels of nucleic acid m6A methylation and histone H3 trimethylation at sites of lysine 4， 9 and 36 in mouse TM4 cells treated by AA2PA， C， E， G： Immunofluorescence staining represents images with scale bar 20 μm； B， D， F， H： Histogram of relative fluorescence intensity. * and ** indicate significant differences at P<0.05 and P<0.001 levels， respectively

Fig. 4 Levels of hormones and lactate in mouse TM4 cells treated by AA2PNote：* and ** indicate significant differences at P<0.05 and P<0.01 levels， respectively.

Fig. 5 RNA-seq identified genes and molecular pathways altered by AA2P treatment of mouse TM4 cellsA： Number of DEGs； B： Heatmap of DEGs； C： Significantly enriched GO term； D： Partial DEGs involved in some key GO terms； E： Significantly enriched KEGG signaling pathways

Fig. 6 Validation of selected DEGsA： Expression and difference of DEGs as detected by RNA-seq； B： Validation of expression and difference of DEGs by RT-qPCR. *， ** and *** indicate significant differences at P<0.05， P<0.01 and P<0.001 levels， respectively

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