Proteomics Study on Spermatogonia Differentiation in Mice

doi:10.13560/j.cnki.biotech.bull.1985.2019-0640

Abstract

Abstract: This work aims to study the quantitative proteomics of undifferentiated spermatogonia and differentiating spermatogonia in mice testis,to explore the difference of proteome expression between the two types of spermatogonia,and to explore the proteins related to spermatogonial differentiation. Thy1+ cells and c-Kit⁺ cells in testis of 7-days male mice were separated as undifferentiated and differentiating spermatogonia by magnetic bead sorting technique combined with Thy1 and c-Kit specific antibodies. Three groups of Thy1 positive cells and four groups of c-Kit positive cells represented undifferentiated spermatogonia and differentiating spermatogonia respectively. High performance liquid chromatography tandem mass spectrometry（LC-MS/MS）was used to analyze the difference of protein expression between the two types of cells. GO function annotation,KEGG metabolic pathway and cluster analysis were performed on the differentially expressed proteins of the two types of cells. A total of 3 228 proteins were identified by mass spectrometry,of which 256 were differentially expressed in two types of cells. Among them,enrichment analysis of differential proteins discovered that in biological processes,annotations showed that differential proteins were mainly enriched in primary metabolic process,cellular metabolic process,macromolecule metabolic process and nitrogen compound metabolic process. In terms of cell components,proteins were mainly concentrated in cell parts,intracellular parts and organelles. In terms of molecular functions,the identified proteins were mainly involved in protein binding,nucleotide binding and nucleic acid binding. KEGG annotation results showed that 88 proteins were functionally annotated in KEGG database and involved in 13 metabolic pathways. The main metabolic pathways were spliceosome and ubiquitin mediated proteolysis. The proteome expression profiles of undifferentiated and differentiating spermatogonia in mouse testis are obtained. The proteome composition of undifferentiated and differentiating spermatogonia is revealed and differential proteins are screened out.

Key words: spermatogonia, differentiation, proteomics

LI Kun, LIU Yue, HUANG Peng, YANG Zhi-fang, HU Qian, ZHANG Ying, LI Zhi-hong, LÜ Ye-hui, LIANG Le. Proteomics Study on Spermatogonia Differentiation in Mice[J]. Biotechnology Bulletin, 2020, 36(3): 168-176.

References

[1] Manku G, Culty M.Mammalian gonocyte and spermatogonia differentiation:recent advances and remaining challenges[J]. Reproduction. 2015, 149(3):139-157.
[2] 冯雅子瑄. 浅谈蛋白质组学的研究进展[J]. 中国科技纵横, 2019(6):194-195.
[3] 朱沙, 张春辉, 李鲜菊. 从基因组学到功能蛋白质组学的研究[J]. 生物学杂志, 2007(1):13-15.
[4] 孙武, 罗静, 王丽, 等. 哺乳动物睾丸蛋白质组学研究进展[J]. 农业生物技术学报, 2018(10):1797-1804.
[5] 刘银凤, 张雷. 生物信息学数据库在医学研究中的应用[J]. 中国病原生物学杂志, 2014, 9(10):1-2, 封3.
[6] Huo R, He Y, Zhao C, et al.Identification of human spermatogenesis-related proteins by comparative proteomic analysis:a preliminary study[J]. Fertil Steril, 2008, 90(4):1109-1118.
[7] Guo X, et al.Proteomic analysis of proteins involved in spermiogene-sis in mouse[J]. J Proteome Res, 2010, 9(3):1246-1256.
[8] Paz M, Morín M, Del Mazo J.Proteome profile changes during mouse testis development[J]. Comp Biochem Physiol Part D Genomics Proteomics, 2006, 1(4):404-415.
[9] Huang XY, Guo XJ, et al.Construction of a proteome profile and functional analysis of the proteins involved in the initiation of mouse spermatogenesis[J]. J Proteome Res, 2008, 7(8):3435-3446.
[10] 王黎熔, 王静, 王富强, 等. iTRAQ技术鉴定小鼠睾丸发育差异表达蛋白的研究[J]. 临床检验杂志, 2010, 28(6):441-443.
[11] Rolland AD, Evrard B, et al.Two-dimensional fluorescence difference gel electrophoresis analysis of spermatogenesis in the rat[J]. J Proteome Res, 2007, 6(2):683-697.
[12] Chen X, Luan X, Zheng Q, et al.Precursor RNA processing 3 is required for male fertility, and germline stem cell self-renewal and differentiation via regulating spliceosome function in Drosophila testes[J]. Sci Rep, 2019, 9(1):9988.
[13] Jin YX, Zheng Z, Yu XF, et al.Autophagy and ubiquitin-mediated proteolysis may not be involved in the degradation of spermatozoon mitochondria in mouse and porcine early embryos[J]. Zygote, 2016, 24(1):31-41.
[14] Liu M, Zhu C, Bai S, et al.An in vivo method to study mouse blood-testis barrier integrity[J]. J Vis Exp, 2018(142), e58512.
[15] Matschurat C, Rode K, Hollenbach J, et al.Impaired spermatoge-nesis, tubular wall disruption, altered blood-testis barrier composition and intratubular lymphocytes in an infertile Beagle dog - a putative case of autoimmune orchitis[J]. Histol Histopathol, 2019, 34(5):525-535.
[16] Gurel C, Kuscu GC, Buhur A, et al.Fluvastatin attenuates doxorubicin-induced testicular toxicity in rats by reducing oxidative stress and regulating the blood-testis barrier via mTOR signaling pathway[J]. Hum Exp Toxicol, 2019, 38(12):1329-1343.
[17] Zhu R, Wang J, Feng T, et al.The alteration of RhoA geranylgera-nylation and Ras farnesylation breaks the integrity of the blood-testis barrier and results in hypospermatogenesis[J]. Cell Death Dis, 2019, 10(6):450.
[18] Wang XX, Zhang Y, Li XY, et al.Kruppel-like factor 6 regulates Sertoli cell blood-testis barrier[J]. Front Biosci(Landmark Ed), 2019, 24:1316-1329.
[19] Li LX, Wu SW, Yan M, et al.Regulation of blood-testis barrier dynamics by the mTORC1/rpS6 signaling complex:An in vitro study[J]. Asian J Androl, 2019, 21(4):365-375.