title: Establishment of a proteome profile and identification of molecular markers for mouse spermatogonial stem cells.
abstract: Spermatogonial stem cells (SSCs) are undifferentiated cells that are required to maintain spermatogenesis throughout the reproductive life of mammals. Although SSC transplantation and culture provide a powerful tool to identify the mechanisms regulating SSC function, the precise signalling mechanisms governing SSC self-renewal and specific surface markers for purifying SSCs remain to be clearly determined. In the present study, we established a steady SSC culture according to the method described by Shinohara's lab. Fertile progeny was produced after transplantation of cultured SSCs into infertile mouse testis, and the red fluorescence exhibited by the culture cell membranes was stably and continuously transmitted to the offspring. Next, via advanced mass spectrometry and an optimized proteomics platform, we constructed the proteome profile, with 682 proteins expressed in SSCs. Furthermore bioinformatics analysis showed that the list contained several known molecules that are regulated in SSCs. Several nucleoproteins and membrane proteins were chosen for further exploration using immunofluorescence and RT-PCR. The results showed that SALL1, EZH2, and RCOR2 are possibly involved in the self-renewal mechanism of SSCs. Furthermore, the results of tissue-specific expression analysis showed that Gpat2 and Pld6 were uniquely and highly expressed in mouse testes and cultured SSCs. The cellular localization of PLD6 was further explored and the results showed it was primarily expressed in the spermatogonial membrane of mouse testes and cultured SSCs. The proteins identified in this study form the basis for further exploring the molecular mechanism of self-renewal in SSCs and for identifying specific surface markers of SSCs.
authors.affiliation: State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Zhou
authors.forename: Yueshuai
authors.initials: Y
authors.lastname: Guo
authors.forename: Bo
authors.initials: B
authors.lastname: Zheng
authors.forename: Binbin
authors.initials: B
authors.lastname: Shao
authors.forename: Min
authors.initials: M
authors.lastname: Jiang
authors.forename: Gaigai
authors.initials: G
authors.lastname: Wang
authors.forename: Tao
authors.initials: T
authors.lastname: Zhou
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Zuomin
authors.initials: Z
authors.lastname: Zhou
authors.forename: Xuejiang
authors.initials: X
authors.lastname: Guo
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Huang
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation*; D000818:Animals; D015415:Biomarkers; D002478:Cells, Cultured; D056970:Co-Repressor Proteins; D019295:Computational Biology; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D020869:Gene Expression Profiling; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000096:biosynthesis; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008822:Mice, Transgenic; D024101:Mitochondrial Proteins / Q000096:biosynthesis; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010739:Phospholipase D / Q000096:biosynthesis; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D013091:Spermatogenesis; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism*; D013737:Testis / Q000166:cytology*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D015415:Biomarkers; D056970:Co-Repressor Proteins; D024101:Mitochondrial Proteins; D009419:Nerve Tissue Proteins; D020543:Proteome; C485650:Rcor2 protein, mouse; D012097:Repressor Proteins; C491254:Sall1 protein, mouse; D014157:Transcription Factors; D000071221:Enhancer of Zeste Homolog 2 Protein; C498410:Ezh2 protein, mouse; D063151:Polycomb Repressive Complex 2; D005992:Glycerol-3-Phosphate O-Acyltransferase; C571223:glycerol-3-phosphate acyltransferase 2, mouse; D010739:Phospholipase D; C558126:mitoPLD protein, mouse
keywords: nucleoproteins; proteome; self-renew; spermatogonial stem cells; surface marker