A novel undifferentiated spermatogonia-speci?c surface protein 1(USSP1) in neonatal mice
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摘要
Mammalian spermatogenesis is maintained by a rare population of spermatogonial stem cells(SSCs),which are important for male fertility. SSCs remain a subset of undifferentiated spermatogonia, which can be isolated by a combination of surface markers. Specific markers to identify and isolate undifferentiated spermatogonia are lacking. Ussp1, a transcript previously annotated as long noncoding RNA(RIKEN cDNA 4933427D06, Gene ID: 232217), virtually encodes a membrane protein, USSP1, in a highly testisspecific manner in mouse. We demonstrate its expression on the membrane of undifferentiated spermatogonia by a homemade polyclonal rabbit antibody against the protein. In vivo, USSP1~+ clusters consist mainly of As, Apr(GFRa1~+) and Aal(PLZF~+) cells. USSP1~+ cells exhibit enrichment of undifferentiated spermatogonia, as shown by increased expression of SSC self-renewal molecular markers and the potential to form SSC clones in vitro and in vivo. However, Ussp1 knockout did not affect the number of SSCs or spermatogenesis in mice. Thy1~+ cells from Ussp1 null mice did not show any defect in the SSC colony formation capacity, indicating that USSP1 is not essential for SSC self-renewal. Our data demonstrate that Ussp1 is specifically expressed in undifferentiated murine spermatogonia, indicating the potential to sort undifferentiated spermatogonia with USSP1 antibodies. Ussp1 might be a good maker for SSC enrichment in neonatal mice.
Mammalian spermatogenesis is maintained by a rare population of spermatogonial stem cells(SSCs),which are important for male fertility. SSCs remain a subset of undifferentiated spermatogonia, which can be isolated by a combination of surface markers. Specific markers to identify and isolate undifferentiated spermatogonia are lacking. Ussp1, a transcript previously annotated as long noncoding RNA(RIKEN cDNA 4933427D06, Gene ID: 232217), virtually encodes a membrane protein, USSP1, in a highly testisspecific manner in mouse. We demonstrate its expression on the membrane of undifferentiated spermatogonia by a homemade polyclonal rabbit antibody against the protein. In vivo, USSP1~+ clusters consist mainly of As, Apr(GFRa1~+) and Aal(PLZF~+) cells. USSP1~+ cells exhibit enrichment of undifferentiated spermatogonia, as shown by increased expression of SSC self-renewal molecular markers and the potential to form SSC clones in vitro and in vivo. However, Ussp1 knockout did not affect the number of SSCs or spermatogenesis in mice. Thy1~+ cells from Ussp1 null mice did not show any defect in the SSC colony formation capacity, indicating that USSP1 is not essential for SSC self-renewal. Our data demonstrate that Ussp1 is specifically expressed in undifferentiated murine spermatogonia, indicating the potential to sort undifferentiated spermatogonia with USSP1 antibodies. Ussp1 might be a good maker for SSC enrichment in neonatal mice.
Mammalian spermatogenesis is maintained by a rare population of spermatogonial stem cells(SSCs),which are important for male fertility. SSCs remain a subset of undifferentiated spermatogonia, which can be isolated by a combination of surface markers. Specific markers to identify and isolate undifferentiated spermatogonia are lacking. Ussp1, a transcript previously annotated as long noncoding RNA(RIKEN cDNA 4933427D06, Gene ID: 232217), virtually encodes a membrane protein, USSP1, in a highly testisspecific manner in mouse. We demonstrate its expression on the membrane of undifferentiated spermatogonia by a homemade polyclonal rabbit antibody against the protein. In vivo, USSP1~+ clusters consist mainly of As, Apr(GFRa1~+) and Aal(PLZF~+) cells. USSP1~+ cells exhibit enrichment of undifferentiated spermatogonia, as shown by increased expression of SSC self-renewal molecular markers and the potential to form SSC clones in vitro and in vivo. However, Ussp1 knockout did not affect the number of SSCs or spermatogenesis in mice. Thy1~+ cells from Ussp1 null mice did not show any defect in the SSC colony formation capacity, indicating that USSP1 is not essential for SSC self-renewal. Our data demonstrate that Ussp1 is specifically expressed in undifferentiated murine spermatogonia, indicating the potential to sort undifferentiated spermatogonia with USSP1 antibodies. Ussp1 might be a good maker for SSC enrichment in neonatal mice.
引文
[1]Brinster RL,Zimmermann JW.Spermatogenesis following male germ-cell transplantation.Proc Natl Acad Sci USA 1994;91:11298-302.
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[3]Wu X,Goodyear SM,Abramowitz LK,et al.Fertile offspring derived from mouse spermatogonial stem cells cryopreserved for more than 14 years.Hum Reprod 2012;27:1249-59.
[4]Olive V,Cuzin F.The spermatogonial stem cell:from basic knowledge to transgenic technology.Int J Biochem Cell Biol 2005;37:246-50.
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[6]de Rooij DG.The nature and dynamics of spermatogonial stem cells.Development 2017;144:3022-30.
[7]Green CD,Ma Q,Manske GL,et al.A Comprehensive Roadmap of Murine Spermatogenesis Defined by Single-Cell RNA-Seq.Dev Cell 2018;46(651-667):e10.
[8]de Rooij DG,Griswold MD.Questions about spermatogonia posed and answered since 2000.J Androl 2012;33:1085-95.
[9]Naughton CK,Jain S,Strickland AM,et al.Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate.Biol Reprod 2006;74:314-21.
[10]He Z,Jiang J,Hofmann MC,et al.GFRa1 silencing in mouse spermatogonial stem cells results in their differentiation via the inactivation of RET tyrosine kinase.Biol Reprod 2007;77:723-33.
[11]Nakagawa T,Sharma M,Nabeshima Y,et al.Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment.Science 2010;328:62-7.
[12]Sada A,Suzuki A,Suzuki H,et al.The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells.Science 2009;325:1394-8.
[13]Oatley JM,Avarbock MR,Telaranta AI,et al.Identifying genes important for spermatogonial stem cell self-renewal and survival.Proc Natl Acad Sci USA2006;103:9524-9.
[14]Wu X,Goodyear SM,Tobias JW,et al.Spermatogonial stem cell self-renewal requires ETV5-mediated downstream activation of Brachyury in mice.Biol Reprod 2011;85:1114-23.
[15]Oatley MJ,Kaucher AV,Racicot KE,et al.Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells in mice.Biol Reprod2011;85:347-56.
[16]Sun F,Xu Q,Zhao D,et al.Id4 marks spermatogonial stem cells in the mouse testis.Sci Rep 2015;5:17594.
[17]Helsel AR,Yang QE,Oatley MJ,et al.ID4 levels dictate the stem cell state in mouse spermatogonia.Development 2017;144:624-34.
[18]Aloisio GM,Nakada Y,Saatcioglu HD,et al.PAX7 expression defines germline stem cells in the adult testis.J Clin Invest 2014;124:3929-44.
[19]Costoya JA,Hobbs RM,Barna M,et al.Essential role of Plzf in maintenance of spermatogonial stem cells.Nat Genet 2004;36:653-9.
[20]Buaas FW,Kirsh AL,Sharma M,et al.Plzf is required in adult male germ cells for stem cell self-renewal.Nat Genet 2004;36:647-52.
[21]Filipponi D,Hobbs RM,Ottolenghi S,et al.Repression of kit expression by Plzf in germ cells.Mol Cell Biol 2007;27:6770-81.
[22]Shinohara T,Avarbock MR,Brinster RL.Beta1-and alpha6-integrin are surface markers on mouse spermatogonial stem cells.Proc Natl Acad Sci USA1999;96:5504-9.
[23]Kubota H,Avarbock MR,Brinster RL.Spermatogonial stem cells share some,but not all,phenotypic and functional characteristics with other stem cells.Proc Natl Acad Sci USA 2003;100:6487-92.
[24]Kubota H,Avarbock MR,Brinster RL.Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells.Biol Reprod 2004;71:722-31.
[25]Huang Y,Xiong Y,Lin Z,et al.Specific Tandem 3’UTR Patterns and Gene Expression Profiles in Mouse Thy1±Germline Stem Cells.PLoS ONE 2015;10:e0145417.
[26]Sander JD,Joung JK.CRISPR-Cas systems for editing,regulating and targeting genomes.Nat Biotechnol 2014;32:347-55.
[27]Kubota H,Brinster RL.Culture of rodent spermatogonial stem cells,male germline stem cells of the postnatal animal.Methods Cell Biol 2008;86:59-84.
[28]Hwang WY,Fu Y,Reyon D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system.Nat Biotechnol 2013;31:227-9.
[29]Kyr?nlahti A,Euler R,Bielinska M,et al.GATA4 regulates Sertoli cell function and fertility in adult male mice.Mol Cell Endocrinol 2011;333:85-95.
[30]Hobbs RM,Seandel M,Falciatori I,et al.Plzf regulates germline progenitor selfrenewal by opposing m TORC1.Cell 2010;142:468-79.
[31]Wang H,Yang H,Shivalila CS,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell 2013;153:910-8.
[32]Liang M,Li W,Tian H,et al.Sequential expression of long noncoding RNA as mRNA gene expression in specific stages of mouse spermatogenesis.Sci Rep2014;4:5966.
[33]Anderson DM,Anderson KM,Chang CL,et al.A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.Cell2015;160:595-606.
[34]Nelson BR,Makarewich CA,Anderson DM,et al.A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle.Science 2016;351:271-5.
[35]Ji Z,Song R,Regev A,et al.Many lncRNAs,5’UTRs,and pseudogenes are translated and some are likely to express functional proteins.Elife 2015;4:e08890.
[36]Hofmann MC,Braydich-Stolle L,Dym M.Isolation of male germ-line stem cells;influence of GDNF.Dev Biol 2005;279:114-24.
[37]Wang S,Wang X,Wu Y,et al.IGF-1R signaling is essential for the proliferation of cultured mouse spermatogonial stem cells by promoting the G2/Mprogression of the cell cycle.Stem Cells Dev 2015;24:471-83.
[38]Peterziel H,Paech T,Strelau J,et al.Specificity in the crosstalk of TGFbeta/GDNF family members is determined by distinct GFR alpha receptors.JNeurochem 2007;103:2491-504.
[2]Kanatsu-Shinohara M,Miki H,Inoue K,et al.Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions.Biol Reprod2005;72:985-91.
[3]Wu X,Goodyear SM,Abramowitz LK,et al.Fertile offspring derived from mouse spermatogonial stem cells cryopreserved for more than 14 years.Hum Reprod 2012;27:1249-59.
[4]Olive V,Cuzin F.The spermatogonial stem cell:from basic knowledge to transgenic technology.Int J Biochem Cell Biol 2005;37:246-50.
[5]de Rooij DG,Russell LD.All you wanted to know about spermatogonia but were afraid to ask.J Androl 2000;21:776-98.
[6]de Rooij DG.The nature and dynamics of spermatogonial stem cells.Development 2017;144:3022-30.
[7]Green CD,Ma Q,Manske GL,et al.A Comprehensive Roadmap of Murine Spermatogenesis Defined by Single-Cell RNA-Seq.Dev Cell 2018;46(651-667):e10.
[8]de Rooij DG,Griswold MD.Questions about spermatogonia posed and answered since 2000.J Androl 2012;33:1085-95.
[9]Naughton CK,Jain S,Strickland AM,et al.Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate.Biol Reprod 2006;74:314-21.
[10]He Z,Jiang J,Hofmann MC,et al.GFRa1 silencing in mouse spermatogonial stem cells results in their differentiation via the inactivation of RET tyrosine kinase.Biol Reprod 2007;77:723-33.
[11]Nakagawa T,Sharma M,Nabeshima Y,et al.Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment.Science 2010;328:62-7.
[12]Sada A,Suzuki A,Suzuki H,et al.The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells.Science 2009;325:1394-8.
[13]Oatley JM,Avarbock MR,Telaranta AI,et al.Identifying genes important for spermatogonial stem cell self-renewal and survival.Proc Natl Acad Sci USA2006;103:9524-9.
[14]Wu X,Goodyear SM,Tobias JW,et al.Spermatogonial stem cell self-renewal requires ETV5-mediated downstream activation of Brachyury in mice.Biol Reprod 2011;85:1114-23.
[15]Oatley MJ,Kaucher AV,Racicot KE,et al.Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells in mice.Biol Reprod2011;85:347-56.
[16]Sun F,Xu Q,Zhao D,et al.Id4 marks spermatogonial stem cells in the mouse testis.Sci Rep 2015;5:17594.
[17]Helsel AR,Yang QE,Oatley MJ,et al.ID4 levels dictate the stem cell state in mouse spermatogonia.Development 2017;144:624-34.
[18]Aloisio GM,Nakada Y,Saatcioglu HD,et al.PAX7 expression defines germline stem cells in the adult testis.J Clin Invest 2014;124:3929-44.
[19]Costoya JA,Hobbs RM,Barna M,et al.Essential role of Plzf in maintenance of spermatogonial stem cells.Nat Genet 2004;36:653-9.
[20]Buaas FW,Kirsh AL,Sharma M,et al.Plzf is required in adult male germ cells for stem cell self-renewal.Nat Genet 2004;36:647-52.
[21]Filipponi D,Hobbs RM,Ottolenghi S,et al.Repression of kit expression by Plzf in germ cells.Mol Cell Biol 2007;27:6770-81.
[22]Shinohara T,Avarbock MR,Brinster RL.Beta1-and alpha6-integrin are surface markers on mouse spermatogonial stem cells.Proc Natl Acad Sci USA1999;96:5504-9.
[23]Kubota H,Avarbock MR,Brinster RL.Spermatogonial stem cells share some,but not all,phenotypic and functional characteristics with other stem cells.Proc Natl Acad Sci USA 2003;100:6487-92.
[24]Kubota H,Avarbock MR,Brinster RL.Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells.Biol Reprod 2004;71:722-31.
[25]Huang Y,Xiong Y,Lin Z,et al.Specific Tandem 3’UTR Patterns and Gene Expression Profiles in Mouse Thy1±Germline Stem Cells.PLoS ONE 2015;10:e0145417.
[26]Sander JD,Joung JK.CRISPR-Cas systems for editing,regulating and targeting genomes.Nat Biotechnol 2014;32:347-55.
[27]Kubota H,Brinster RL.Culture of rodent spermatogonial stem cells,male germline stem cells of the postnatal animal.Methods Cell Biol 2008;86:59-84.
[28]Hwang WY,Fu Y,Reyon D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system.Nat Biotechnol 2013;31:227-9.
[29]Kyr?nlahti A,Euler R,Bielinska M,et al.GATA4 regulates Sertoli cell function and fertility in adult male mice.Mol Cell Endocrinol 2011;333:85-95.
[30]Hobbs RM,Seandel M,Falciatori I,et al.Plzf regulates germline progenitor selfrenewal by opposing m TORC1.Cell 2010;142:468-79.
[31]Wang H,Yang H,Shivalila CS,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell 2013;153:910-8.
[32]Liang M,Li W,Tian H,et al.Sequential expression of long noncoding RNA as mRNA gene expression in specific stages of mouse spermatogenesis.Sci Rep2014;4:5966.
[33]Anderson DM,Anderson KM,Chang CL,et al.A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.Cell2015;160:595-606.
[34]Nelson BR,Makarewich CA,Anderson DM,et al.A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle.Science 2016;351:271-5.
[35]Ji Z,Song R,Regev A,et al.Many lncRNAs,5’UTRs,and pseudogenes are translated and some are likely to express functional proteins.Elife 2015;4:e08890.
[36]Hofmann MC,Braydich-Stolle L,Dym M.Isolation of male germ-line stem cells;influence of GDNF.Dev Biol 2005;279:114-24.
[37]Wang S,Wang X,Wu Y,et al.IGF-1R signaling is essential for the proliferation of cultured mouse spermatogonial stem cells by promoting the G2/Mprogression of the cell cycle.Stem Cells Dev 2015;24:471-83.
[38]Peterziel H,Paech T,Strelau J,et al.Specificity in the crosstalk of TGFbeta/GDNF family members is determined by distinct GFR alpha receptors.JNeurochem 2007;103:2491-504.