GDNF Up-Regulates c-Myc Transcription via the PI3K/Akt Pathway to Promote Dairy Goat Male Germline Stem Cells (mGSC) Proliferation

doi:10.1016/S2095-3119(13)60263-4

Journal of Integrative Agriculture ›› 2013, Vol. 12 ›› Issue (6): 1054-1065.DOI: 10.1016/S2095-3119(13)60263-4

GDNF Up-Regulates c-Myc Transcription via the PI3K/Akt Pathway to Promote Dairy Goat Male Germline Stem Cells (mGSC) Proliferation

SUN Jun-wei, ZHU Hai-jing, LIU Chao, LI Ming-zhao , HUA Jin-lian

College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology/Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling 712100, P.R.China

收稿日期:2012-03-08 出版日期:2013-06-01 发布日期:2013-06-09
通讯作者: Correspondence HUA Jin-lian, Tel: +86-29-87080068, Fax: +86-29-87080068, E-mail: jinlianhua@nwsuaf.edu.cn
基金资助:
This work was supported by the National Natural Science Foundation of China (30972097, 31272518), the Program for the New Century Excellent Talents of State Ministry of Education of P.R. China (NCET-09-0654), the Doctoral Fund of Ministry of Education of P.R. China (RFDP, 20120204110030), the Scientific Research Program of Shaanxi Province, China (2011K02-06), and the Fundamental Research Funds for the Central Universities, China (QN2011012).

GDNF Up-Regulates c-Myc Transcription via the PI3K/Akt Pathway to Promote Dairy Goat Male Germline Stem Cells (mGSC) Proliferation

SUN Jun-wei, ZHU Hai-jing, LIU Chao, LI Ming-zhao , HUA Jin-lian

College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology/Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling 712100, P.R.China

Received:2012-03-08 Online:2013-06-01 Published:2013-06-09
Contact: Correspondence HUA Jin-lian, Tel: +86-29-87080068, Fax: +86-29-87080068, E-mail: jinlianhua@nwsuaf.edu.cn
Supported by:
This work was supported by the National Natural Science Foundation of China (30972097, 31272518), the Program for the New Century Excellent Talents of State Ministry of Education of P.R. China (NCET-09-0654), the Doctoral Fund of Ministry of Education of P.R. China (RFDP, 20120204110030), the Scientific Research Program of Shaanxi Province, China (2011K02-06), and the Fundamental Research Funds for the Central Universities, China (QN2011012).

摘要/Abstract

摘要： Studies have demonstrated that regulation of GDNF on male germline stem cells (mGSCs) mainly through Ras/Erk1/2, Src family kinase and PI3K/Akt signaling pathways, but the signaling pathways GDNF-mediated are different when the species and cell lines varied. Whether GDNF regulates self-renewal of mGSCs isolated from livestock has not been reported. Here, we purified mGSCs from dairy goat testis using mixed enzymes and fibronectin. Immunofluoresce staining revealed the cultured dairy mGSCs expressed Vasa, Nanos2, Ngn3, Tert, Dazl, Lin28, Oct4, CD49f, Stra8 and GFRa1, reflecting that these cells were mGSCs phenotype. Then we cultured these dairy goat mGSCs in different concentrations of GDNF (0, 5, 10, or 20 ng mL-1) to optimize the best concentration of GDNF to sustain the dairy goat mGSCs self-renewal, after that the inhibitor of PI3K (LY294002, 10 μmol L-1) was added to the medium which contains the optimal concentration of GDNF we obtained by experiments. The mGSCs cultured in different media were compared through the population doubling time (PDT), capacity of cell proliferation evaluated by PCNA and BrdU immunofluorescence staining, RT-PCR, QRT-PCR, Western blotting and flow cytometry. Results showed that 10 ng mL-1 was the optimal concentration of GDNF to maintain goat mGSCs self-renewal and GDNF up-regulates c-Myc transcription via the PI3K/Akt pathway to promote goat mGSCs proliferation. This study provides us an efficient model to study the mechanism in mGSCs proliferation and differentiation in goat, and has important implications in unveiling signaling pathways in livestock GSCs.

关键词: GDNF, male germline stem cells (mGscs), PI3K/Akt, c_Myc

Abstract: Studies have demonstrated that regulation of GDNF on male germline stem cells (mGSCs) mainly through Ras/Erk1/2, Src family kinase and PI3K/Akt signaling pathways, but the signaling pathways GDNF-mediated are different when the species and cell lines varied. Whether GDNF regulates self-renewal of mGSCs isolated from livestock has not been reported. Here, we purified mGSCs from dairy goat testis using mixed enzymes and fibronectin. Immunofluoresce staining revealed the cultured dairy mGSCs expressed Vasa, Nanos2, Ngn3, Tert, Dazl, Lin28, Oct4, CD49f, Stra8 and GFRa1, reflecting that these cells were mGSCs phenotype. Then we cultured these dairy goat mGSCs in different concentrations of GDNF (0, 5, 10, or 20 ng mL-1) to optimize the best concentration of GDNF to sustain the dairy goat mGSCs self-renewal, after that the inhibitor of PI3K (LY294002, 10 μmol L-1) was added to the medium which contains the optimal concentration of GDNF we obtained by experiments. The mGSCs cultured in different media were compared through the population doubling time (PDT), capacity of cell proliferation evaluated by PCNA and BrdU immunofluorescence staining, RT-PCR, QRT-PCR, Western blotting and flow cytometry. Results showed that 10 ng mL-1 was the optimal concentration of GDNF to maintain goat mGSCs self-renewal and GDNF up-regulates c-Myc transcription via the PI3K/Akt pathway to promote goat mGSCs proliferation. This study provides us an efficient model to study the mechanism in mGSCs proliferation and differentiation in goat, and has important implications in unveiling signaling pathways in livestock GSCs.

Key words: GDNF, male germline stem cells (mGscs), PI3K/Akt, c_Myc

SUN Jun-wei, ZHU Hai-jing, LIU Chao, LI Ming-zhao , HUA Jin-lian. GDNF Up-Regulates c-Myc Transcription via the PI3K/Akt Pathway to Promote Dairy Goat Male Germline Stem Cells (mGSC) Proliferation[J]. Journal of Integrative Agriculture, 2013, 12(6): 1054-1065.

参考文献

[1]Ahmed N N, Grimes H L, Bellacosa A, Chan T O, Tsichlis PN. 1997. Transduction of interleukin-2 antiapoptotic andproliferative signals via Akt protein kinase Proceedingsof the National Academy of Sciences of the UnitedStates of America, 94, 3627-3632

[2]Aponte P M, Soda T, Teerds K J, Mizrak S C, van de KantH J, de Rooij D G. 2008. Propagation of bovinespermatogonial stem cells in vitro. Reproduction, 136,543-557

[3]Beumer T L, Roepers-Gajadien H L, Gademan I S, Kal H B,de Rooij D G. 2000. Involvement of the D-type cyclinsin germ cell proliferation and differentiation in themouse. Biology of Reproduction, 63, 1893-1898

[4]Braydich-Stolle L, Kostereva N, Dym M, Hofmann M C.2007. Role of Src family kinases and N-Myc inspermatogonial stem cell proliferation. DevelopmentBiology, 304, 34-45

[5]Brinster R L. 2002. Germline stem cell transplantation andtransgenesis. Science, 29, 2174-2176

[6]Cao H, Chu Y, Zhu H, Sun J, Pu Y, Gao Z, Yang C, Peng S,Dou Z, Hua J. 2011. Characterization of immortalizedmesenchymal stem cells derived from foetal porcinepancreas. Cell Proliferation, 44, 19-32

[7]Cardoso M C, Leonhardt H, Nadal-Ginard B. 1993. Reversalof terminal differentiation and control of DNAreplication: cyclin A and Cdk2 specifically localize atsubnuclear sites of DNA replication. Cell, 74, 979-992

[8]Chen B, Wang Y B, Zhang Z L, Xia W L, Wang H X, XiangZ Q, Hu K, Han Y F, Wang Y X, Huang Y R, et al. 2009.Xeno-free culture of human spermatogonial stem cellssupported by human embryonic stem cell-derivedfibroblast-like cells. Asian Journal of Andrology, 11,557-565

[9]Conrad S, Renninger M, Hennenlotter J, Wiesner T, Just L,Bonin M, Aicher W, Bühring H J, Mattheus U, Mack A,et al. 2008. Generation of pluripotent stem cells fromadult human testis. Nature, 456, 344-349

[10]Dadoune J P. 2007. New insights into male gametogenesis:what about the spermatogonial stem cell niche? FoliaHistochemica et Cytobiologica, 45, 141-147

[11]Dolci S, Pellegrini M, DiAgostino S, Geremia R, Rossi P.2001. Signaling through extra cellular signal-regulatedkinase is required for spermatogonial proliferativeresponse to stem cell factor. The Journal of BiologicalChemistry, 276, 40225-40233

[12]Du H J, Tang N, Liu B C, You B R, Shen F H, Ye M, Gao A,Huang C. 2006. Benzo[a]pyrene-induced cell cycleprogression is through ERKs/cyclin D1 pathway andrequires the activation of JNKs and p38 mapk in humandiploid lung fibroblasts. Molecular and CellularBiochemistry, 287, 79-89

[13]Dyce P W, Zhu H, Craig J, Li J. 2004. Stem cells withmultilineage potential derived from porcine skin.B i o c h e m i c a l a n d B i o p h y s i c a l R e s e a r c hCommunications, 316, 651-658

[14]Dym M, He Z, Jiang J, Pant D, Kokkinaki M. 2009.Spermatogonial stem cells: unlimited potential.Reproduction, Fertility, and Development, 21, 15-21

[15]Ebata K T, Zhang X, Nagano M C. 2005. Expression patternsof cell-surface molecules on male germ line stem cellsduring postnatal mouse development. MolecularReproduction and Development, 72, 171-181

[16]Ebata K T, Yeh J R, Zhang X, Nagano M C. 2011. Solublegrowth factors stimulate spermatogonial stem celldivisions that maintain a stem cell pool and produceprogenitors in vitro. Experimental Cell Research, 317,1319-1329

[17]Gassei K, Ehmcke J, Schlatt S. 2009. Efficient enrichment ofundifferentiated GFR alpha 1+ spermatogonia fromimmature rat testis by magnetic activated cell sorting.Cell and Tissue Research, 337, 177-183

[18]Gassei K, Ehmcke J, Dhir R, Schlatt S. 2010. Magneticactivated cell sorting allows isolation of spermatogoniafrom adult primate testes and reveals distinct GFRa1-positive subpopulations in men. Journal of MedicalPrimatology, 39, 83-91

[19]Gille H, Downward J. 1999. Multiple ras effector pathwayscontribute to G(1) cell cycle progression. The Journalof Biological Chemistry, 274, 22033-22040

[20]Girard F, Strausfeld U, Fernandez A, Lamb N J. 1991. CyclinA is required for the onset of DNA replication inmammalian fibroblasts. Cell, 67, 1169-1179

[21]Golestaneh N, Kokkinaki M, Pant D, Jiang J, DeStefano D,Fernandez-Bueno C, Rone J D, Haddad B R, GallicanoG I, Dym M. 2009. Pluripotent stem cells derived fromadult human testes. Stem Cells and Development, 18,1115-1126

[22]Groschel B, Bushman F. 2005. Cell cycle arrest in g2/mpromotes early steps of infection by humanimmunodeficiency virus. Journal of Virology, 79, 5695-5704

[23]He Z, Kokkinaki M, Dym M. 2009. Signaling molecules andpathways regulating the fate of spermatogonial stemcells. Microscopy Research and Technique, 72, 586-595

[24]He Z, Jiang J, Kokkinaki M, Golestaneha N, Hofmannb MC, Dym M. 2008. GDNF up-regulates c-fos transcriptionvia the Ras/Erk1/2 pathway to promote mousespermatogonial stem cell proliferation. Stem Cells, 26,266-278

[25]Hofmann M C. 2008. Gdnf signaling pathways within themammalian spermatogonial stem cell niche. Molecularand Cellular Endocrinology, 288, 95-103

[26]Hua J, Zhu H, Pan S, Liu C, Sun J, Ma X, Dong W, Liu W,Li W. 2011. Pluripotent male germline stem cells fromgoat fetal testis and their survival in mouse testis.Cellular Reprogramming (Cloning and Stem Cells), 13,133-144

[27]Huleihel M, Abuelhija M, Lunenfeld E. 2007. In vitro cultureof testicular germ cells: regulatory factors andlimitations. Growth Factors, 25, 236-252

[28]Izadyar F, Wong J, Maki C, Pacchiarotti J, Ramos T,Howerton K, Yuen C, Greilach S, Zhao H H, Chow M,Chow Y C, Rao J, Barritt J, Bar-Chama N, Copperman A.2011. Identification and characterization of repopulatingspermatogonial stem cells from the adult human testis.Hum Reproduction, 26, 1296-1306

[29]Jijiwa M, Kawai K, Fukihara J, Nakamura A, Hasegawa M,Suzuki C, Sato T, Enomoto A, Asai N, Murakumo Y, etal. 2008. GDNF-mediated signaling via RET tyrosine1062 is essential for maintenance of spermatogonial stemcells. Genes to Cells, 13, 365-374

[30]Kanatsu-Shinohara M, Lee J, Inoue K, Ogonuki N, Miki H,Toyokuni S, Ikawa M, Nakamura T, Ogura A, ShinoharaT. 2008. Pluripotency of a single spermatogonial stemcell in mice. Biology of Reproduction, 78, 681-687

[31]Kossack N, Meneses J, Shefi S, Nguyen H N, Chavez S,Nicholas C, Gromoll J, Turek P J, Reijo-Pera R A. 2009.Isolation and characterization of pluripotent humanspermatogonial stem cell-derived cells. Stem Cells, 27,138-149

[32]Kubota H, Avarbock M R, Brinster R L. 2004a. Growthfactors essential for self-renewal and expansion ofmouse spermatogonial stem cells. Proceedings of theNational Academy of Sciences of the United States ofAmerica, 101, 16489-16494

[33]Kubota H, Avarbock M R, Brinster R L. 2004b. Cultureconditions and single growth factors affect fatedetermination of mouse spermatogonial stem cells.Biology of Reproduction, 71, 722-731

[34]Kubota H, Wu X, Goodyear S M, Avarbock M R, Brinster RL. 2011. Glial cell line-derived neurotrophic factor andendothelial cells promote self-renewal of rabbit germcells with spermatogonial stem cell properties. TheJournal of Federation of Ameican Socieities forExperimental Biology, 25, 2604-2614

[35]Kuijk E W, Colenbrander B, Roelen B A. 2009. The effectsof growth factors on in vitro-cultured porcine testicularcells. Reproduction, 138, 721-731

[36]Lee J, Kanatsu-Shinohara M, Ogonuki N, Miki H, Inoue K,Morimoto T, Morimoto H, Ogura A, Shinohara T. 2009.Heritable imprinting defect caused by epigeneticabnormalities in mouse spermatogonial stem cells.Biology of Reproduction, 80, 518-527

[37]Meng X, Lindahl M, Hyvönen M E, Parvinen M, de RooijD G, Hess M W, Raatikainen-Ahokas A, Sainio K,Rauvala H, Lakso M, et al. 2000. Regulation of cell fatedecision of undifferentiated spermatogonia by GDNF.Science, 287, 1489-1493

[38]Mizrak S C, Chikhovskaya J V, Sadri-Ardekani H, van DaalenS, Korver C M, Hovingh S E, Roepers-Gajadien H L,Raya A, Fluiter K, de Reijke T M, et al. 2010. Embryonicstem cell-like cells derived from adult human testis.Human Reproduction, 25, 158-167

[39]Nagano M, Ryu B Y, Brinster C J, Avarbock M R, Brinster RL. 2003. Maintenance of mouse male germ line stemcells in vitro. Biology of Reproduction, 68, 2207-2214

[40]Naughton C K, Jain S, Strickland A M, Gupta A, MilbrandtJ. 2006. Glial cell-line derived neurotrophic factormediatedRET signaling regulates spermatogonial stemcell fate. Biology of Reproduction, 74, 314-321

[41]Oatley J M, Avarbock M R, Brinster R L. 2007. Glial cellline-derived neurotrophic factor regulation of genesessential for self-renewal of mouse spermatogonial stemcells is dependent on Src family kinase signaling. TheJournal of Biological Chemistry, 282, 25842-25851

[42]Oatley J M, Avarbock M R, Telaranta A I, Fearon D T,Brinster R L. 2006. Identifying genes important forspermatogonial stem cell self-renewal and survival.Proceedings of the National Academy of Sciences ofthe United States of America, 103, 9524-9529

[43]Oatley J M, Brinster R L. 2008. Regulation of spermatogonialstem cell self-renewal in mammals. Annual Review ofCell and Developmental Biology, 24, 263-286

[44]Pagano M, Pepperkok R, Lukas J, Baldin V, Ansorge W,Bartek J, Draetta G. 1993. Regulation of the cell cycle bythe cdk2 protein kinase in cultured human fibroblasts.The Journal of Cell Biology, 121, 101-111

[45]Ryu B Y, Kubota H, Avarbock M R, Brinster R L. 2005.Conservation of spermatogonial stem cell self-renewalsignaling between mouse and rat. Proceedings of theNational Academy of Sciences of the United States ofAmerica, 102, 14302-14307

[46]Sariola H, Immonen T. 2008. GDNF maintains mousespermatogonial stem cells in vivo and in vitro. Methodsin Molecular Biology, 450, 127-135

[47]Sariola H, Saarma M. 2003. Novel functions and signallingpathways for GDNF. Journal of Cell Science, 116, 3855-3862

[48]Schmidt J A, Avarbock M R, Tobias J W, Brinster R L. 2009.Identification of glial cell line-derived neurotrophicfactor-regulated genes important for spermatogonialstem cell self-renewal in the rat. Biology ofReproduction, 81, 56-66

[49]Simon L, Ekman G C, Tyagi G, Hess RA, Murphy K M,Cooke P S. 2007. Common and distinct factors regulateexpression of mRNA for ETV5 and GDNF, Sertoli cellproteins essential for spermatogonial stem cellmaintenance. Experimental Cell Research, 313, 3090-3099

[50]Spinnler K, Köhn F M, Schwarzer U, Mayerhofer A. 2010.Glial cell line-derived neurotrophic factor isconstitutively produced by human testicularperitubular cells and may contribute to thespermatogonial stem cell niche in man. HumanReproduction, 25, 2181-2187

[51]Tadokoro Y, Yomogida K, Ohta H, Tohda A, Nishimune Y.2002. Homeostatic regulation of germinal stem cellproliferation by the GDNF/FSH pathway. Mechanismsof Development, 113, 29-39

[52]Tang X D, Zhou X, Zhou K Y. 2009. Dauricine inhibitsinsulin-like growth factor-I-induced hypoxia induciblefactor 1alpha protein accumulation and vascularendothelial growth factor expression in human breastcancer cells. Acta Pharmacologica Sinica, 30, 605-616

[53]Tyagi G, Carnes K, Morrow C, Kostereva N V, Ekman G C,Meling D D, Hostetler C, Griswold M, Murphy K M,Hess R A, et al. 2009. Loss of Etv5 decreasesproliferation and RET levels in neonatal mouse testicular germ cells and causes an abnormal first wave ofspermatogenesis. Biology of Reproduction, 81, 258-266

[54]Wang F, Liu C, Zhang S, Liu W, Hua J. 2013. Transplantationof goat bone marrow mesenchymal stem cells (gMSCs)help restore spermatogenesis in endogenous germ cellsdepletedmouse models. Journal of IntegrativeAgriculture, 12, 101-108

[55]Wu X, Oatley J M, Oatley M J, Kaucher A V, Avarbock MR, Brinster R L. 2010. The POU domain transcriptionfactor POU3F1 is an important intrinsic regulator ofGDNF-induced survival and self-renewal of mousespermatogonial stem cells. Biology of Reproduction,82, 1103-1111

[56]Wu X, Schmidt J A, Avarbock M R, Tobias J W, Carlson CA, Kolon T F, Ginsberg J P, Brinster R L. 2009.Prepubertal human spermatogonia and mousegonocytes share conserved gene expression of germlinestem cell regulatory molecules. Proceedings of theNational Academy of Sciences of the United States ofAmerica, 106, 21672-21677

[57]Yomogida K, Yagura Y, Tadokoro Y, Nishimune Y. 2003.Dramatic expansion of germinal stem cells by ectopicallyexpressed human glial cell line-derived neurotrophicfactor in mouse Sertoli cells. Biology of Reproduction,69, 1303-1307

[58]Zhang H, Li D, Cao H, Lv X, Chu Y, Bai Y, Jin Y, Peng S,Dou Z, Hua J. 2013. Conophylline promotes theproliferation of immortalized mesenchymal stem cellsderived from fetal porcine pancreas (iPMSCs). Journalof Integrative Agriculture, 12, 678-686

[59]Zhang S, Sun J, Pan S, Zhu H, Wang L, Hu Y, Cao H, Yan X,Hua J. 2011. Retinol (vitamin A) maintains self-renewalof pluripotent male germline stem cells (mGSCs) fromadult mouse testis. Journal of Cellular Biochemistry,12, 1009-1021

[60]Zhu H, Liu C, Li M, Sun J, Song W, Hua J. 2013. Optimizationof the conditions of isolation and culture of dairy goatmale germline stem cells (mGSC). Animal ReproductionScience, 137, 45-52.

GDNF Up-Regulates c-Myc Transcription via the PI3K/Akt Pathway to Promote Dairy Goat Male Germline Stem Cells (mGSC) Proliferation

GDNF Up-Regulates c-Myc Transcription via the PI3K/Akt Pathway to Promote Dairy Goat Male Germline Stem Cells (mGSC) Proliferation

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics