Spermatogonial Stem Cells: Biology, Isolation, Culture, Characterization, and Practical Perspectives

Document Type: Review Article


1 Assistant Professor, Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran

2 Professor, Department of Biology, Faculty of Science & Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

3 Assistant Professor, Department of Biology, Faculty of Science & Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

4 Assistant Professor, Department of Biology, Faculty of Science, Hakim Sabzevari University, Sabzevar, Iran

5 Assistant Professor, Department of Biology, Faculty of Basic Science, Gonbad Kavous University, Gonbad Kavous, Iran


Spermatogonial stem cells (SSCs) also known as germ stem cells (GSCs) are the basis of spermatogenesis process in the testis. Furthermore, they are also valuable cells with different applications in developmental biology, transgenesis technology, and clinic. Understanding the new findings related to the cell and molecular biology of SSCs and the methods for isolation and maintenance of these cells are important and essential for their applications in medicine to treat some infertility problems and also in biotechnology to produce transgenic animals. The present review was conducted to describe the cell and molecular basis of development, self-renewal, and differentiation of mammalian and poultry SSCs in vivo (natural niche) and in vitro. Moreover, this study represents specific molecular markers to characterize SSCs. We also introduce methods to isolate, cultivate and enrich these cells, which are important for their applications. Finally, the significance of SSCs in different fields and their practical perspectives, and also the differentiation potential of other stem cells into spermatogonial- and spermatic-like cells are discussed.


  1. Phillips BT, Gassei K, Orwig KE. Spermatogonial stem cell regulation and spermatogenesis. Philos Trans R Soc Lond B Biol Sci 2010; 365(1546): 1663-1678.
  2. Huckins C. The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat Rec 1971; 169(3): 533-557.
  3. Oatley JM, Brinster RL. Regulation of spermatogonial stem cell self-renewal in mammals. Annu Rev Cell Dev Biol 2008; 24: 263-286.
  4. Griswold MD. The central role of Sertoli cells in spermatogenesis. Semin Cell Dev Biol 1998; 9(4): 411-416.
  5. McCarrey JR. Development of the germ cell. In Cell and molecular biology of the testis New York, NY: Oxford University Press; 1993; 58-89.
  6. Kanatsu-Shinohara M, Shinohara T. Spermatogonial stem cell self-renewal and development. Annu Rev Cell Dev Biol 2013; 29: 163-187.
  7. Chiarini-Garcia H, Russell LD. High-resolution light microscopic characterization of mouse spermatogonia. Biol Reprod 2001; 65(4): 1170-1178.
  8. Chiarini-Garcia H, Russell LD. Characterization of mouse spermatogonia by transmission electron microscopy. Reproduction 2002; 123(4): 567-577.
  9. Chiarini-Garcia H, Meistrich ML. High-resolution light microscopic characterization of spermatogonia. Methods Mol Biol 2008; 450: 95-107.
  10. deRooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask. J Androl 2000; 21(6): 776-798.
  11. Hamer G, Roepers-Gajadien HL, Gademan IS, Kal HB, De Rooij DG. Intercellular bridges and apoptosis in clones of male germ cells. Int J Androl 2003; 26(6): 348-353.
  12. Zhou Q, Griswold MD. Regulation of spermatogonia 2008; 1-17.
  13. de Rooij DG, Mizrak SC. Deriving multipotent stem cells from mouse spermatogonial stem cells: a new tool for developmental and clinical research. Development2008; 135(13): 2207-2213.
  14. de Rooij DG. Stem cells in the testis. Int J Exp Pathol 1998; 79(2): 67-80.
  15. Yoshida S, Nabeshima Y, Nakagawa T. Stem cell heterogeneity: actual and potential stem cell compartments in mouse spermatogenesis. Ann N Y Acad Sci 2007; 1120: 47-58.
  16. Rossi P, Sette C, Dolci S, Geremia R. Role of c-kit in mammalian spermatogenesis. J Endocrinol Invest 2000; 23(9): 609-615.
  17. Hermann BP, Sukhwani M, Hansel MC, Orwig KE. Spermatogonial stem cells in higher primates: are there differences from those in rodents? Reproduction 2010; 139(3): 479-493.
  18. Dym M, Kokkinaki M, He Z. Spermatogonial stem cells: mouse and human comparisons. Birth Defects Res C Embryo Today 2009; 87(1): 27-34.
  19. Amann RP. The cycle of the seminiferous epithelium in humans: a need to revisit? J Androl 2008; 29(5): 469-487.
  20. Ehmcke J, Schlatt S. A revised model for spermatogonial expansion in man: lessons from non-human primates. Reproduction 2006; 132(5): 673-680.
  21. Ehmcke J, Wistuba J, Schlatt S. Spermatogonial stem cells: questions, models and perspectives. Hum Reprod Update 2006; 12(3): 275-282.
  22. Lavial F, Pain B. Chicken embryonic stem cells as a non-mammalian embryonic stem cell model. Dev Growth Differ 2010; 52(1): 101-114.
  23. Eyal-Giladi H, Kochav S. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Dev Biol 1976; 49(2): 321-337.
  24. Mozdziak PE, Angerman-Stewart J, Rushton B, Pardue SL, Petitte JN. Isolation of chicken primordial germ cells using fluorescence-activated cell sorting. Poult Sci 2005; 84(4):594-600.
  25. Ukeshima A, Yoshinaga K, Fujimoto T. Scanning and transmission electron microscopic observations of chick primordial germ cells with special reference to the extravasation in their migration course. J Electron Microsc (Tokyo) 1991; 40(2): 124-128.
  26. Nakamura Y, Yamamoto Y, Usui F, et al. Migration and proliferation of primordial germ cells in the early chicken embryo. Poult Sci 2007; 86(10): 2182-2193.
  27. Han JY. Germ cells and transgenesis in chickens. Comp Immunol Microbiol Infect Dis 2009; 32(2): 61-80.
  28. Doitsidou M, Reichman-Fried M, Stebler J, et al. Guidance of primordial germ cell migration by the chemokine SDF-1. Cell. 2002; 111(5): 647-659.
  29. Weidinger G, Stebler J, Slanchev K, et al. dead end, a novel vertebrategerm plasm component, is required for zebrafish primordial germ cell migration and survival. Curr Biol 2003; 13(16): 1429-1434.
  30. Tegelenbosch RA, de Rooij DG. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H101/F1 hybrid mouse. Mutat Res 1993; 290(2): 193-200.
  31. Jung JG, Lee YM, Park TS, Park SH, Lim JM, Han JY. Identification, culture, and characterization of germline stem cell-like cells in chicken testes. Biol Reprod 2007; 76(1): 173-182.
  32. Wu X, Schmidt JA, Avarbock MR, et al. Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules. Proc Natl Acad Sci U S A 2009; 106(51): 21672-21677.
  33. de Rooij DG. The spermatogonial stem cell niche. Microsc Res Tech 2009; 72(8): 580-585.
  34. Oatley JM, Brinster RL. The germline stem cell niche unit in mammalian testes. Physiol Rev 2012; 92(2): 577-595.
  35. Smith JF, Yango P, Altman E, et al. Testicular niche required for human spermatogonial stem cell expansion. Stem cells translational medicine 2014; 3(9): 1043-1054.
  36. Baratelli GM, Lanzani A, Sacco RN. Biography of Enrico Sertoli. Urology 2002; 60(1): 196-198.
  37. Steinberger A, Steinberger E. Replication pattern of Sertoli cells in maturing rat testis in vivo and in organ culture. Biol Reprod 1971; 4(1): 84-87.
  38. Kluin PM, Kramer MF, de Rooij DG. Proliferation of spermatogonia and Sertoli cells in maturing mice. Anat Embryol (Berl) 1984; 169(1): 73-78.
  39. Wing TY, Christensen AK. Morphometric studies on rat seminiferous tubules. Am J Anat 1982; 165(1): 13-25.
  40. Petersen C, Soder O. The sertoli cell-a hormonal target and 'super' nurse for germ cells that determines testicular size. Horm Res 2006; 66(4): 153-161.
  41. Carr I, Clegg EJ, Meek GA. Sertoli cells as phagocytes: an electron microscopic study. J Anat 1968; 102(Pt 3): 501-509.
  42. Plotton I, Garby L, Morel Y, Lejeune H. Decrease of anti-Mullerian hormone in genetic spermatogenic failure. Andrologia 2012; 44(5): 349-354.
  43. Barakat B, O'Connor AE, Gold E, de Kretser DM, Loveland KL. Inhibin, activin, follistatin and FSH serum levels and testicular production are highly modulated during the first spermatogenic wave inmice. Reproduction 2008; 136(3): 345-359.
  44. Cai K, Hua G, Ahmad S, et al. Action mechanism of inhibin alpha-subunit on the development of Sertoli cells and first wave of spermatogenesis in mice. PLoS One 2011; 6(10): e25585.
  45. Caneguim BH, Beltrame FL, da Luz JS, Valentini SR, Cerri PS, Sasso-Cerri E. Primordial germ cells (spermatogonial stem cells) of bullfrogs express sex hormone-binding globulin and steroid receptors during seasonal spermatogenesis. Cells Tissues Organs 2013; 197(2): 136-144.
  46. Lardone MC, Castillo P, Valdevenito R, et al. P450-aromatase activity and expression in human testicular tissues with severe spermatogenic failure. Int J Androl  2010; 33(4): 650-660.
  47. Morrow CM, Tyagi G, Simon L, et al. Claudin 5 expression in mouse seminiferous epithelium is dependent upon the transcription factor ets variant 5 and contributes to blood-testis barrier function. Biol Reprod 2009; 81(5): 871-879.
  48. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Shinohara T. Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells. Hum Reprod 2003; 18(12): 2660-2667.
  49. Kanatsu-Shinohara M, Miki H, Inoue K, et al. Germline niche transplantation restores fertility in infertile mice. Hum Reprod 2005; 20(9): 2376-2382.
  50. Yoshida S, Sukeno M, Nabeshima Y. A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science 2007; 317(5845): 1722-1726.
  51. Kanatsu-Shinohara M, Morimoto T, Toyokuni S, Shinohara T. Regulation of mouse spermatogonial stem cell self-renewing division by the pituitary gland. Biol Reprod 2004; 70(6): 1731-1737.
  52. Momeni-Moghaddam M, Matin MM, Boozarpour S, Sisakhtnezhad S, et al. A simple method for isolation, culture and in vitro maintenance of chicken spermatogonial stem cells. In Vitro Cell Dev Biol Anim 2014; 50: 155-161.
  53. Meng X, Lindahl M, Hyvonen ME, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000; 287(5457): 1489-1493.
  54. Zhou Q, Li Y, Nie R, et al. Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro. Biol Reprod 2008; 78(3): 537-545.
  55. Oatley JM, Avarbock MR, Brinster RL. Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. J Biol Chem 2007; 282(35): 25842-25851.
  56. He Z, Jiang J, Kokkinaki M, Golestaneh N, Hofmann MC, Dym M. Gdnf upregulates c-Fos transcription via the Ras/Erk1/2 pathway to promote mouse spermatogonial stem cell proliferation. Stem Cells 2008; 26(1): 266-278.
  57. Loveland KL, Schlatt S. Stem cell factor and c-kit in the mammalian testis: lessons originating from Mother Nature's gene knockouts. J Endocrinol 1997; 153(3): 337-344.
  58. Kubota H, Avarbock MR, Brinster RL. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 2004; 101(47): 16489-16494.
  59. Yoon KA, Chae YM, Cho JY. FGF2 stimulates SDF-1 expression through the Erm transcription factor in Sertoli cells. J Cell Physiol 2009; 220(1): 245-256.
  60. Raverot G, Weiss J, Park SY, Hurley L, Jameson JL. Sox3 expression in undifferentiated spermatogonia is required for the progression of spermatogenesis. Dev Biol 2005; 283(1): 215-225.
  61. Ballow D, Meistrich ML, Matzuk M, Rajkovic A. Sohlh1 is essential for spermatogonial differentiation. Dev Biol 2006; 294(1): 161-167.
  62. Hao J, Yamamoto M, Richardson TE, et al. Sohlh2 knockout mice are male-sterile because of degeneration of differentiating type A spermatogonia. Stem Cells 2008; 26(6): 1587-1597.
  63. Choi Y-J, Yoon J-W, Pyo C-W, Kim J-A, Bae S-H, Park S-S. A possible role of STRA8 as a transcriptional factor. Genes Genom 2010; 32: 521-526.
  64. Oatley JM, Avarbock MR, Telaranta AI, Fearon DT, Brinster RL. Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc Natl Acad Sci U S A 2006; 103(25): 9524-9529.
  65. Kaucher AV, Oatley MJ, Oatley JM. NEUROG3 is a critical downstream effector for STAT3-regulated differentiation of mammalian stem and progenitor spermatogonia. Biol Reprod 2012; 86(5): 164, 1-11.
  66. Chen JX, Xu LL, Wang XC, Qin HY, Wang JL. Involvement of c-Src/STAT3 signal in EGF-induced proliferation of rat spermatogonial stem cells. Mol Cell Biochem 2011; 358(1-2): 67-73.
  67. Mirzapour T, Movahedin M, Tengku Ibrahim TA, et al. Effects of basic fibroblast growth factor and leukaemia inhibitory factor on proliferation and short-term culture of human spermatogonial stem cells. Andrologia 2012; 44(1):41-55.
  68. Schrans-Stassen BH, Saunders PT, Cooke HJ, de Rooij DG. Nature of the spermatogenic arrest in Dazl -/- mice. Biol Reprod 2001; 65(3): 771-776.
  69. Boettger-Tong HL, Johnston DS, Russell LD, Griswold MD, Bishop CE. Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis. Biol Reprod 2000; 63(4): 1185-1191.
  70. Lolicato F, Marino R, Paronetto MP, et al. Potential role of Nanos3 in maintaining the undifferentiated spermatogonia population. Dev Biol 2008; 313(2): 725-738.
  71. Falender AE, Freiman RN, Geles KG, et al. Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID. Genes Dev 2005; 19(7): 794-803.
  72. Buaas FW, Kirsh AL, Sharma M, et al. Plzf is required in adult male germ cells for stem cell self-renewal. NatGenet 2004; 36(6): 647-652.
  73. Holdcraft RW, Braun RE. Hormonal regulation of spermatogenesis. Int J Androl 2004; 27(6): 335-342.
  74. Takubo K, Ohmura M, Azuma M, et al. Stem cell defects in ATM-deficient undifferentiated spermatogonia through DNAdamage-induced cell-cycle arrest. Cell Stem Cell 2008; 2(2): 170-182.
  75. Shetty G, Weng CC. Cryptorchidism rescues spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mice. Endocrinology 2004; 145(1): 126-133.
  76. Atchison FW, Means AR. A role for Pin1 in mammalian germ cell development and spermatogenesis. Front Biosci 2004; 9: 3248-3256.
  77. Brehm R, Zeiler M, Ruttinger C, et al. A sertoli cell-specific knockout of connexin43 prevents initiation of spermatogenesis. Am J Pathol 2007; 171(1): 19-31.
  78. Izadyar F, Creemers LB, van Dissel-Emiliani FM, van Pelt AM, de Rooij DG. Spermatogonial stem cell transplantation. Mol Cell Endocrinol 2000; 169(1-2): 21-26.
  79. Xueming Z, Liangxue L, Dexue L, et al. eparationand purification of spermatogonia in mouse. Acta Anatomica Sinica 2000; 31(3): 235-238.
  80. Liu S, Tang Z, Xiong T, Tang W. Isolation and characterization of human spermatogonial stem cells. Reprod Biol Endocrinol 2011; 9: 141.
  81. Yu F, Ding LJ, Sun GB, et al. Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells. Mol Reprod Dev 2010; 77(4): 340-347.
  82. Hong W, Bi-chun L, Guan-yue Z, Si-yu S, Jie Q, Guo-hong C. Isolation,purificationand culture of spermatogonia in chicken. Acta Vet et Zootechnica Sinica 2006; 37: 1173-1178.
  83. Wu XS, Wu H, Li BC, et al. Isolation, purification and culture of spermatogonia in chicken. J Animal Vet Adv 2009; 8(12): 2418-2423.
  84. Trefil P, Micakova A, Mucksova J, et al. Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken. Biol Reprod 2006; 75(4): 575-581.
  85. 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 U S A 2003; 100(11): 6487-6492.
  86. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996; 183(4): 1797-1806.
  87. von Schonfeldt V, Krishnamurthy H, Foppiani L, Schlatt S. Magnetic cell sorting is a fast and effective method of enriching viable spermatogonia from Djungarian hamster, mouse, and marmoset monkey testes. Biol Reprod 1999; 61(3): 582-589.
  88. Kanatsu-Shinohara M, Ogonuki N, Inoue K, et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003; 69(2): 612-616.
  89. Kubota H, Avarbock MR, Brinster RL. Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod 2004; 71(3): 722-731.
  90. Hamra FK, Chapman KM, Nguyen DM, Williams-Stephens AA, Hammer RE, Garbers DL. Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proc Natl Acad Sci U S A 2005; 102(48): 17430-17435.
  91. Sadri-Ardekani H, Akhondi MA, van der Veen F, Repping S, van PeltAM. In vitro propagation of human prepubertal spermatogonial stem cells. JAMA 2011; 305(23): 2416-2418.
  92. Kanatsu-Shinohara M, Inoue K, Ogonuki N, Morimoto H, Ogura A, Shinohara T. Serum- and feeder-free culture of mouse germline stem cells. Biol Reprod 2011; 84(1): 97-105.
  93. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A 1994; 91(24): 11303-11307.
  94. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994; 91(24): 11298-11302.
  95. Ohta H, Tohda A, Nishimune Y. Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis. Biol Reprod 2003; 69(6): 1815-1821.
  96. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nat Med 2000; 6(1): 29-34.
  97. Nakamura T, Yao R, Ogawa T, et al. Oligo-astheno-teratozoospermia in mice lacking Cnot7, a regulator of retinoid X receptor beta. Nat Genet 2004; 36(5): 528-533.
  98. Dobrinski I, Ogawa T, Avarbock MR, Brinster RL. Computer assisted image analysis to assess colonization of recipient seminiferous tubules by spermatogonial stem cells from transgenic donor mice. Mol Reprod Dev 1999; 53(2): 142-148.
  99. Izadyar F, Den Ouden K, Stout TA, et al. Autologous and homologous transplantation of bovine spermatogonial stem cells. Reproduction 2003; 126(6): 765-774.
100. Nagano M, McCarrey JR, Brinster RL. Primate spermatogonial stem cells colonize mouse testes. Biol Reprod 2001; 64(5): 1409-1416.

101. Nagano M, Patrizio P, Brinster RL. Long-term survival of human spermatogonial stem cells in mouse testes. Fertil Steril 2002; 78(6): 1225-1233.

102. Dobrinski I, Avarbock MR, Brinster RL. Germ cell transplantation from large domestic animals into mouse testes. Mol Reprod Dev 2000; 57(3): 270-279.

103. Dastpak M, Matin MM, Farshchian M, et al. Construction and quantitative evaluation of a dual specific promoter system for monitoring the expression status of Stra8 and c-kit genes. Mol Biotechnol 2014; 56(12): 1100-1119.

104. Shinohara T, Avarbock MR, Brinster RL. beta1- and alpha6-integrin are surface markers on mouse spermatogonialstem cells. Proc Natl Acad Sci U S A 1999; 96(10): 5504-5509.

105. Lacham-Kaplan O. In vivo and in vitro differentiation of male germ cells in the mouse. Reproduction 2004; 128(2): 147-152.

106. Huang YH, Chin CC, Ho HN, et al. Pluripotency of mouse spermatogonial stem cells maintained by IGF-1- dependent pathway. FASEB J 2009; 23(7): 2076-2087.

107. van Pelt AM, Roepers-Gajadien HL, Gademan IS, Creemers LB, de Rooij DG, van Dissel-Emiliani FM. Establishment of cell lines with rat spermatogonial stem cell characteristics. Endocrinology 2002; 143(5): 1845-1850.

108. Seandel M, James D, Shmelkov SV, et al. Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature 2007; 449(7160): 346-350.

109. YehJR, Nagano MC. Spermatogonial stem cell biomarkers: improved outcomes of spermatogonial transplantation in male fertility restoration? Expert Rev Mol Diagn 2009; 9(2): 109-114.

110. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proc Natl Acad Sci U S A 2000; 97(15): 8346-8351.

111. Pramod RK, Mitra A. In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus). J Assist Reprod Genet 2014; 31(8): 993-1001.

112. Eildermann K, Gromoll J, Behr R. Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture. Hum Reprod 2012; 27(6): 1754-1767.

113. Lavial F, Acloque H, Bachelard E, Nieto MA, Samarut J, Pain B. Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate. Dev Biol 2009; 330(1): 73-82.

114. Heo YT, Lee SH, Yang JH, Kim T, Lee HT. Bone marrow cell-mediated production of transgenic chickens. Lab Invest 2011; 91(8): 1229-1240.

115. Clark AT, Bodnar MS, Fox M, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum Mol Genet 2004; 13(7): 727-739.

116. Yoshinaga K, Nishikawa S, Ogawa M, Hayashi S, Kunisada T, Fujimoto T. Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 1991; 113(2): 689-699.

117. Goertz MJ, Wu Z, Gallardo TD, Hamra FK, Castrillon DH. Foxo1 is required in mouse spermatogonial stem cells for their maintenance and the initiation of spermatogenesis. J Clin Invest 2011; 121(9): 3456-3466.

118. Hermann BP, Sukhwani M, Lin CC, et al. Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques. Stem Cells 2007; 25(9): 2330-2338.

119. Ryu BY, Kubota H, Avarbock MR, Brinster RL. Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc Natl Acad Sci U S A 2005; 102(40): 14302-14307.

120. Kanatsu-Shinohara M, Muneto T, Lee J, Takenaka M, et al. Long-term culture of male germline stem cells from hamster testes. Biol Reprod 2008; 78(4): 611-617.

121. Kanatsu-Shinohara M, Toyokuni S, Shinohara T. CD9 is a surface marker on mouse and rat male germline stem cells. Biol Reprod 2004; 70(1): 70-75.

122. Zheng Y, Thomas A, Schmidt CM, Dann CT. Quantitative detection of human spermatogonia for optimization of spermatogonial stem cell culture. Hum Reprod 2014; 29(11): 2497-2511.

123. Ebata KT, Zhang X, Nagano MC. Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development. Mol Reprod Dev 2005; 72(2): 171-181.

124. Martin LA,Seandel M. Propagation of Adult SSCs: From Mouse to Human. BioMed Res Int 2013; 2013: 1-9.

125. Conrad S, Renninger M, Hennenlotter J, et al. Generation of pluripotent stem cells from adult human testis. Nature 2008; 456(7220): 344-349.

126. Sisakhtnezhad S, Bahrami AR, Matin MM, et al. The molecular signature and spermatogenesis potential of newborn chicken spermatogonial stem cells in vitro. In Vitro Cell Dev Biol Anim 2015; 51(4):415-25. 

127. Feng LX, Chen Y, Dettin L, et al. Generation and in vitro differentiation of a spermatogonial cell line. Science 2002; 297(5580): 392-395.

128. Sato T, Katagiri K, Yokonishi T, et al. In vitro production of fertile sperm from murine spermatogonial stem cell lines. Nat Commun 2011; 2: 472.

129. Abu Elhija M, Lunenfeld E, Schlatt S, Huleihel M. Differentiation of murine male germ cells to spermatozoa in a soft agar culture system. Asian J Androl 2012; 14(2): 285-293.

130. Sisakhtnezhad S, Matin MM. Transdifferentiation: a cell and molecular reprogramming process. Cell Tissue Res 2012; 348(3): 379-396.

131. Kanatsu-Shinohara M, Inoue K, Lee J, et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell 2004; 119(7): 1001-1012.

132. Guan K, Nayernia K, Maier LS, et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006; 440(7088): 1199-1203.

133. Mizrak SC, Chikhovskaya JV, Sadri-Ardekani H, et al. Embryonic stem cell-like cells derived from adult human testis. Hum Reprod 2010; 25(1): 158-167.

134. Golestaneh N, Kokkinaki M, Pant D, et al. Pluripotent stem cells derived from adult human testes. Stem Cells Dev 2009; 18(8): 1115-1126.

135. Orwig KE, Schlatt S. Cryopreservation and transplantation of spermatogonia and testicular tissue for preservation of male fertility. J Natl Cancer Inst Monogr 2005; 34: 51-56.

136. Goossens E, Geens M, De Block G, Tournaye H. Spermatogonial survival in long-term human prepubertal xenografts. Fertil Steril 2008; 90(5): 2019-2022.

137. Virant-Klun I, Skutella T, Bhartiya D, Jin X. Stem cells in reproductive tissues: from the basics to clinics. BioMed Res Int 2013; 2013: 357102.

138. Schlatt S, Ehmcke J, Jahnukainen K. Testicular stem cells forfertility preservation: preclinical studies on male germ cell transplantation and testicular grafting. Pediatr Blood Cancer 2009; 53(2): 274-280.

139. Ginsberg JP, Carlson CA, Lin K, et al. An experimental protocol for fertility preservation in prepubertal boys recently diagnosed with cancer: a report of acceptability and safety. Hum Reprod 2010; 25(1): 37-41.

140. Azizollahi S, Aflatoonian R, Sedigi-Gilani MA, et al. Recruiting testicular torsion introduces an azoospermic mouse model for spermatogonial stem cell transplantation. Urol J 2014; 11(3): 1648-1655.

141. Honaramooz A, Snedaker A, Boiani M, Scholer H, Dobrinski I, Schlatt S. Sperm from neonatal mammalian testes grafted in mice. Nature 2002; 418(6899): 778-781.

142. Ivarie R. Competitive bioreactor hens on the horizon. Trends Biotechnol 2006; 24(3):99-101.

143. Li JJ, Lu LZ. Recent progress on technologies and applications of transgenic poultry. Afr J Biotechnol 2010; 9(24): 3481-3488.

144. Raju TS, Briggs JB, Borge SM, Jones AJ. Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 2000; 10(5): 477-486.

145. Rapp JC, Harvey AJ, Speksnijder GL, Hu W, Ivarie R. Biologically active human interferon alpha-2b produced in the egg white of transgenic hens. Transgenic Res 2003; 12(5): 569-575.

146. Zhu L, van de Lavoir MC, Albanese J, et al. Production of human monoclonal antibody in eggs of chimeric chickens. Nat Biotechnol 2005; 23(9): 1159-1169.

147. Lillico SG, Sherman A, McGrew MJ, et al. Oviduct-specific expression of two therapeutic proteins in transgenic hens. Proc Natl Acad Sci U S A 2007; 104(6): 1771-1776.

148. Li B, Sun G, Sun H, et al. Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection. Sci China C Life Sci 2008; 51(8): 734-742.

149. Jung JG, Lee YM, Kim JN, et al. The reversible developmental unipotency of germ cells in chicken. Reproduction 2010; 139(1): 113-119.

150. Min S, Qing SQ, Hui YY, et al. Generation of antiviral transgenic chicken usingspermatogonial stem cell transfected in vivo. African J Biotechnol 2011; 10(70): 15678-15683.

151. Stukenborg JB, Schlatt S, Simoni M, et al. New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells. Mol Hum Reprod 2009; 15(9): 521-529.

152. Nayernia K, Lee JH,  Drusenheimer N, et al. Derivation of male germ cells from bone marrow stem cells. Lab Invest 2006; 86(7): 654-63.

153. Hua J,  Pan S, Yang C, et al. Derivation of male germ cell-like lineage from human fetal bone marrow stem cells. Reprod Biomed Online 2009; 19(1): 99-105.

154. Kaviani M, Ezzatabadipour M, Nematollahi-Mahani SN, et al. Evaluation of gametogenic potential of vitrified human umbilical cord Wharton's jelly-derived mesenchymal cells. Cytotherapy 2014; 16(2): 203-212.

155. Hassan AI, Alam SS. Evaluation of mesenchymal stem cells in treatment of infertility in male rats. Stem Cell Res Ther 2014; 5(6): 131.

156. Ghasemzadeh-Hasankolaei M, Eslaminejad MB, Sedighi-Gilani M. Derivation of male germ cells from ram bone marrow mesenchymal stem cells by three different methods and evaluation of their fate after transplantation into the testis. In Vitro Cell Dev Biol Anim 2015.

157. Mazaheri Z,  Movahedin M, Rahbarizadeh F, Amanpour S. Generation of In-vitro spermatogonial stem cells following genetic manipulation of primordial germ-like cells. Avicenna J Med Biotechnol 2012; 4(2): 55-63.

158. Easley CAT, Phillips BT, McGuire MM, et al. Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells. Cell Rep 2012; 2(3): 440-446.