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Proliferation of small number of human spermatogonial stem cells obtained from azoospermic patients

  • Stem Cell Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

This study aims to proliferate spermatogonial stem cells (SSCs) and compare the in-vitro effects of laminin and growth factors on the proliferation of adult human SSC.

Methods

Isolated testicular cells were cultured in DMEM supplemented with 5 % fetal calf serum (FCS). During the culture, enriched spermatogonial cells were treated with a combination of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and mouse leukemia inhibitory factor (LIF) in the presence or absence of human placental laminin-coated dishes. Cluster assay was performed during culture. Presence of spermatogonia was determined by an ultrastructural study of the cell clusters, reverse transcription polymerase chain reaction (RT-PCR) for spermatogonial markers and xenotransplantation to the testes of busulfan-treated recipient mice. Statistical significance between mean values was determined using statistical ANOVA tests.

Results

The findings indicated that the addition of GDNF, bFGF, EGF and LIF on laminin-coated dishes significantly increased in-vitro spermatogonial cell cluster formation in comparison with the control group (p ≤ 0.001). The expression of spermatogonial markers was maintained throughout the culture period. Furthermore, a transplantation experiment showed the presence of SSC among the cultured cells. In addition, a transmission electron microscopy (TEM) study suggested the presence of spermatogonial cells of typical morphology among the cluster cells.

Conclusions

It can be concluded that human SSCs obtained from non-obstructive azoospermic (NOA) patients had the ability to self-renew in the culture system. This system can be used for the propagation of a small number of these cells from small biopsies.

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Abbreviations

SSCs:

spermatogonial stem cells

GDNF:

glial cell line-derived neurotrophic factor

bFGF:

basic fibroblast growth factor

EGF:

epidermal growth factor

LIF:

leukemia inhibitory factor

NOA:

non-obstructive azoospermic

RT-PCR:

reverse transcription polymerase chain reaction

TESE:

Testis biopsies

PBS:

phosphate buffered saline solution

FCS:

fetal calf serum

PLZF:

promyelocytic leukaemia zinc-finger

DAZL:

deleted in azoospermia-like

Oct4:

Octamer-binding transcription factor 4

ITGB1:

β1-integrin

ITGA6:

α6-integrin

TEM:

transmission electron microscopy

BrdU:

5-Bromo-2-deoxyuridine

ES-like:

embryonic stem cell-like

GSC:

germ-line stem cells

References

  1. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A. 1994;91(24):11298–302.

    Article  PubMed  CAS  Google Scholar 

  2. Brinster RL. Male germline stem cells: from mice to men. Science (New York, NY). 2007;316(5823):404–5. doi:10.1126/science.1137741.

    Article  CAS  Google Scholar 

  3. Radford J, Shalet S, Lieberman B. Fertility after treatment for cancer. Questions remain over ways of preserving ovarian and testicular tissue. BMJ. 1999;319(7215):935–6.

    Article  PubMed  CAS  Google Scholar 

  4. Honaramooz A, Behboodi E, Blash S, Megee SO, Dobrinski I. Germ cell transplantation in goats. Mol Reprod Dev. 2003;64(4):422–8.

    Article  PubMed  CAS  Google Scholar 

  5. Izadyar F, Den Ouden K, Stout TA, Stout J, Coret J, Lankveld DP, et al. Autologous and homologous transplantation of bovine spermatogonial stem cells. Reproduction. 2003;126(6):765–74.

    Article  PubMed  CAS  Google Scholar 

  6. Schlatt S, Kim SS, Gosden R. Spermatogenesisand steroidogenesis in mouse, hamster and monkey testicular tissue after cryopreservation and heterotopic grafting to castrated hosts. Reproduction. 2002;124(3):339–46.

    Article  PubMed  CAS  Google Scholar 

  7. Lim JJ, Sung SY, Kim HJ, Song SH, Hong JY, Yoon TK, et al. Long-term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non-obstructive azoospermia under exogenous feeder-free culture conditions. Cell Prolif. 2010;43(4):405–17. doi:10.1111/j.1365-2184.2010.00691.x.

    Article  PubMed  CAS  Google Scholar 

  8. Tegelenbosch RA, de Rooij DG. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat Res. 1993;290(2):193–200.

    Article  PubMed  CAS  Google Scholar 

  9. Aponte PM, Soda T, Teerds KJ, Mizrak SC, van de Kant HJ, de Rooij DG. Propagation of bovine spermatogonial stem cells in vitro. Reproduction. 2008;136(5):543–57.

    Article  PubMed  CAS  Google Scholar 

  10. Brinster RL, Nagano M. Spermatogonial stem cell transplantation, cryopreservation and culture. Semin Cell Dev Biol. 1998;9(4):401–9.

    Article  PubMed  CAS  Google Scholar 

  11. Mirzapour T, Movahedin M, Ibrahim TABT, Haron AW, Makulati Z, Nowroozi M. Effect of donor cells concentration on colonization of human spermatogonial stem cells in recipient mouse testes. J Biol Sci. 2010;10:730–8.

    Article  Google Scholar 

  12. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod. 2003;69(2):612–6.

    Article  PubMed  CAS  Google Scholar 

  13. Sadri-Ardekani H, Mizrak SC, van Daalen SK, Korver CM, Roepers-Gajadien HL, Koruji M, et al. Propagation of Human Spermatogonial Stem Cells In Vitro. JAMA. 2009;302(19):2127–34. doi:10.1001/jama.2009.1689.

    Article  PubMed  CAS  Google Scholar 

  14. Anjamrooz SH, Movahedin M, Tiraihi T, Mowla SJ. Graft efficiency of co-cultured spermatogonial cells using sperm assay in epididymal lumen of recipient mice. Cell J (Yakhteh). 2006;7:242–9.

    Google Scholar 

  15. Kanatsu-Shinohara M, Inoue K, Lee J, Miki H, Ogonuki N, Toyokuni S, et al. Anchorage-independent growth of mouse male germline stem cells in vitro. Biol Reprod. 2006;74(3):522–9. doi:10.1095/biolreprod.105.046441.

    Article  PubMed  CAS  Google Scholar 

  16. Ebata KT, Yeh JR, Zhang X, Nagano MC. Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro. Exp Cell Res. 2011;317(10):1319–29. doi:10.1016/j.yexcr.2011.03.013.

    Article  PubMed  CAS  Google Scholar 

  17. Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Ogura A, et al. Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions. Biol Reprod. 2005;72(4):985–91. doi:10.1095/biolreprod.104.036400.

    Article  PubMed  CAS  Google Scholar 

  18. Kubota H, Avarbock MR, Brinster RL. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA. 2004;101(47):16489–94.

    Article  PubMed  CAS  Google Scholar 

  19. Meng X, Lindahl M, Hyvonen ME, Parvinen M, de Rooij DG, Hess MW, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science (New York, NY). 2000;287(5457):1489–93.

    Article  CAS  Google Scholar 

  20. Kuijk E, Colenbrander B, Roelen B. The effects of growth factors on in vitro-cultured porcine testicular cells. Reproduction. 2009. doi:10.1530/REP-09-0138.

  21. Sadri-Ardekani H, Akhondi MA, van der Veen F, Repping S, van Pelt AM. In vitro propagation of human prepubertal spermatogonial stem cells. JAMA. 2011;305(23):2416–8. doi:10.1001/jama.2011.791.

    Article  PubMed  CAS  Google Scholar 

  22. Aponte PM, van Bragt MP, de Rooij DG, van Pelt AM. Spermatogonial stem cells: characteristics and experimental possibilities. APMIS. 2005;113(11–12):727–42.

    Article  PubMed  Google Scholar 

  23. Huang YH, Chin CC, Ho HN, Chou CK, Shen CN, Kuo HC, et al. Pluripotency of mouse spermatogonial stem cells maintained by IGF-1- dependent pathway. FASEB J. 2009;23(7):2076–87. doi:10.1096/fj.08-121939.

    Article  PubMed  CAS  Google Scholar 

  24. Kim J, Seandel M, Falciatori I, Wen D, Rafii S. CD34+ testicular stromal cells support long-term expansion of embryonic and adult stem and progenitor cells. Stem cells (Dayton, Ohio). 2008;26(10):2516–22. doi:10.1634/stemcells.2008-0379.

    Article  Google Scholar 

  25. Koruji M, Movahedin M, Mowla SJ, Gourabi H, Arfaee AJ. Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions. Vitro Cell Dev Biol Anim. 2009;45(5–6):281–9. doi:10.1007/s11626-008-9169-y.

    Article  CAS  Google Scholar 

  26. Mohamadi SM, Movahedin M, Koruji SM, Jafarabadi MA, Makoolati Z. Comparison of colony formation in adult mouse spermatogonial stem cells developed in Sertoli and STO coculture systems. Andrologia. 2012;44(1):431–437. doi:10.1111/j.1439-0272.2011.01201.x.

    Google Scholar 

  27. Liu S, Tang Z, Xiong T, Tang W. Isolation and characterization of human spermatogonial stem cells. Reprod Biol Endocrinol. 2011;9:141. doi:10.1186/1477-7827-9-141.

    Article  PubMed  Google Scholar 

  28. Mirzapour T, Movahedin M, Tengku Ibrahim TA, Koruji M, Haron AW, Nowroozi MR, 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. doi:10.1111/j.1439-0272.2010.01135.x.

  29. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad SciU S A. 1994;91(24):11303–7.

    Article  CAS  Google Scholar 

  30. Rowley MJ, Berlin JD, Heller CG. The ultrastructure of four types of human spermatogonia. Z Zellforsch Mikrosk Anat. 1971;112(2):139–57.

    Article  PubMed  CAS  Google Scholar 

  31. Hofmann MC, Braydich-Stolle L, Dym M. Isolation of male germ-line stem cells; influence of GDNF. Dev Biol. 2005;279(1):114–24.

    Article  PubMed  CAS  Google Scholar 

  32. Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stem cells and their niche. Cell. 2004;116(6):769–78.

    Article  PubMed  CAS  Google Scholar 

  33. Li L, Xie T. Stem cell niche: structure and function. Annu Rev Cell Dev Biol. 2005;21:605–31. doi:10.1146/annurev.cellbio.21.012704.131525.

    Article  PubMed  CAS  Google Scholar 

  34. Simon L, Ekman GC, Tyagi G, Hess RA, Murphy KM, Cooke PS. Common and distinct factors regulate expression of mRNA for ETV5 and GDNF, Sertoli cell proteins essential for spermatogonial stem cell maintenance. Exp Cell Res. 2007;313(14):3090–9. doi:10.1016/j.yexcr.2007.05.002.

    Article  PubMed  CAS  Google Scholar 

  35. Aponte P, de Rooij D. Biomanipulation of bovine spermatogonial stem cells. Anim Reprod. 2008;5:16–22.

    Google Scholar 

  36. Dirami G, Ravindranath N, Pursel V, Dym M. Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM. Biol Reprod. 1999;61(1):225–30.

    Article  PubMed  CAS  Google Scholar 

  37. Shinohara T, Avarbock MR, Brinster RL. beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci USA. 1999;96(10):5504–9.

    Article  PubMed  CAS  Google Scholar 

  38. Orwig KE, Shinohara T, Avarbock MR, Brinster RL. Functional analysis of stem cells in the adult rat testis. Biol Reprod. 2002;66(4):944–9.

    Article  PubMed  CAS  Google Scholar 

  39. Shinohara T, Avarbock MR, Brinster RL. Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models. Dev Biol. 2000;220(2):401–11. doi:10.1006/dbio.2000.9655.

    Article  PubMed  CAS  Google Scholar 

  40. Kokil SN, Patil PR, Mahadik KR, Paradkar AR. Effect of molecular weight of hydrolyzed gelatin on its binding properties in tablets: a technical note. AAPS PharmSciTech. 2004;5(3):38–42. doi:10.1208/pt050341.

    Article  Google Scholar 

  41. Conrad S, Renninger M, Hennenlotter J, Wiesner T, Just L, Bonin M, et al. Generation of pluripotent stem cells from adult human testis. Nature. 2008;456(7220):344–9.

    Article  PubMed  CAS  Google Scholar 

  42. Kanatsu-Shinohara M, Lee J, Inoue K, Ogonuki N, Miki H, Toyokuni S, et al. Pluripotency of a single spermatogonial stem cell in mice. Biol Reprod. 2008;78(4):681–7. doi:10.1095/biolreprod.107.066068.

    Article  PubMed  CAS  Google Scholar 

  43. Hermann B, Sukhwani M, Hansel M, Orwig K. Spermatogonial stem cells in higher primates: are there differences to those in rodents? Reproduction. 2009. doi:10.1530/REP-09-0255.

  44. Ohbo K, Yoshida S, Ohmura M, Ohneda O, Ogawa T, Tsuchiya H, et al. Identification and characterization of stem cells in prepubertal spermatogenesis in mice small star, filled. Dev Biol. 2003;258(1):209–25.

    Article  PubMed  CAS  Google Scholar 

  45. Ohmura M, Yoshida S, Ide Y, Nagamatsu G, Suda T, Ohbo K. Spatial analysis of germ stem cell development in Oct-4/EGFP transgenic mice. Arch Histol Cytol. 2004;67(4):285–96.

    Article  PubMed  CAS  Google Scholar 

  46. Bhartiya D, Kasiviswanathan S, Unni SK, Pethe P, Dhabalia JV, Patwardhan S, et al. Newer insights into premeiotic development of germ cells in adult human testis using Oct-4 as astem cell marker. J Histochem Cytochem. 2010;58(12):1093–106. doi:10.1369/jhc.2010.956870.

    Article  PubMed  CAS  Google Scholar 

  47. Izadyar F, Wong J, Maki C, Pacchiarotti J, Ramos T, Howerton K, et al. Identification and characterization of repopulating spermatogonial stem cells from the adult human testis. Hum Reprod. 2011;26(6):1296–306. doi:10.1093/humrep/der026.

    Article  PubMed  Google Scholar 

  48. Dann CT, Alvarado AL, Molyneux LA, Denard BS, Garbers DL, Porteus MH. Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation. Stem cells (Dayton, Ohio). 2008;26(11):2928–37. doi:10.1634/stemcells.2008-0134.

    Article  CAS  Google Scholar 

  49. 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(11):6487–92.

    Article  PubMed  CAS  Google Scholar 

  50. Reijo R, Seligman J, Dinulos MB, Jaffe T, Brown LG, Disteche CM, et al. Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty. Genomics. 1996;35(2):346–52. doi:10.1006/geno.1996.0366.

    Article  PubMed  CAS  Google Scholar 

  51. Hermann BP, Sukhwani M, Lin CC, Sheng Y, Tomko J, Rodriguez M, et al. Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques. Stem cells (Dayton, Ohio). 2007;25(9):2330–8. doi:10.1634/stemcells.2007-0143.

    Article  CAS  Google Scholar 

  52. Brinster RL. Germline stem cell transplantation and transgenesis. Science (New York, NY. 2002;296(5576):2174–6.

    Article  CAS  Google Scholar 

  53. McLean DJ. Spermatogonial stem cell transplantation, testicular function, and restoration of male fertility in mice. Methods Mol Biol (Clifton, NJ). 2008;450:149–62.

    Article  CAS  Google Scholar 

  54. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proc Natl Acad Sci USA. 2000;97(15):8346–51.

    Article  PubMed  CAS  Google Scholar 

  55. Bellve AR, Cavicchia JC, Millette CF, O’Brien DA, Bhatnagar YM, Dym M. Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J Cell Biol. 1977;74(1):68–85.

    Article  PubMed  CAS  Google Scholar 

  56. Chiarini-Garcia H, Russell LD. High-resolution light microscopic characterization of mouse spermatogonia. Biol Reprod. 2001;65(4):1170–8.

    Article  PubMed  CAS  Google Scholar 

  57. Morena AR, Boitani C, Pesce M, De Felici M, Stefanini M. Isolation of highly purified type A spermatogonia from prepubertal rat testis. J Androl. 1996;17(6):708–17.

    PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank all the patients who donated tissues for research to the lab of Royan institute. We also appreciate the contributions of H. Sadri-ardekani, S.C. Mizrak, R. Aflatounian, and MR. Hadjighassem for comments; M. Moraveji, H. Baharvand, H. Azizi, P. Eftekhari-Yazdi, T. Mirzapour, M. Soleimani, M. Lotfipanah for technical and administrative support; and MR. Akhoond for data analyzing. This work was supported by a grant from Royan Institute, Tehran, Iran (Number: 158-3).

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Authors and Affiliations

  1. Cellular and Molecular Research Center and Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Hemmat Highway, P.O. Box 14155-5983, Tehran, Iran

    Morteza Koruji

  2. Department of Stem Cells and Developmental Biology, Cell Sciences Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box 19395-4644, Tehran, Iran

    Morteza Koruji & Abdulhossein Shahverdi

  3. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

    Arghavan Janan & Mohammad Reza Lakpour

  4. Cell and Molecular biology Research Center and Department of Biology and Anatomical Sciences, School of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran

    Abbas Piryaei

  5. Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

    Mohammad Ali Gilani Sedighi

  6. Department of Urology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Mohammad Ali Gilani Sedighi

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  1. Morteza Koruji
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  2. Abdulhossein Shahverdi
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  3. Arghavan Janan
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  4. Abbas Piryaei
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  6. Mohammad Ali Gilani Sedighi
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Correspondence to Morteza Koruji.

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Proliferation of SSCs obtained from NOA patients are increased by GDNF, bFGF, EGF and LIF in the presence or absence of laminin.

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Koruji, M., Shahverdi, A., Janan, A. et al. Proliferation of small number of human spermatogonial stem cells obtained from azoospermic patients. J Assist Reprod Genet 29, 957–967 (2012). https://doi.org/10.1007/s10815-012-9817-8

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  • DOI: https://doi.org/10.1007/s10815-012-9817-8

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