Isolation and Culture of Immature Rat Type A Spermatogonial Stem Cells

  • G. Dirami
  • N. Ravindranath
  • M. C. Jia
  • M. Dym
Part of the Ernst Schering Research Foundation Workshop book series (SCHERING FOUND, volume 2/1996)


Spermatogenesis is a complex process of cellular renewal and differentiation that begins with the divisions of the type A spermatogonial stem cells and ends as late spermatids are released into the seminiferous tubule lumen as spermatozoa (Dym 1983). In vivo spermatogonial stem cells can be directed to one of three fates: (1) renew themselves into other stem cells; (2) differentiate into type B spermatogonia and eventually spermatocytes and more mature germ cells; or (3) degenerate. Despite abundant studies on the morphology and kinetics of spermatogonial cell renewal and differentiation (Clermont 1966, 1969; Dym and Clermont 1970; Huckins 1971; Oakberg 1971), very little is known about the regulation of spermatogonial cell proliferation or degeneration in mammals. Attempts to study spermatogonial proliferation in organ cultures have been made by several investigators (Martinovitch 1937, 1939; Steinberger et al. 1964; Ghatnekar et al. 1974; Aizawa and Nishimune 1979; Curtis 1981; Boitani et al. 1993). In all these studies, differentiation stopped at the pachytene spermatocyte stage. Similarly, spermatogonial proliferation and differentiation up to pachytene spermatocytes have been observed in cocultures of spermatogenic cells with Sertoli cells in serum-free defined medium supplemented with hormone and growth factors (Tres and Kierszenbaum 1983; Hadley et al. 1985). However, differentiation beyond pachytene spermatocytes into young spermatids was observed in seminiferous tubule segments cultured for 4–6 days (Parvinen et al. 1983). Thus, it is likely that cellular interactions are important for spermatogonial proliferation and differentiation. However, the Sertoli cell factors which are responsible for regulating this process are not known. It has been observed that up to 75% of spermatogonia undergo spontaneous degeneration before maturation (Abe 1987; Huckins 1978). Although the withdrawal of gonadotropins by hypophysectomy or immunoneutralization enhances the degeneration of germ cells (Raj and Dym 1976; Russell and Clermont 1977), other factors promoting degeneration of germ cells in intact animals have not been identified. In order to examine the regulation of spermatogonial renewal, differentiation, and degeneration under controlled culture conditions, it is important to develop an in vitro model system. In this direction, we have isolated rat type A spermatogonial cells from 9-day-old rats (Dym et al. 1995) and purified them to greater than 95% purity by sedimentation velocity at unit gravity followed by differential plating in fetal bovine serum (FBS)-supplemented medium. The present study was undertaken to standardize culture conditions which would allow the type A spermatogonia to survive for longer periods of time.


Sedimentation Electrophoresis Selenium Germinal Testosterone 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abe SI (1987) Differentiation of spermatogenic cells from vertebrates in vitro. Int Rev Cytol 109:159–209.PubMedCrossRefGoogle Scholar
  2. Aizawa S, Nishimime Y (1979) In-vitro differentiation of type A spermatogonia in mouse cryporchid testis. J Reprod Fertil 56:99–104.PubMedCrossRefGoogle Scholar
  3. Allan DJ, Harmon BV, Roberts SA (1992) Spermatogonial apoptosis has three morphologically recognizable phases and shows no circadian rhythm during normal spermatogenesis in the rat. Cell Prolif 25:241–250.PubMedCrossRefGoogle Scholar
  4. Bellvé AR, Cavicchia JC, Millette CF, O’Brien DA, Bhatnagar YM, Dym M (1977) Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization. J Cell Biol 74:68–85.PubMedCrossRefGoogle Scholar
  5. Boitani C, Politi MG, Menna T (1993) Spermatogonial cell proliferation in organ culture of immature rat testis. Biol Reprod 48:761–767.PubMedCrossRefGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.PubMedCrossRefGoogle Scholar
  7. Clermont Y (1966) Renewal of spermatogonia in man. Am J Anat 118:509–524.PubMedCrossRefGoogle Scholar
  8. Clermont Y (1969) Two classes of spermatogonial stem cells in the monkey (Cercopithecus aethiops). Am J Anat 126:57–72.PubMedCrossRefGoogle Scholar
  9. Compton MM (1992) A biochemical hallmark of apoptosis: internucleosomal degradation of the genome. Cancer Metastasis Rev 11:105–119.PubMedCrossRefGoogle Scholar
  10. Curtis D (1981) In-vitro differentiation of diakinesis figures in human testis. Hum Genet 59:406–411.PubMedCrossRefGoogle Scholar
  11. Dirami G, Ravindranath N, Kleinman HK, Dym M (1995) Evidence that basement membrane prevents apoptosis of Sertoli cells in vitro in the absence of known regulators of Sertoli cell function. Endocrinology 136:4439–4447.PubMedCrossRefGoogle Scholar
  12. Dolci S, Williams DE, Ernst MK, Resnick JL, Brannan CI, Lock LF, Lyman SD, Boswell HS, Donovan PJ (1991) Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature 352:809–811.PubMedCrossRefGoogle Scholar
  13. Dym M (1983) The male reproductive system. In: Weiss L (ed) Histology: cell and tissue biology. Elsevier Biomedical, New York, pp 1000–1053.Google Scholar
  14. Dym M (1994) Spermatogonial stem cells of the testis. Proc Natl Acad Sci USA 91:11287–11289.PubMedCrossRefGoogle Scholar
  15. Dym M, Clermont Y (1970) Role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of the rat testis. Am J Anat 128:265–282.PubMedCrossRefGoogle Scholar
  16. Dym M, Fawcett DW (1970) The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod 3:308–326.PubMedGoogle Scholar
  17. Dym M, Jia MC, Dirami G, Price JM, Rabin SJ, Mocchetti I, Ravindranath N (1995) Expression of c-kit receptor and its phosphorylation in immature rat type A spermatogonia. Biol Reprod 52:8–19.PubMedCrossRefGoogle Scholar
  18. Ghatnekar R, Lima-De-Faria A, Rubin S, Menander K (1974) Development of human male meiosis in vitro. Hereditas 78:265–272.PubMedCrossRefGoogle Scholar
  19. Hadley MA, Byers SW, Suarez-Quian CA, Kleinman HK, Dym M (1985) Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation, and germ cell development in vitro. J Cell Biol 101:1511–1522.PubMedCrossRefGoogle Scholar
  20. Huang E, Nocka K, Beier DR, Chu T-Y, Buck J, Lahm H-W, Wellner D, Leder P, Besmer P (1990) The hematopoietic growth factor KL is encoded by the S1 locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell 63:225–233.PubMedCrossRefGoogle Scholar
  21. Huckins C (1971) The spermatogonial stem cell population in adult rats. 1. Their morphology, proliferation and maturation. Anat Rec 169:533–558.PubMedCrossRefGoogle Scholar
  22. Huckins C (1978) The morphology and kinetics of spermatogonial degeneration in normal adult rats: an analysis using a simplified classification of the germinal epithelium. Anat Rec 190:905–926.PubMedCrossRefGoogle Scholar
  23. Lam D, Furrer R, Bruce WR (1970) The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse. Proc Natl Acad Sci USA 65:192–199.PubMedCrossRefGoogle Scholar
  24. Leigh BR, Khan W, Hancock SL, Knox SJ (1995) Stem cell factor enhances the survival of murine intestinal stem cells after photon irradiation. Radiat Res 142:12–15.PubMedCrossRefGoogle Scholar
  25. Manova K, Nocka K, Besmer P, Bachvarova RF (1990) Gonadal expression of c-kit encoded at the W locus of the mouse. Development 110:1057–1069.PubMedGoogle Scholar
  26. Martinovitch PN (1937) Development in-vitro of mamalian gonad. Nature 139:413–415.CrossRefGoogle Scholar
  27. Martinovitch PN (1939) Development in-vitro of mamalian gonad. Arch Exp Zellforsch 22:74–76.Google Scholar
  28. McNiece IK, Briddell RA (1995) Stem cell factor. J Leukoc Biol 57:14–22.Google Scholar
  29. Meistrich ML (1972) Separation of mouse spermatogenic cells by velocity sedimentation. J Cell Physiol 80:299–312.PubMedCrossRefGoogle Scholar
  30. Mosman TR, Fong TAT (1989) Specific assays for cytokine production by T cells. J Immunol Methods 116:151–158.CrossRefGoogle Scholar
  31. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival. J Immunol Methods 65:55–63.PubMedCrossRefGoogle Scholar
  32. Oakberg EF (1971) Spermatogonial stem-cell renewal in the mouse. Am J Anat 169:515–532.Google Scholar
  33. Ohno M, Abe T (1995) Rapid colonmetric assay for the quantification of leukemia inhibility factor (LIF) and interleukin-6 (IL-6). J Immunol Methods 145:199–203.CrossRefGoogle Scholar
  34. Parvinen M, Wright WW, Phillips DM, Mather JP, Musto NA, Bardin CW (1983) Spermatogenesis in vitro: completion of meiosis and early sper-miogenesis. Endocrinology 112:1150–1152.PubMedCrossRefGoogle Scholar
  35. Pesce M, Farrace MG, Piacentini M, Dolci S, De Felici M (1993) Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death. Development 118:1089–1094.PubMedGoogle Scholar
  36. Raj HGM, Dym M (1976) The effects of selective withdrawal of FSH or LH on spermatogenesis in the immature rat. Biol Reprod 14:489–494.Google Scholar
  37. Romrell LJ, Bellve AR, Fawcett DW (1976) Separation of mouse sperma-togenic cells by sedimentation velocity. A morphological characterization. Dev Biol 49:119–131.PubMedCrossRefGoogle Scholar
  38. Rossi P, Albanesi C, Grimaldi P, Geremia R (1991) Expression of the mRNA for the ligand of c-kit in mouse Sertoli cells. Biochem Biophys Res Commun 176:910–914.PubMedCrossRefGoogle Scholar
  39. Rossi P, Dolci S, Albanesi C, Grimaldi P, Ricca R, Geremia R (1993) Follicle-stimulating hormone induction of steel factor (SLF) mRNA in mouse Sertoli cells and stimulation of DNA synthesis in spermatogonia by soluble SLF. Dev Biol 155:68–74.PubMedCrossRefGoogle Scholar
  40. Russell LD (1980) Sertoli-germ cell interrelations: a review. Gamete Res 3:179–302.CrossRefGoogle Scholar
  41. Russell LD (1995) Morphological and functional evidence for Sertoli-germ cell relationships. In: Russell LD, Griswold MD (eds) The Sertoli cell. Cache River Press, Clearwater, pp 365–390.Google Scholar
  42. Russell LD, Clermont Y (1977) Degeneration of germ cells in normal, hypo-physectomized, and hormone treated hypophysectomized rats. Anat Rec 187:347–366.PubMedCrossRefGoogle Scholar
  43. Skinner MK (1993) Secretion of growth factors and other regulator factors. In: Rüssel LD, Griswold MD (eds) The Sertoli cells. Cache River Press, Clearwater, pp 237–248.Google Scholar
  44. Sorrentino V, Giorgi M, Geremia R, Besmer P, Rossi P (1991) Expression of the c-kit proto-oncogene in the murine male germ cells. Oncogene 6:149–151.PubMedGoogle Scholar
  45. Steinberger A, Steinberger E (1966) In-vitro culture of rat testicular cells. Exp Cell Res 44:443–452.PubMedCrossRefGoogle Scholar
  46. Steinberger A, Steinberger E, Perloff WH (1964) Mammalian testes in organ culture. Exp Cell Res 36:19–27.PubMedCrossRefGoogle Scholar
  47. Telford WG, King LE, Franker PJ (1992) Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 13:137–143.PubMedCrossRefGoogle Scholar
  48. Tres LL, Kierszenbaum AL (1983) Viability of rat spermatogenic cells is facilitated by their coculture with Sertoli cells in serum-free hormone supplemented medium. Proc Natl Acad Sci USA 80:3377–3381.PubMedCrossRefGoogle Scholar
  49. Tres LL, Smith EP, VanWyk JJ, Kierszenbaum AL (1986) Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures. Exp Cell Res 162:33–50.PubMedCrossRefGoogle Scholar
  50. Vistica DT, Skehan P, Scudiero D, Monks A, Pittman A, Boyd MR (1991) Te-trazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res 51:2515–2520.PubMedGoogle Scholar
  51. Williams DE, Eisenman J, Baird A, Rauch C, VanNess K, March CJ, Park LS, Martin U, Mochizuki DY, Boswell HS, Burgess GS, Cosman D, Lyman SD (1990) Identification of a ligand for the c-kit proto-oncogene. Cell 63:167–174.PubMedCrossRefGoogle Scholar
  52. Yee NS, Paek I, Besmer P (1994) Role of kit-ligand in proliferation and suppression of apoptosis in mast cells: basis for radiosurvivability of white spotting and steel mutant mice. J Exp Med 179:1777–1787.PubMedCrossRefGoogle Scholar
  53. Yoshinaga K, Nishikawa S, Ogawa M, Hayashi S, Kunisada T, Fujimoto T (1991) Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 113:689–699.PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1996

Authors and Affiliations

  • G. Dirami
  • N. Ravindranath
  • M. C. Jia
  • M. Dym

There are no affiliations available

Personalised recommendations