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Développementin vitro de la lignée germinale foetale mâle chez le rat, la souris et l’homme

In vitro development of foetal male germ cells in rats, mice and humans

Resume

Le potentiel reproducteur de l’adulte dépend, en partie, de la mise en place de la lignée germinale pendant la vie fœtale et néonatale. Une hypothèse récente assez largement partagée suggère que l’augmentation des altérations de la reproduction masculine observée au cours des dernières décennies, comme la diminution de la production spermatique et l’augmentation de l’incidence des cancers testiculaires, résulterait de modifications du développement de la lignée germinale pendant la vie fœtale et néonatale en réponse à l’augmentation de la pollution environnementale. Cependant peu d’outils sont disponibles pour étudier le développement de la lignée germinale fœtale et néonatale.

Nous décrivons ici un système de culture organotypique dans lequel le testicule se développe sur un filtre flottant à la surface d’un milieu synthétique ne contenant ni sérum ni facteurs biologiques. Chez le rat et la souris, nous avons comparé le développement des cellules de Sertoli et des cellules germinates dans ce système avec leur développement observéin vivo. Ces cellules se développent normalement chez le rat sur une période de deux semaines. Moins de cellules sont produites qu’in vivo mais les fonctions de chaque cellule sont comparables. Des résultats similaires ont été observés chez la souris, mais la durée de maintienin vitro est plus courte que chez le rat et ce sont les stades fœtaux les plus jeunes qui donnent les meilleurs résultats. En utilisant ce modèle, nous avons pu étudier le développement de la lignée germinale de testicules prélevés immédiatement avant la naissance sur des fœtus invalidés pour p63, un gène requis pour la survie postnatale, et montrer que p63 est impliqué dans le contrôle de l’apoptose néonatale de la lignée germinale. Enfin, nous avons étendu ce modèle de culture à l’espèce humaine (6 à 12 semaines de grossesse) et montré que l’on peut maintenir l’architecture testiculaire et les cellules germinates pendant 4 jours avec une efficacité supérieure pour les stades jeunes (moins de 8 semaines).

En conclusion, ce modèle est potentiellement très intéressant pour étudier l’effet de facteurs physiologiques ou toxiques sur la mise en place de la lignée germinale chez le mâle.

Abstract

The key role of the foetal germ cell line in the reproductive capacity of the adult has been known for a long time. More recently, the observed increase in male reproductive disorders such as the decline of sperm count and quality and the increased incidence of testicular cancer has been postulated to be due to alterations of foetal and neonatal testicular development in response to increasing environmental pollution. However, few tools are available to study foetal and neonatal germ cell line development and the effects of physiological or toxic substances on this process.

The authors have developed an organ culture system in which foetal or neonatal testis is grown on a filter floating on a synthetic medium free of serum, hormones or biological factors. This study, using rats and mice, first compared the long-term morphological and functional development of Sertoli and germ cells in thisin vivo system. In rats, these cells developed normally over a period of two weeksin vitro. Fewer cells were produced thanin vivo, but a similar level of differentiated function was observed. Germ cells, which are difficult to maintainin vitro, resumed mitosis after a quiescent period, at the same time asin vivo. Similar results were obtained with mouse fetuses, but this model was less efficient.

This culture model can be used to study post-natal development of the germ cell lineage in testes derived from foetuses on the last day of foetal life and invalidated for P63, that do not survive after birth. This gene was found to be involved in the regulation of germ apoptosis which resumes after birth in the mouse. Lastly, this model applied to the human species (from 6 to 12 weeks of gestation) showed that testicular architecture and germ cells can be maintained for 4 days with better efficiency at younger stages than at older stages. p]In conclusion, testicular architecture and intercellular communications are sufficiently preserved to sustain gametogenesisin vitro with no added factors. This method is potentially useful to study the effects of various factors, particularly xenobiotics.

References

  1. 1.

    ABERCOMBRIE M.: Estimation of nuclear population from microtome sections. Anat. Rec., 1946, 94: 238–248.

    Google Scholar 

  2. 2.

    ANDERSON R., GARCIA-CASTRO M., HEASMAN J., WYLIE C.: Early stages in males germ cell differentiation in the mouse. APMIS, 1998, 106: 127–133.

    PubMed  CAS  Google Scholar 

  3. 3.

    ANWAY M.D., MEMON M.A., UZUMCU M., SKINNER M.K.: Transgenerational effect of the endocrine disruptor vinclozolin on male spermatogenesis. J. Androl., 2006.

  4. 4.

    AUGER J., KUNSTMANN J.M., CZYGLIK F., JOUANNET P.: Decline in semen quality among fertile men in Paris during the past 20 years. N. Engl. J. Med., 1995, 332: 281–285.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    BAKER P.J., O’SHAUGHNESSY P.J.: Role of gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development in mice. Reproduction, 2001, 122:227–234.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    BEAUMONT H.M., MANDL A.M.: A quantitative study of primordial germ cells in the rat. J. Embryol. Exp. Morph., 1963, 11: 715–740.

    PubMed  CAS  Google Scholar 

  7. 7.

    BEGEOT M., LANGLOIS D., PENHOAT A., SAEZ J.M.: Variations in guanine-binding proteins (Gs, Gi) in cultured bovine adrenal cells. Consequences on the effects of phorbol ester and angiotensin II on adrenocorticotropin-induced and cholera-toxin-induced cAMP production. Eur. J. Biochem., 1988, 174:317–321.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    BENDSEN E., BYSKOV A.G., LAURSEN S.B., LARSEN H.P., ANDERSEN C.Y., WESTERGAARD L.G.: Number of germ cells and somatic cells in human fetal testes during the first weeks after sex differentiation. Hum. Reprod.,2003,18: 13–18.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    BENDSEN E., LAURSEN S., OLESEN C., WESTERGAARD L., ANDERSEN C., BYSKOV A.: Effect of 4-ocylphenol on germ cell number in cultured human fetal gonads. Hum. Reprod., 2001, 16:336–343.

    Article  Google Scholar 

  10. 10.

    BOITANI C., GIUDITTA POLITI M., MENNA T.: Spermatogonial cell proliferation in organ culture of immature rat testis. Biol. Reprod., 1993, 48: 761–767.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    BOULOGNE B., HABERT R., LEVACHER C.: Regulation of the proliferation of cocultured gonocytes and Sertoli cells by retinoids, triiodothyronine, and intracellular signaling factors: differences between fetal and neonatal cells. Mol. Reprod. Dev., 2003, 65: 194–203.

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    BOULOGNE B., OLASO R., LEVACHER C., DURAND P., HABERT R.: Apoptosis and mitosis in gonocytes of the rat testis during foetal and neonatal development. Int. J. Androl., 1999,22:356–365.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    BUEHR M., GU S., MCLAREN A.: Mesonephric contribution to testis differentiation in the fetal mouse. Development, 1993, 117:273–281.

    PubMed  CAS  Google Scholar 

  14. 14.

    CUPPA A., DUFOUR J., KIM G., SKINNER M., KIM K.: Action of retinoids on embryonic and early postnatal testis development. Endocrinology, 1999, 140: 2343–2352.

    Article  Google Scholar 

  15. 15.

    De SOUSA LOPES S.M., ROELEN B.A., MONTEIRO R.M. et al.: BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes Dev., 2004, 18:1838–1849.

    PubMed  Article  Google Scholar 

  16. 16.

    DELBES G., LEVACHER C., DUQUENNE C., HABERT R.: Is fetal testis in danger? Med. Sci., 2005, 21: 1083–1088.

    Google Scholar 

  17. 17.

    DELBES G., LEVACHER C., DUQUENNE C., RACINE C., PAKARINEN P., HABERT R.: Endogenous estrogens inhibit mouse fetal Leydig cell development via estrogen receptor alpha. Endocrinology, 2005, 146: 2454–2461.

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    DELBES G., LEVACHER C., HABERT R.: Estrogen effects on fetal and neonatal testicular development. Reproduction, 2006, in press.

  19. 19.

    DELBES G., LEVACHER C., PAIRAULT C. et al.: Estrogen receptor (beta)-mediated inhibition of male germ cell line development in mice by endogenous estrogens during perinatal life. Endocrinology, 2004, 145: 3395–3403.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    ENDERS G.C., MAY J.J. 2ND.: Developmentally regulated expression of a mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and female mice. Dev. Biol., 1994, 163:331–340.

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    EVTOUCHENKO L., STUDER L., SPENGER C., DREHER E., SEILER R.W.: A mathematical model for the estimation of human embryonic and fetal age. Cell Transplant., 1996, 5: 453–464.

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    FUKUDA T., HEDINGER C., GROSCURTH P.: Ultrastructure of developing germ cells in the fetal human testis. Cell Tissue Res., 1975, 161: 55–70.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    GASKELL T.L., ESNAL A., ROBINSON L.L., ANDERSON R.A., SAUNDERS P.T.: Immunohistochemical profiling of germ cells within the human fetal testis: identification of three subpopulations. Biol. Reprod., 2004, 71: 2012–2021.

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    GONDOS B.: Development and differentiation of the testis and male reproductive tract. In: Steinberger A., Steinberger E. eds. Testicular development, structure, and function. New York, Raven Press, 1980: 3–20.

    Google Scholar 

  25. 25.

    HABERT R., DEVIF I., GANGNERAU M.N., LECERF L.: Ontogenesis of the in vitro response of rat testis to gonadotropin-releasing hormone. Mol. Cell. Endocrinol., 1991, 82: 199–206.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    HABERT R., LEJEUNE H., SAEZ J.M.: Origin, differentiation and regulation of fetal and adult Leydig cells. Mol. Cell. Endocrinol., 2001, 179: 47–74.

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    HILSCHER B., HILSCHER W., BULTHOFF-OBNOLZ B. et al.: Kinetics of gametogenesis. I. Comparative histological and autoradiographic studies of oocytes and transitional prospermatogonia during oogenesis and prespermatogenesis. Cell Tissue Res., 1974, 154: 443–470.

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    JOHNSTON H., BAKER P.J., ABEL M. et al.: Regulation of Sertoli cell number and activity by follicle-stimulating hormone and androgen during postnatal development in the mouse. Endocrinology, 2004, 145: 318–329.

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    JOST A.: Données préliminaires sur les stades initiaux de la différenciation du testicule chez le rat. Arch. Anat. Micr. Morphol. Exp., 1972, 61: 415–438.

    Google Scholar 

  30. 30.

    JOST A.: Hormonal and genetic factors affecting the development of the male genital system. Andrologia, 1976, 8: 17–33.

    Google Scholar 

  31. 31.

    JOST A., MAGRE S.: Sexual differentiation. In: Thibault C., Levasseur M., Hunter R.H.F. eds. Reproduction in Mammals and Man. Paris, Ellipses, 1993: 197–226.

    Google Scholar 

  32. 32.

    LAMBROT R., COFFIGNY H., PAIRAULT C. et al.: Use of organ culture to study the human fetal testis development: effect of retinoic acid. J. Clin. Endocrinol. Metab., 2006, 91: 2696–2703.

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Le GNMAGUERESSE B., PINEAU C., GUILLOU F., JEGOU B.: Influence of germ cells upon transferrin secretion by rat Sertoli cells in vitro. J. Endocrinol., 1988, 118: R13-R16.

    Article  Google Scholar 

  34. 34.

    LECERF L., ROUILLER-FABRE V., LEVACHER C., GAUTIER C., SAEZ J., HABERT R.: Stimulatory effect of follicle-stimulating hormone on basal and luteinizing hormone-stimulated testosterone secretion by fetal rat testis in vitro. Endocrinology, 1993, 133: 2313–2318.

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    LI H., KIM K.H.: Effects of mono-(2-ethylhexyl) phthalate on fetal and neonatal rat testis organ cultures. Biol. Reprod., 2003, 69: 1964–1972.

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    LI H., PAPADAPOULOS V., VIDIC B., DYM M., CULTY M.: Regulation of rat testis gonocyte proliferation by platelet-derived growth factor and estradiol: identification of signaling mechanisms involved. Endocrinology, 1997,138:1289–1298.

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    LIVERA G., DELBES G., PAIRAULT C., ROUILLER-FABRE V., HABERT R.: Organotypic culture, a powerful model for studying rat and mouse fetal testis development. Cell Tissue Res., 2006, 324:507–521.

    PubMed  Article  Google Scholar 

  38. 38.

    LIVERA G., PAIRAULT C., LAMBROT R. et al.: Retinoid-sensitive steps in steroidogenesis in fetal and neonatal rat testes: in vitro and in vivo studies. Biol. Reprod.,2004, 70: 1814–1821.

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    LIVERA G., ROUILLER-FABRE V., DURAND P., HABERT R.: Multiple effects of retinoids on the development of Sertoli, germ and Leydig cells of fetal and neonatal rat testis in culture. Biol. Reprod., 2000, 62: 1303–1314.

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    LIVERA G., ROUILLER-FABRE V., HABERT R.: Retinoid receptors involved in the effects of retinoic acid on rat testis development. Biol. Reprod., 2001, 64: 1307–1314.

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    MAGRE S., JOST A.: The initial phases of testicular organogenesis in the rat. An electron microscopy study. Arch. Anat. Micr. Morphol. Exp., 1980, 69: 297–318.

    CAS  Google Scholar 

  42. 42.

    MANNAERTS B., DELEEUV R., GEELEN J. et al.: Comparative in vitro and in vivo studies on biological characteristics of recombinant human Follicle-Stimulating Hormone. Endocrinology, 1991, 129: 2623–2630.

    PubMed  CAS  Google Scholar 

  43. 43.

    MIGRENNE S., RACINE C., GUILLOU F., HABERT R.: Pituitary hormones inhibit the function and differentiation of fetal Sertoli cells. Endocrinology, 2003, 144: 2617–2622.

    PubMed  Article  CAS  Google Scholar 

  44. 44.

    MILLS A.A., ZHENG B., WANG X.J., VOGEL H., ROOP D.R., BRADLEY A.: p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature, 1999, 398: 708–713.

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    NAGANO R., TABATA S., NAKANISHI Y., OHSAKO S., KUROHMARU M., HAYASHI Y.: Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis. Anat. Rec., 2000, 258: 210–220.

    PubMed  CAS  Google Scholar 

  46. 46.

    O’SHAUGHNESSY P., BAKER U., SOHNIUS U., HAAVISTO A.M., CHARLTON H., HUHTANIEMI I.: Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function. Endocrinology, 1998, 139:1141–1146.

    PubMed  Article  Google Scholar 

  47. 47.

    OHTA H., WAKAYAMA T., NISHIMUNE Y.: Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development. Biol. Reprod., 2004, 70: 1286–1291.

    PubMed  Article  CAS  Google Scholar 

  48. 48.

    OLASO R., HABERT R.: Genetic and cellular analysis of male germ cell development. J. Androl., 2000, 21: 497–511.

    PubMed  CAS  Google Scholar 

  49. 49.

    OLASO R., PAIRAULT C., BOULOGNE B., DURAND P., HABERT R.: Transforming Growth Factor β1 and β2 reduce the number of gonocytes by increasing apoptosis. Endocrinology, 1998, 139:733–740.

    PubMed  Article  CAS  Google Scholar 

  50. 50.

    OMEZZINE A., CHATER S., MAUDUIT C. et al.: Long-term apoptotic cell death process with increased expression and activation of caspase-3 and -6 in adult rat germ cells exposed in utero to flutamide. Endocrinology, 2003, 144: 648–661.

    PubMed  Article  CAS  Google Scholar 

  51. 51.

    ORTH J.: The role of follicle-stimulating hormone in controlling Sertoli cell proliferation in testes of fetal rats. Endocrinology, 1984, 115: 1248–1255.

    PubMed  CAS  Article  Google Scholar 

  52. 52.

    ORTH J.M.: Proliferation of Sertoli cells in fetal and postnatal rats: a quantitative autoradiographic study. Anat. Rec., 1982, 203:485–492.

    PubMed  Article  CAS  Google Scholar 

  53. 53.

    PAZ G.F., THLIVERIS J.A., WINTER J.S., REYES I.F., FAIMAN C.: Hormonal control of testosterone secretion by the fetal rat testis in organ culture. Biol. Reprod., 1980, 23: 1087–1095.

    PubMed  Article  CAS  Google Scholar 

  54. 54.

    PETRE-LAZAR B., LIVERA G., MORENO S.G. et al.: The role of p63 in germ cell apoptosis in the developing testis. J. Cell Physiol., 2006, in press.

  55. 55.

    POINTIS G., MAHOUDEAU J.A.: [Testosterone production by embryonic testis of mouse in organ culture]. C. R. Acad. Sci. Hebd. Seances Acad. Sci. D, 1974, 279: 1197–1200.

    PubMed  CAS  Google Scholar 

  56. 56.

    ROBINSON L.L., TOWNSEND J., ANDERSON R.A.: The human fetal testis is a site of expression of neurotrophins and their receptors: regulation of the germ cell and peritubular cell population. J. Clin. Endocrinol. Metab., 2003, 88: 3943–3951.

    PubMed  Article  CAS  Google Scholar 

  57. 57.

    ROSS A.J., CAPEL B.: Signaling at the crossroads of gonad development. Trends Endocrinol. Metab., 2005, 16: 19–25.

    PubMed  Article  CAS  Google Scholar 

  58. 58.

    ROUILLER-FABRE V., LECERF L., GAUTIER C., SAEZ J.M., HABERT R.: Expression and effect of Insulin-like Growth Factor I on rat fetal Leydig cell function and differentiation. Endocrinology, 1998, 139: 2926–2934.

    PubMed  Article  CAS  Google Scholar 

  59. 59.

    SCHLATT S., ZHENGWEI Y., MEEHAN T., De KRETSER D.M., LOVELAND K.L.: Application of morphometric techniques to postnatal rat testes in organ culture: insights into testis growth. Cell. Tissue Res., 1999, 298: 335–343.

    PubMed  Article  CAS  Google Scholar 

  60. 60.

    SCHLUTER C., DUCHROW M., WOHLENBERG C. et al.: The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J. Cell. Biol., 1993, 123: 513–522.

    PubMed  Article  CAS  Google Scholar 

  61. 61.

    SHARPE R.M., IRVINE D.S.: How strong is the evidence of a link between environmental chemicals and adverse effects on human reproductive health? Br. Med. J., 2004, 328:447–451.

    Article  CAS  Google Scholar 

  62. 62.

    SHARPE R.M., SKAKKEBAEK N.E.: Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet, 1993, 341: 1392–1395.

    PubMed  Article  CAS  Google Scholar 

  63. 63.

    SKAKKEBAEK N.E., RAJPERT-DE MEYTS E., MAIN K.M.: Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum. Reprod., 2001, 16:972–978.

    PubMed  Article  CAS  Google Scholar 

  64. 64.

    STORGAARD L., BONDE J.R., OLSEN J.: Male reproductive disorders in humans and prenatal indicators of estrogen exposure. A review of published epidemiological studies. Reprod. Toxicol., 2006, 21: 4–15.

    PubMed  Article  CAS  Google Scholar 

  65. 65.

    TOPPARI J., LARSEN J., CHRISTIANSEN P. et al.: Male reproductive health and environmental xenoestrogens. Environ. Health Perspect., 1996, 104: 741–803.

    PubMed  Article  CAS  Google Scholar 

  66. 66.

    VAN DISSEL-EMILIANI F.M.F., DE BOER-BROUWER M., DE ROOIJ D.G.: Effect of Fibroblast Growth Factor-2 on Sertoli cells and gonocytes in coculture during the perinatal period. Endocrinology, 1996, 137: 647–654.

    PubMed  Article  Google Scholar 

  67. 67.

    VAN DISSEL-EMILIANI F.M.F., DE BOER-BROUWER M., SPEK E.R., VAN DER DONK J.A., DE ROOIJ D.G.: Survival and proliferation of rat gonocytes in vitro. Cell. Tissue Res., 1993,273:141–147.

    PubMed  Article  Google Scholar 

  68. 68.

    VERGOUWEN R.P., JACOBS S.G., HUISKAMP R., DAVIDS J.A., DE ROOIJ D.G.: Proliferative activity of gonocytes, Sertoli cells and interstitial cells during testicular development in mice. J. Reprod. Fertil., 1991, 93: 233–243.

    PubMed  CAS  Google Scholar 

  69. 69.

    VIGIER B., TRAN D., DU mesnil du buisson F., HEYMAN Y., JOSSO N.: Use of monoclonal antibody techniques to study the ontogeny of bovine anti-Müllerian hormone. J. Reprod. Fert., 1983, 69: 207–214.

    CAS  Article  Google Scholar 

  70. 70.

    VOS J.G., DYBING E., GREIM H.A. et al.: Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit. Rev. Toxicol., 2000, 30:71–133.

    PubMed  Article  CAS  Google Scholar 

  71. 71.

    WARTENBERG H.: Differentiation and development of the testes. New York, Raven Press, 1989.

    Google Scholar 

  72. 72.

    WENIGER J.P.: Steroid secretion by foetal mammal gonads and its regulation by gonadotrophins. Reprod. Nutr. Dev., 1986,26:921–932.

    PubMed  Article  CAS  Google Scholar 

  73. 73.

    WOLFF E.:Sur la différenciation sexuelle des gonades de souris explantées in vitro. C. R. Hebd. Seances Acad. Sci., 1952,234: 1712–1714

    PubMed  CAS  Google Scholar 

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Correspondence to Gabriel Livera or Romain Lambrot or René Frydman or Hervé Coffigny or Catherine Pairault or Béetrice Petre-Lazar or StéPhanie G. Moreno or Virginie Rouiller-Fabre or René Habert.

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Ce travail est dédié à la mémoire de José Maria Saez

Communication présentée au XXIIeme Congrès de la SALF,

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Livera, G., Lambrot, R., Frydman, R. et al. Développementin vitro de la lignée germinale foetale mâle chez le rat, la souris et l’homme. Androl. 17, 25–41 (2007). https://doi.org/10.1007/BF03041153

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Mots clés

  • cellules germinates
  • fœtus
  • culture
  • développement
  • testicule

Key-Words

  • germ cells
  • foetus
  • culture
  • development
  • testis