- Article Original
- Lignée germinale fœTale mÂle
- Published:
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
Andrologie volume 17, pages 25–41 (2007)
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
ABERCOMBRIE M.: Estimation of nuclear population from microtome sections. Anat. Rec., 1946, 94: 238–248.
ANDERSON R., GARCIA-CASTRO M., HEASMAN J., WYLIE C.: Early stages in males germ cell differentiation in the mouse. APMIS, 1998, 106: 127–133.
ANWAY M.D., MEMON M.A., UZUMCU M., SKINNER M.K.: Transgenerational effect of the endocrine disruptor vinclozolin on male spermatogenesis. J. Androl., 2006.
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.
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.
BEAUMONT H.M., MANDL A.M.: A quantitative study of primordial germ cells in the rat. J. Embryol. Exp. Morph., 1963, 11: 715–740.
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.
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.
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.
BOITANI C., GIUDITTA POLITI M., MENNA T.: Spermatogonial cell proliferation in organ culture of immature rat testis. Biol. Reprod., 1993, 48: 761–767.
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.
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.
BUEHR M., GU S., MCLAREN A.: Mesonephric contribution to testis differentiation in the fetal mouse. Development, 1993, 117:273–281.
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.
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.
DELBES G., LEVACHER C., DUQUENNE C., HABERT R.: Is fetal testis in danger? Med. Sci., 2005, 21: 1083–1088.
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.
DELBES G., LEVACHER C., HABERT R.: Estrogen effects on fetal and neonatal testicular development. Reproduction, 2006, in press.
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.
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.
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.
FUKUDA T., HEDINGER C., GROSCURTH P.: Ultrastructure of developing germ cells in the fetal human testis. Cell Tissue Res., 1975, 161: 55–70.
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.
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.
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.
HABERT R., LEJEUNE H., SAEZ J.M.: Origin, differentiation and regulation of fetal and adult Leydig cells. Mol. Cell. Endocrinol., 2001, 179: 47–74.
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.
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.
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.
JOST A.: Hormonal and genetic factors affecting the development of the male genital system. Andrologia, 1976, 8: 17–33.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
MIGRENNE S., RACINE C., GUILLOU F., HABERT R.: Pituitary hormones inhibit the function and differentiation of fetal Sertoli cells. Endocrinology, 2003, 144: 2617–2622.
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.
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.
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.
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.
OLASO R., HABERT R.: Genetic and cellular analysis of male germ cell development. J. Androl., 2000, 21: 497–511.
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.
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.
ORTH J.: The role of follicle-stimulating hormone in controlling Sertoli cell proliferation in testes of fetal rats. Endocrinology, 1984, 115: 1248–1255.
ORTH J.M.: Proliferation of Sertoli cells in fetal and postnatal rats: a quantitative autoradiographic study. Anat. Rec., 1982, 203:485–492.
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.
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.
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.
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.
ROSS A.J., CAPEL B.: Signaling at the crossroads of gonad development. Trends Endocrinol. Metab., 2005, 16: 19–25.
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.
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.
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.
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.
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.
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.
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.
TOPPARI J., LARSEN J., CHRISTIANSEN P. et al.: Male reproductive health and environmental xenoestrogens. Environ. Health Perspect., 1996, 104: 741–803.
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.
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.
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.
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.
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.
WARTENBERG H.: Differentiation and development of the testes. New York, Raven Press, 1989.
WENIGER J.P.: Steroid secretion by foetal mammal gonads and its regulation by gonadotrophins. Reprod. Nutr. Dev., 1986,26:921–932.
WOLFF E.:Sur la différenciation sexuelle des gonades de souris explantées in vitro. C. R. Hebd. Seances Acad. Sci., 1952,234: 1712–1714
Author information
Authors and Affiliations
Corresponding author
Additional information
Ce travail est dédié à la mémoire de José Maria Saez
Communication présentée au XXIIeme Congrès de la SALF,
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF03041153