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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

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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.

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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.

Additional information

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) doi:10.1007/BF03041153

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Mots clés

  • cellules germinates
  • fœtus
  • culture
  • développement
  • testicule

Key-Words

  • germ cells
  • foetus
  • culture
  • development
  • testis