Hervé Coffigny

Phthalates Impair Germ Cell Development in the Human Fetal Testis in Vitro without Change in Testosterone Production

Background Several studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. Phthalate esters represent a class of environmental endocrine-active chemicals known to disrupt development of the male reproductive tract by decreasing testosterone production in the fetal rat. Objectives Using the organ culture system we developed previously, we investigated the effects on the development of human fetal testis of one phthalate—mono-2-ethylhexyl phthalate (MEHP)—an industrial chemical found in many products, which has been incriminated as a disruptor of male reproductive function. Methods Human fetal testes were recovered during the first trimester (7–12 weeks) of gestation, a critical period for testicular differentiation, and cultured for 3 days with or without MEHP in basal conditions or stimulated with luteinizing hormone (LH). Results Whatever the dose, MEHP treatment had no effect on basal or LH-stimulated testosterone produced by the human fetal testis in vitro, although testosterone production can be modulated in our culture system. MEHP (10−4 M) did not affect proliferation or apoptosis of Sertoli cells, but it reduced the mRNA expression of anti-Müllerian hormone. MEHP (10−4 M) reduced the number of germ cells by increasing their apoptosis, measured by the detection of caspase-3–positive germ cells, without modification of their proliferation. Conclusions This is the first experimental demonstration that phthalates alter the development of the germ cell lineage in humans. However, in contrast to results observed in the rat, phthalates did not affect steroidogenesis.

p63 null mutation protects mouse oocytes from radio-induced apoptosis.

Female fertility in mammals is determined by the pool of primordial follicles and low doses of radiation induce a major loss of primordial follicles in the ovary. We investigated the expression of p53 and its homologues, p63 and p73, in the normal and irradiated neonatal ovary. p63 was the only member of the p53 family detected in oocyte nucleus. No p63 transcripts or protein were detected in the early foetal ovary. p63 production began in late pachytene-stage oocytes and peaked in diplotene oocytes in mice and humans. The production of p63 was correlated with meiotic DNA double-strand break repair. Only transactivation (TA) isoforms were present in the ovary, with TAp63 alpha by far the most abundant in terms of mRNA and protein levels. Complete p63 null mutation did not affect normal ovary development. Irradiation rapidly triggered p63 phosphorylation. p63 null mutation prevented the cleavage of caspases-9 and -3 and the follicle loss induced by ionising radiation. Thus, our results evidence that irradiation-induced depletion of the primordial follicle pool results from the activation of p63 in quiescent oocytes.

Cadmium Increases Human Fetal Germ Cell Apoptosis

Background Cadmium (Cd) is a common environmental pollutant and a major constituent of tobacco smoke. Adverse effects of this heavy metal on reproductive function have been identified in adults; however, no studies have examined its effects on human reproductive organs during development. Objectives Using our previously developed organ culture system, we investigated the effects of cadmium chloride on human gonads at the beginning of fetal life, a critical stage in the development of reproductive function. Methods Human fetal gonads were recovered during the first trimester (7–11 weeks postconception) and cultured with or without Cd. We used different concentrations of Cd and compared results with those obtained with mouse fetal gonads at similar stages. Results Cd, at concentrations as low as 1 μM, significantly decreased the germ cell density in human fetal ovaries. This correlated with an increase in germ cell apoptosis, but there was no effect on proliferation. Similarly, in the human fetal testis, Cd (1 μM) reduced germ cell number without affecting testosterone secretion. In mouse fetal gonads, Cd increased only female germ cell apoptosis. Conclusions This is the first experimental demonstration that Cd, at low concentrations, alters the survival of male and female germ cells in humans. Considering data demonstrating extensive human exposure, we believe that current environmental levels of Cd could be deleterious to early gametogenesis.

The role of p63 in germ cell apoptosis in the developing testis

The fetal and neonatal development of male germ cells (gonocytes) is a poorly understood but crucial process for establishing fertility. In rodents, gonocytes go through two phases of proliferation accompanied by apoptosis and separated by a quiescent period during the end of fetal development. P63 is a member of the P53 gene family that yields six isoforms. We detected only the p63 protein and no p53 and p73 in the nucleus of the gonocytes of mouse testes. We report for the first time the ontogeny of each p63 mRNA isoform during testis development. We observed a strong expression of p63γ mRNA and protein when gonocytes are in the quiescent period. In vitro treatment with retinoic acid prevented gonocytes from entering the quiescent period and was correlated with a reduced production of p63γ isoform mRNA. We investigated the function of p63 by studying the testicular phenotype of P63‐null mice. P63 invalidation slightly, but significantly increased the number of gonocytes counted during the quiescent period. As P63‐null animals die at birth we used an original organ culture that mimicked neonatal in vivo development to study further the testicular development. P63 invalidation resulted in a sharply increased number of gonocytes during the culture period due to a decrease in spontaneous apoptosis with no change in proliferation. P63 invalidation also caused abnormal morphologies in the germ cells that were also found in P63+/− adult male mice. Thus, p63 appears as an important regulator of germ cell development. J. Cell. Physiol. 210: 87–98, 2007.

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

ResumeLe 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.AbstractThe 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.