Romain Lambrot

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.

Adverse effects of endocrine disruptors on the foetal testis development: focus on the phthalates.

There are great concerns about the increasing incidence of abnormalities in male reproductive function. Human sperm counts have markedly dropped and the rate of testicular cancer has clearly augmented over the past four decades. Moreover, the prevalence rates of cryptorchidism and hypospadias are also probably increasing. It has been hypothesized that all these adverse trends in male reproduction result from abnormalities in the development of the testis during foetal and neonatal life. Furthermore, many recent epidemiological, clinical and experimental data suggest that these male reproductive disorders could be due to the effects of xenobiotics termed endocrine disruptors, which are becoming more and more concentrated and prevalent in our environment. Among these endocrine disruptors, we chose to focus this review on the phthalates for different reasons: 1) they are widespread in the environment; 2) their concentrations in many human biological fluids have been measured; 3) the experimental data using rodent models suggesting a reprotoxicity are numerous and are the most convincing; 4) their deleterious effects on the in vivo and in vitro development and function of the rat foetal testis have been largely studied; 5) some epidemiological data in humans suggest a reprotoxic effect at environmental concentrations at least during neonatal life. However, the direct effects of phthalates on human foetal testis have never been explored. Thus, as we did for the rat in the 1990s, we recently developed and validated an organ culture system which allows maintenance of the development of the different cell types of human foetal testis. In this system, addition of 10-4 M MEHP (mono-2-ethylhexyl phthalate), the most produced phthalate, had no effect on basal or LH-stimulated production of testosterone, but it reduced the number of germ cells by increasing their apoptosis, without modification of their proliferation. This is the first experimental demonstration that phthalates alter the development of the foetal testis in humans. Using our organotypic culture system, we and others are currently investigating the effect of MEHP in the mouse and the rat, and it will be interesting to compare the results between these species to analyse the relevance of toxicological tests based on rodent models.

Ontogenesis of testicular function in humans.

The two major functions of the testis, steroidogenesis and gametogenesis, take place during fetal life. These two functions have been extensively studied in rodents and adult humans. However, their onset during fetal life is poorly documented in humans. In the first part of this work we presented both our experimental data and some data of literature concerning the development of the human fetal testis. In the second part of this article, using the organ culture system we previously developed, we have investigated the regulations or perturbations of fetal testis development both in rodent and human models. Our findings provide important insight into the potential role of exposure to environmental pollutants (physical factors, in particular ionizing radiation, cadmium and endocrine disruptors such as phthalates) during fetal testicular development and their potential deleterious effects on male fertility in adulthood. Our results highlight the specificity of the human model compared with rodent models.

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.