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

Concerns about the widespread use of rodent models for human risk assessments of endocrine disruptors.

International audienceFetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk

A new chapter in the bisphenol A story: bisphenol S and bisphenol F are not safe alternatives to this compound

Bisphenol A (BPA) is a widely studied typical endocrine-disrupting chemical, and one of the major new issues is the safe replacement of this commonly used compound. Bisphenol S (BPS) and bisphenol F (BPF) are already or are planned to be used as BPA alternatives. With the use of a culture system that we developed (fetal testis assay [FeTA]), we previously showed that 10 nmol/L BPA reduces basal testosterone secretion of human fetal testis explants and that the susceptibility to BPA is at least 100-fold lower in rat and mouse fetal testes. Here, we show that addition of LH in the FeTA system considerably enhances BPA minimum effective concentration in mouse and human but not in rat fetal testes. Then, using the FeTA system without LH (the experimental conditions in which mouse and human fetal testes are most sensitive to BPA), we found that, as for BPA, 10 nmol/L BPS or BPF is sufficient to decrease basal testosterone secretion by human fetal testes with often nonmonotonic dose-response curves. In fetal mouse testes, the dose-response curves were mostly monotonic and the minimum effective concentrations were 1,000 nmol/L for BPA and BPF and 100 nmol/L for BPS. Finally, 10,000 nmol/L BPA, BPS, or BPF reduced Insl3 expression in cultured mouse fetal testes. This is the first report describing BPS and BPF adverse effects on a physiologic function in humans and rodents

Effects of environmental Bisphenol A exposures on germ cell development and Leydig cell function in the human fetal testis

<div><p>Background</p><p>Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models.</p><p>Methods</p><p>Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks.</p><p>Results</p><p>With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice.</p><p>Conclusions</p><p>Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures.</p></div

Sorting and Manipulation of Human PGC-LC Using PDPN and Hanging Drop Cultures

The generation of oocytes from induced pluripotent stem cells (iPSCs) was proven efficient with mouse cells. However, no human iPSCs have yet been reported to generate cells able to complete oogenesis. Additionally, efficient sorting of human Primordial Germ Cell-like Cells (hPGC-LCs) without genomic integration of fluorescent reporter for their downstream manipulation is still lacking. Here, we aimed to develop a model that allows human germ cell differentiation in vitro in order to study the developing human germline. The hPGC-LCs specified from two iPS cell lines were sorted and manipulated using the PDPN surface marker without genetic modification. hPGC-LCs obtained remain arrested at early stages of maturation and no further differentiation nor meiotic onset occurred when these were cultured with human or mouse fetal ovarian somatic cells. However, when cultured independently of somatic ovarian cells, using BMP4 and the hanging drop-transferred EBs system, early hPGC-LCs further differentiate efficiently and express late PGC (DDX4) and meiotic gene markers, although no SYCP3 protein was detected. Altogether, we characterized a tool to sort hPGC-LCs and an efficient in vitro differentiation system to obtain pre-meiotic germ cell-like cells without using a gonadal niche

Different exposure windows to low doses of genistein and/or vinclozolin result in contrasted disorders of testis function and gene expression of exposed rats and their unexposed progeny

International audienceLiving species including humans are continuously exposed to low levels of a myriad of endocrine active compounds that may affect their reproductive function. In contrast, experimental designs scrutinizing this question mostly consider the gestational/lactational period, select high unrealistic doses and, have rarely investigated the possible reproductive consequences in the progeny. The present study aimed at assessing comparatively a set of male reproductive endpoints according to exposure windows, gestational/lactational versus pre-pubertal to adulthood, using low doses of endocrine active substances in male rats as well as their unexposed male progeny. Animals were orally exposed to 1 mg/kg bw/d of genistein and/or vinclozolin, from conception to weaning or from prepuberty to young adulthood. A number of reproductive endpoints were assessed as well as testicular mRNA expression profiles, in the exposed rats and their unexposed progeny. Overall, the low dosage used only affected weakly most of classical reproductive endpoints. However, the gestational/lactational exposure to vinclozolin alone or combined to genistein significantly delayed the puberty onset. Contrasting with the gestational/lactational exposure, a decreased sperm production was found in the animals exposed to genistein and vinclozolin from the pre-pubertal period but also in their progeny for vinclozolin and the mixture. The expression level of several genes involved in meiosis, apoptosis and steroidogenesis was also affected differentially as a function of the exposure window in both exposed rats and unexposed offspring. We also provide further evidence that doses of endocrine active substances relevant with human exposure may affect the male reproductive phenotype and testicular transcriptome in the exposed generation as well as in the indirectly exposed offspring

Unexpected Interacting Effects of Physical (Radiation) and Chemical (Bisphenol A) Treatments on Male Reproductive Functions in Mice

International audienceFor decades, numerous chemical pollutants have been described to interfere with endogenous hormone metabolism/signaling altering reproductive functions. Among these endocrine disrupting substances, Bisphenol A (BPA), a widely used compound, is known to negatively impact germ and somatic cells in the testis. Physical agents, such as ionizing radiation, were also described to perturb spermatogenesis. Despite the fact that we are constantly exposed to numerous environmental chemical and physical compounds, very few studies explore the impact of combined exposure to chemical and physical pollutants on reproductive health. The aim of this study was to describe the impact of fetal co-exposure to BPA and IR on testicular function in mice. We exposed pregnant mice to 10 µM BPA (corresponding to 0.5 mg/kg/day) in drinking water from 10.5 dpc until birth, and we irradiated mice with 0.2 Gy (γ-ray, RAD) at 12.5 days post-conception. Co-exposure to BPA and γ-ray induces DNA damage in fetal germ cells in an additive manner, leading to a long-lasting decrease in germ cell abundance. We also observed significant alteration of adult steroidogenesis by RAD exposure independently of the BPA exposure. This is illustrated by the downregulation of steroidogenic genes and the decrease of the number of adult Leydig cells. As a consequence, courtship behavior is modified, and male ultrasonic vocalizations associated with courtship decreased. In conclusion, this study provides evidence for the importance of broadening the concept of endocrine disruptors to include physical agents, leading to a reevaluation of risk management and regulatory decisions

Divergent roles of CYP26B1 and endogenous retinoic acid in mouse fetal gonads

International audienceIn female mammals, germ cells enter meiosis in the fetal ovaries, while in males, meiosis is prevented until postnatal development. Retinoic acid (RA) is considered the main inducer of meiotic entry, as it stimulates Stra8 which is required for the mitotic/meiotic switch. In fetal testes, the RA-degrading enzyme CYP26B1 prevents meiosis initiation. However, the role of endogenous RA in female meiosis entry has never been demonstrated in vivo. In this study, we demonstrate that some effects of RA in mouse fetal gonads are not recapitulated by the invalidation or up-regulation of CYP26B1. In organ culture of fetal testes, RA stimulates testosterone production and inhibits Sertoli cell proliferation. In the ovaries, short-term inhibition of RA-signaling does not decrease $Stra8$ expression. We develop a gain-of-function model to express CYP26A1 or CYP26B1. Only CYP26B1 fully prevents STRA8 induction in female germ cells, confirming its role as part of the meiotic prevention machinery. CYP26A1, a very potent RA degrading enzyme, does not impair the formation of STRA8-positive cells, but decreases Stra8 transcription. Collectively, our data reveal that CYP26B1 has other activities apart from metabolizing RA in fetal gonads and suggest a role of endogenous RA in amplifying Stra8, rather than being the initial inducer of $Stra8$. These findings should reactivate the quest to identify meiotic preventing or inducing substances