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

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

Antagonistic effects of gestational dietary exposure to low-dose vinclozolin and genistein on rat fetal germ cell development

International audienceContinuous, low-dose exposure to a phytoestrogen (1 mg/kg/day genistein) and/or to an antiandrogenic food contaminant (1 mg/kg/day vinclozolin) has been recently reported to affect male reproductive tract and fertility [1] in adults. We investigated whether alterations of the testis are already present at the end of in utero exposure using the same rat model and doses following exposure from conception to delivery. After vinclozolin exposure, we observed in the neonate a slight but significant alteration of steroidogenesis and gametogenesis with a reduction of testosterone secretion and of the number of gonocytes. In contrast, genistein exposure had no effect. While the vinclozolin-genistein mixture acts in a synergistic manner to induce the most significant alterations in the adult, interestingly, genistein antagonized the deleterious effect of vinclozolin on germ cells in the neonate. This difference emphasizes the importance of studying the effects of endocrine disruptors during various developmental stages to understand their effects

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

Phenotypic and functional similarity of gut intraepithelial and systemic T cells in a teleost fish

International audienceGut-associated lymphocytes were described in fish, but their involvement in immune responses is still unknown. In rainbow trout, intraepithelial lymphocytes (IELs) are scattered between gut epithelial cells, but neither Peyer’s patches nor mesenteric lymph nodes were identified. Rainbow trout IELs contain mainly T cells, because they expressed transcripts of T cell marker homologs of CD8, CD4, CD28, CD3_, TCR_, TCR_, and TCR_ and lacked IgM. However, trout IELs did not show specific homing to the gut mucosa, which in mammals defines IELs as a distinctive mucosal population. A detailed analysis of the TCR_ repertoire of rainbow trout IELs was performed in both naive and virus-infected animals. TCR_ transcripts of rainbow trout IELs were highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals. Significant modifications of the trout IEL TCR_ repertoire were observed after a systemic infection with a fish rhabdovirus and were especially marked for V_4-bearing receptors as previously reported for spleen cells. Thus, we could not find any specific properties of the trout IEL TCR_ repertoire compared with the spleen and pronephros TCR_ repertoire, which questions the reality of a distinct IEL compartment in teleosts. Our findings suggest that a highly diversified __ _CR repertoire is maintained in fish IELs in the absence of Peyer’s patches and mesenteric lymph nodes, whereas the restricted diversity of mouse __ IELs is attributed to multiple cycles of activation and recirculation, allowing a progressive narrowing of the repertoire

TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

AbstractDuring testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence.These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis

DNA methylation dynamics during spermatogenesis in ruminants

International audienceDNA methylation of cytosines is a critical epigenetic modification in mammals that plays crucial roles in transcriptional regulation, chromatin remodelling and genomic imprinting. Dynamic erasure and reestablishment of DNA methylation marks are required for spermatogenesis and the normal function of mature sperm. DNA methylation is catalysed by DNA methyltransferase enzymes (DNMT) providing either maintenance (DNMT1) or de novo (DNMT3A/B/L) DNA methylation processes. The DNA methylation dynamics during spermatogenesis has been previously described in mice and humans but nothing is known in productive livestock. However, these investigations in ruminants could be helpful to determine epigenetic events likely to be crucial for male fertility. Interestingly, a recent study from our laboratory pointed DNA undermethylation of bull spermatozoa compared to other mammals such as humans, mice, sheep or goats [1]. This result raises the question of the dynamic of DNA methylation in bovine male germline. We thus propose to define and compare the methylation dynamics of bovine and caprine germ cells during spermatogenesis. Analyses of 5mC immunohistochemistry were performed on adult testis sections to illustrate the presence of DNA methylation marks in various spermatogenic cells. As purification of the different spermatogenic populations is required to characterize DNA methylation more precisely, we firstly developed a flow cytometry-based method (Hoechtst-FACS) for cell sorting from goat testis. Fraction enrichments were evaluated using stage-specific markers by real time qPCR analyses. In addition, de novo DNA methyltransferase expression was studied on the different fractions of purified spermatogenic cells, giving some clues on DNA methylation dynamics. In the future, spermatogenic cells will be purified from both caprine and bovine testes, and together with gene expression analyses (RNA-sequencing), DNA methylation will be determined for each cell types by Reduced-Representation Bisulfite Sequencing (RRBS

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