Human Primordial Germ Cell Formation Is Diminished by Exposure to Environmental Toxicants Acting through the AHR Signaling Pathway

Department of Obstetrics and Gynecology, Center for Human Embryonic Stem Cell Research and Education, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford University, Palo Alto, California, USA.
Toxicological Sciences (Impact Factor: 3.85). 09/2010; 117(1):218-24. DOI: 10.1093/toxsci/kfq179
Source: PubMed


Historically, effects of environmental toxicants on human development have been deduced via epidemiological studies because direct experimental analysis has not been possible. However, in recent years, the derivation of human pluripotent stem cells has provided a potential experimental system to directly probe human development. Here, we used human embryonic stem cells (hESCs) to study the effect of environmental toxicants on human germ cell development, with a focus on differentiation of the founding population of primordial germ cells (PGCs), which will go on to form the oocytes of the adult. We demonstrate that human PGC numbers are specifically reduced by exposure to polycyclic aromatic hydrocarbons (PAHs), a group of toxicants common in air pollutants released from gasoline combustion or tobacco smoke. Further, we demonstrate that the adverse effects of PAH exposure are mediated through the aromatic hydrocarbon receptor (AHR) and BAX pathway. This study demonstrates the utility of hESCs as a model system for direct examination of the molecular and genetic pathways of environmental toxicants on human germ cell development.

Download full-text


Available from: Renee A Reijo Pera, Dec 05, 2014
  • Source
    • "In addition to zebrafish embryo test (ZET) model, two other developmental toxicity systems have been used: the mouse embryonic stem cell (ESC) test and the rat whole embryo culture (WEC) which can both be used for chemical classification and have been reviewed for regulatory validation by the European Center for the Validation of Alternative methods (ECVAM) (Scialli, 2008; Robinson et al., 2012b). Although, so far, PAHs have not been studied in these test systems, PAH have been shown to have an adverse effect on the development of human embryonic cells (Kee et al., 2010) suggesting that the ESC model might be applicable to study the PAH present in petroleum substances as well. The ESC test makes use of mouse cells and looks at gene expression profiles related to developmental toxicity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The REACH legislation requires chemicals - including petroleum substances - that are put on the EU market in quantities greater than 1000 tonnes/year, to be tested for prenatal developmental toxicity. This will require large numbers of animals since prenatal development toxicity testing is animal-intensive. The application of toxicogenomic technologies might reduce the number of animals to study prenatal developmental toxicity of petroleum substances by allowing their grouping into categories with the same toxicological properties. This substance categorization may be supported by similarities in molecular fingerprints. The developmental toxicity effects observed in some oil products are most likely related to polycyclic aromatic hydrocarbons (PAHs) with high-molecular weight. However, the current review indicates that even though the available studies provide clues regarding the HOX, FOX, SHH and PAX family genes, which regulate functions in skeleton development, single individual genes cannot be used as biomarkers of PAHs exposure and subsequent prenatal developmental toxicity. Furthermore, it should be considered that toxicogenomic technologies applied to specific tissues/organs testing might lead to unreliable results regarding developmental toxicity due to induction of tissue-specific pathways. Thus, an approach which applies a battery of in vitro tests including the zebrafish embryo test, embryonic stem cells, and the whole embryo culture is suggested as it would be more relevant for studying developmental effects in the terms of substances categorization. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Toxicology in Vitro 12/2014; 29(2). DOI:10.1016/j.tiv.2014.11.005 · 2.90 Impact Factor
  • Source
    • "in an increase in germ cell apoptosis (Coutts et al., 2007). Human embryonic stem cells induced to differentiate towards the germ cell lineage also express the AhR, and are sensitive to PAHs (Kee et al., 2010). In that model, DMBA-DHD resulted in reduced expression of primordial germ cell genes, and increased apoptosis, although the suitability of the ES cell system as a model for human ovarian germ cell development in vivo remains to be determined. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fetal life is a critical time for female fertility, when germ cells complete proliferation, initate meiosis and ultimately form the lifetime stock of primordial follicles. Female fertility may be reduced by in utero exposure to cigarette smoke, which contains ligands for the aryl hydrocarbon receptor (AhR). The AhR is a critical regulator of ovarian germ cell survival in mice, thus activation of this receptor in the ovaries of fetuses exposed to maternal cigarette smoke in utero may provide a mechanism by which female fertility is reduced in later life. We have therefore investigated AhR expression in the human fetal ovary, and examined the effects of an AhR ligand present in cigarette smoke, on germ cells in human fetal ovaries cultured in vitro. The results showed that AHR mRNA expression increased 2-fold between first and late second trimester (p=0.008). AhR protein was confined to germ cells at all gestations, but varied from expression in most germ cells during the first trimester, to only patchy expression by clusters of germ cells at later gestations. Culture of human fetal ovaries with the AhR ligand 9,10-dimethyl-1,2-benzanthracene-3,4-dihydrodiol (DMBA-DHD; a component of cigarette smoke) did not affect germ cell number in vitro, but significantly reduced the proportion of proliferating germ cells by 29% (as assessed by phospho-histone H3 staining (p=0.04)). Germ cell apoptosis was not significantly affected. These results reveal that germ cells in the human fetal ovary express AhR from the proliferative stage of development through entry into meiosis and beyond, and demonstrate that AhR ligands found in cigarette smoke have the capacity to impair human fetal ovarian germ cell proliferation.
    Molecular Human Reproduction 08/2013; 20(1). DOI:10.1093/molehr/gat059 · 3.75 Impact Factor
  • Source
    • "). The extra band observed for AHR–RL may arise from posttranslational modification of AHR in HEK293-related cells, as previously reported (Kee et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Abstract Dioxin and dioxin-like compounds are among the most prevalent and toxic environmental pollutants. At present, analytical chemical techniques are considered the gold standard for detection of dioxins. Here, we describe a highly sensitive and cost-effective alternative, based on bioluminescence and bioluminescence resonance energy transfer (BRET). Upon binding to dioxin, Aryl hydrocarbon receptor (AHR) dissociates from HSP90 and subsequently translocates to the nucleus, where it interacts with AHR nuclear translocator (ARNT). We generated cell lines that stably co-express a fusion protein of AHR and Renilla luciferase (AHR-RL) and either HSP90 or ARNT tagged with yellow fluorescent protein (HSP90-YFP or ARNT-YFP). The fluorescent signals of YFP are activated by the emission of RL while the interactions between AHR and HSP90 (or ARNT) were monitored. Application of 3-methylcholanthrene (3MC), the AHR agonist, enhances BRET signals in cells co-expressing AHR-RL, AIP-HIS, P23-HIS and ARNT-YFP (AAPA cells), while suppressing BRET signals in cells co-expressing AHR-RL, AIP-HIS, P23-HIS and HSP90-YFP (AAPH cells). In addition, dioxin treatment reduced Renilla luminescence in AAPH cells in a concentration-dependent manner, due to degradation of AHR. Intriguingly, the detection limit for dioxin in our AHR degradation assay was as low as 10(-17) M. This work highlights the potential of AHR-RL degradation assays to detect dioxin-like pollutants.
    Toxicology mechanisms and methods 11/2012; 23(4). DOI:10.3109/15376516.2012.745105 · 1.52 Impact Factor
Show more