Examining the Developmental Trajectory of an in Vitro Model of Mouse Primordial Germ Cells following Exposure to Environmentally Relevant Bisphenol A Levels

Abstract

Background: Animal-based studies indicate that bisphenol A (BPA) exposure is detrimental to reproductive health, but its impact on the earliest stages of germ cell development remains poorly defined. Objectives: Using a murine in vitro model of early germ cell specification and differentiation, we sought to assess whether exposure to low levels of BPA prior to formation of primordial germ cells (PGCs) alters their differentiation trajectory and unique molecular program. Methods: We used an established method of in vitro differentiation of mouse embryonic stem cells (ESCs) into epiblast-like cells (EpiLCs) followed by PGC-like cells (PGCLCs), which together recapitulate defined stages of early germ cell development. Cellular consequences were determined using hemocytometer-based cell counting, fixation, and intracellular staining, followed by flow cytometry/fluorescence-activated cell sorting (FACS) of cells exposed to increasing concentrations (range: ) of BPA. To interrogate and characterize gene expression differences resulting from BPA exposure, we also generated RNA-seq libraries from RNA extracted from FACS-purified PGCLCs and performed transcriptome analysis using bioinformatics-based approaches. Results: Exposure of EpiLCs to BPA resulted in higher numbers of cells that were associated with a higher proportion of cells in S-phase as well as a lower proportion undergoing apoptosis; this difference occurred in a concentration-dependent manner. Exposure also resulted in a greater fraction of EpiLCs showing signs of DNA damage. Remarkably, EpiLC exposure did not negatively affect PGC specification and resulted in a concentration-dependent effect on PGCLC proliferation in XX but not XY cells. PGCLC transcriptome analysis revealed an aberrant program with significant deregulation of X-linked genes and retrotransposon expression. Differential gene expression analysis also revealed the deregulation of genes associated with lipid metabolism as well as deregulated expression of genes associated with later stages of gametogenesis. Conclusions: To the best of our knowledge our findings represent the first characterization of the consequences of early BPA exposure on a model of mammalian PGC development, highlighting altered cell behavior, altered underlying pathways, and altered molecular processes. https://doi.org/10.1289/EHP8196

2,4-Dichlorophenol Increases Primordial Germ Cell Numbers via ESR2a-Dependent Pathway in Zebrafish Larvae

“…The sustained upregulation of vasa and dnd could serve as important indicators of increased PGC number by 2,4-DCP. Our finding that 2,4-DCP exposure increases PGC numbers at 15 dpf is in line with a previous study reporting a similar estrogen chemical BPA-promoted proliferation of PGCs in mice . This finding is also in accord with the model that the molecular factors, specifically vasa and dnd , driving germ-line proliferation are sensitive to estrogenic compounds. − Notably, the metabolites of 2,4-DCP, such as 2,4-dichloro-6-nitrophenol (DCNP) and hydroquinone, have shown endocrine disruption in zebrafish. , Therefore, further research is needed to explore their action on disrupting sex differentiation in zebrafish.…”

Off to a Rough Start: Environmental Exposures May Alter Germ Cell Development

“… 2 In an in vitro study recently published in Environmental Health Perspectives , researchers show for the first time how toxicant exposures at environmentally relevant concentrations can influence the earliest stages of germ cell specification. 3 The results add to mounting evidence that the timing of exposure is critical when assessing chemical toxicities.…”

Early Gonadal Development and Sex Determination in Mammal

“…Similarly, the cell adhesion microenvironment was found to contribute to the differentiation of ESCs, which provide new ideas for PGCs derivation in vitro, where mesh substrates were found to induce self-organize and differentiation of ESCs, transiting to a PGCs-like state without the addition of biochemical inducers [ 207 ]. Interestingly, sex differences were found in the associations between Bisphenol A and PGCLC proliferation, with downregulated X-linked gene expression and PGCLC proliferation inhibited in XX cells but not in XY cells [ 208 ], providing a theoretical basis for intervening in the fate of different gender PGCs.…”