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Physiologically-relevant Bisphenol A levels alter the developmental trajectory of an in vitro model of mouse Primordial Germ Cells

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 remain poorly defined. Objective: 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 Primordial Germ Cell (PGC) formation 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 recapitulates defined stages of early germ cell development. Cellular consequences were determined using hemocytometer-based cell counting, fixation and intracellular staining followed by flow cytometry/FACS on cells exposed to increasing doses of BPA. We also generated RNA-seq libraries from RNA extracted from FACS-purified PGCLCs and performed transcriptome analysis using bioinformatics-based approaches, to interrogate and characterise gene expression changes resulting from BPA exposure. Results: Exposure of EpiLCs to BPA resulted in a dose-dependent increase in cell number, associated with an increase proportion of cells in S-phase as well as a reduced proportion undergoing apoptosis. Exposure also resulted in a greater proportion of EpiLCs showing signs of DNA damage. Remarkably, EpiLC exposure did not negatively impact PGC specification and resulted in dose-dependent increase in PGCLC proliferation. However, 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: Our findings represent the first characterisation of the consequences of early BPA exposure on a model of PGC development, highlighting altered cell behaviour and underlying pathways and molecular processes.

ORGANISM(S): Mus musculus

PROVIDER: GSE157570 | GEO | 2021/09/01

REPOSITORIES: GEO

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Project description:The generation of properly functioning gametes in vitro, a key goal in developmental/reproductive biology, requires multi-step reconstitutions of complex germ cell development. Based on the logic of primordial germ cell (PGC)-specification, we demonstrate here the generation of PGC-like cells (PGCLCs) in mice with robust capacity for spermatogenesis from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pre-gastrulating epiblasts, but distinct from epiblast stem cells (EpiSCs). The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs are a meticulous capture of those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-beta 3 and SSEA1 as markers that purify PGCLCs with spermatogenic capacity free from tumorigenic undifferentiated cells. With the reconstitution of PGC specification pathway from the naive inner cell mass state, our study defines a paradigm for the essential step of in vitro gametogenesis. We performed this analysis to reveal the characters of the cells that we created in this study, epiblast-like cells (EpiLCs) and primordial germ cells-like cells (PGCLCs). Because EpiLCs were induced from embryonic stem cells (ESCs), and equivalent to pre-gastrulating epiblast (embryonic day [E] 5.5-6.0) in vivo (embryo), ESCs and epiblast were included in this analysis. Epiblast stem cells (EpiSCs) are a culture cell type derived from epiblast, and were also included. PGCLCs were supposed to be equivalent to E9.5 PGCs based on reporter fluorescent transgene expressions and epigenetic properties, and therefore E9.5 PGCs were also inckuded in this analysis. Because epiblast and E9.5 PGCs are of a small number of cells in embryos (a few hundred to thousand cells), cDNAs were amplified with a quantitative global PCR method (Kurimoto et al., 2006, Nucleic Acids Research) for microarray analyses. We took two biological replicate for each cell type.

Project description:Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGC) in mice1, where its precise role is yet unclear2-4. We investigated this in an in vitro model, where naÃ¯ve pluripotent embryonic stem cells (ESCs) cultured in bFGF/ActivinA develop as epiblast-like cells (EpiLCs), and gain competence for PGC-like fate5. Consequently, bone morphogenetic protein (BMP4), or ectopic expression of key germline transcription factors Prdm1/ Prdm14/ Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ESCs6-8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that following the dissolution of the naÃ¯ve ESC pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG binding pattern between ESCs and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ESCs, they show contrasting roles in EpiLCs since Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development. To characterize Nanog-induced Primordial Germ cell-like cells (PGCLCs), we performed expression analysis of Nanog-induced Day4 PGCLCs compared to male mouse Embryonic Stem Cells (mESCs) and male Day4 PGCLCs which were induced by cytokines. mESCs were maintained in N2B27 2i(CHIR99021 3 ÂµM, PD0325901 1 ÂµM) LIF(1000 U/ml) medium and Day4 PGCLCs were induced in GK15 medium with Nanog induction (0.7 Âµg/ml) or cytokines (BMP4 500 ng/ml, BMP8A 500 ng/ml, SCF 100 ng/ml, EGF 50 ng/ml and LIF 1000U/ml) as previously reported (Hayashi K et al., Cell, 2011).

Project description:The germ cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have established a culture system that recapitulates the mouse germ-cell specification pathway: Using cytokines, embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) are induced into epiblast-like cells (EpiLCs) and then into primordial germ cell-like cells (PGCLCs) with capacity both for spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous over-expression of three transcription factors (TFs), Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), directs EpiLCs, but not ESCs, swiftly and highly efficiently into a PGC state with endogenous transcription circuitry. The induction of the PGC state on EpiLCs minimally requires Prdm14 but not Blimp1 or Tfap2c. The TF-induced PGC state reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC specification in vivo and in vitro by cytokines including BMP4. Importantly, the TF-induced PGC-like cells robustly contribute to spermatogenesis and fertile offspring. Our findings provide not only a novel insight into the transcriptional logic that creates a germ cell state, but also a foundation for the TF-based reconstitution and regulation of mammalian gametogenesis. Aim of this analysis is characterization of transcription factor-induced primordial germ cells (TF-PGCLCs) compared with cytokine-induced primordial germ cells (Ck-PGCLCs) (Hayashi et al., 2011, Cell), epiblast-like cells (EpiLCs) (Hayashi et al., 2011, Cell), and embryonic stem cells (ESCs) and identification of genes differentially expressed among them. TF-PGCLCs induced by multiple combinations of TFs (Blimp1 (B), Prdm14 (P14), and Tfap2c (A) (BP14A), BP14, P14A, P14) on day 2 and 4 (for BP14A cells) of the induction were also compared. Parental clone without exogenous TFs cultured with doxycycline, are also included as a negative control. Ck-PGCLCs day 2 and day 4 samples, which are previously unreported, EpiLCs and ESCs used in this study were also included. Overexpression of exogenous three TFs in ESCs yields stella-ECFP (SC) positive cells, which were sorted and included in the analysis. cDNA samples, prepared from approximately 20,000 cells, were amplified with a quantitative global PCR method (Kurimoto et al., 2006, Nucleic Acids Research). Two biological duplicates for each cell type were analyzed. Samples from GSE30056 were also included and reanalysed (GSM1070855-GSM1070864).

Project description:The germ cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have established a culture system that recapitulates the mouse germ-cell specification pathway: Using cytokines, embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) are induced into epiblast-like cells (EpiLCs) and then into primordial germ cell-like cells (PGCLCs) with capacity both for spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous over-expression of three transcription factors (TFs), Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2?), directs EpiLCs, but not ESCs, swiftly and highly efficiently into a PGC state with endogenous transcription circuitry. The induction of the PGC state on EpiLCs minimally requires Prdm14 but not Blimp1 or Tfap2c. The TF-induced PGC state reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC specification in vivo and in vitro by cytokines including BMP4. Importantly, the TF-induced PGC-like cells robustly contribute to spermatogenesis and fertile offspring. Our findings provide not only a novel insight into the transcriptional logic that creates a germ cell state, but also a foundation for the TF-based reconstitution and regulation of mammalian gametogenesis. Aim of this analysis is identification of genes whose expression was altered by each of key transcription factor for transcription factor-induced primordial germ cells (TF-PGCLCs) induction (Blimp1 (B), Prdm14 (P14), and Tfap2c (A)). Both of epiblast-like cells (EpiLCs) (Hayashi et al., 2011, Cell) and aggregates of EpiLCs cultured with doxycycline on day 1 were harvested for 5 cell lines, including BP14A (Clone #3-3), B (#2-4), P14 (#7-109), A (#8-2), and the parental clone (BVSCR26rtTA embryonic stem cells). Total RNA was isolated and analyzed. Two biological duplicates for each cell type were included.

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Physiologically-relevant Bisphenol A levels alter the developmental trajectory of an in vitro model of mouse Primordial Germ Cells

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