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Genome-wide transcriptome analysis of Epiblast Like Cells (EpiLC), early Primordial Germ Cells (PGCLCd3) and late Primordial Germ Cells (PGCLCd5) with WT or EsrrbKO background

ABSTRACT: We report the application of genome-wide RNA-sequencing analysis for WT or EsrrbKO mES cells after transition towards EpiLC and subsequent differentiation in early (d3) or late (d5) PGCLC. We find that formative early and formative late gene signatures were significantly reduced in EsrrbKO EpiLC compared to WT EpiLC. Furthermore, both early and late PGC gene sets were significantly downregulated in EsrrbKO PGCLC, while somatic markers were robustly increased. This indicates that the developmental program is not properly activated during transition towards PGCLC in EsrrbKO cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE184136 | GEO | 2023/01/25

REPOSITORIES: GEO

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Project description: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.

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Genome-wide transcriptome analysis of Epiblast Like Cells (EpiLC), early Primordial Germ Cells (PGCLCd3) and late Primordial Germ Cells (PGCLCd5) with WT or EsrrbKO background

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