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: $1\text{\hspace{0.17em}nM}–10\text{\hspace{0.17em}μM}$) 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

中文翻译：

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检查暴露于环境相关双酚 A 水平后小鼠原始生殖细胞体外模型的发育轨迹

摘要

背景：

基于动物的研究表明，双酚 A (BPA) 暴露对生殖健康有害，但其对生殖细胞发育早期阶段的影响仍不清楚。

目标：

使用早期生殖细胞规范和分化的小鼠体外模型，我们试图评估在原始生殖细胞 (PGC) 形成之前暴露于低水平的 BPA 是否会改变它们的分化轨迹和独特的分子程序。

方法：

我们使用了一种既定的方法，将小鼠胚胎干细胞 (ESCs)体外分化为外胚层样细胞 (EpiLCs)，然后是 PGC 样细胞 (PGCLCs)，它们共同概括了早期生殖细胞发育的特定阶段。使用基于血细胞计数器的细胞计数、固定和细胞内染色确定细胞后果，然后对暴露于增加浓度（范围：$1\text{\hspace{0.17em}纳米}–10\text{\hspace{0.17em}微米}$) 的双酚 A。为了询问和表征 BPA 暴露导致的基因表达差异，我们还从 FACS 纯化的 PGCLC 中提取的 RNA 生成 RNA-seq 文库，并使用基于生物信息学的方法进行转录组分析。

结果：

EpiLCs 暴露于 BPA 导致更多的细胞数量与更高比例的 S 期细胞以及更低比例的细胞凋亡相关；这种差异以浓度依赖性方式发生。暴露还导致更大比例的 EpiLCs 显示出 DNA 损伤的迹象。值得注意的是，EpiLC 暴露不会对 PGC 规格产生负面影响，并且会对 XX 而不是 XY 细胞中的 PGCLC 增殖产生浓度依赖性影响。PGCLC 转录组分析揭示了一个异常程序，其中 X 连锁基因和反转录转座子表达显着失调。差异基因表达分析还揭示了与脂质代谢相关的基因的失调以及与配子发生后期相关的基因的失调表达。

结论：

据我们所知，我们的研究结果首次表征了早期 BPA 暴露对哺乳动物 PGC 发育模型的影响，突出了细胞行为的改变、潜在途径的改变和分子过程的改变。https://doi.org/10.1289/EHP8196