UCLA Institute for Society & Genetics, University of California, Los Angeles, Los Angeles, California, USA.
Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA.
Environ Health Perspect. 2021 Sep;129(9):97013. doi: 10.1289/EHP8196. Epub 2021 Sep 29.
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.
Using a murine 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.
We used an established method of 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.
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.
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.
动物研究表明，双酚 A（BPA）暴露对生殖健康有害，但它对生殖细胞早期发育阶段的影响仍未得到明确界定。
本研究使用一种早期生殖细胞特化和分化的鼠模型，旨在评估在原始生殖细胞（PGCs）形成之前暴露于低水平 BPA 是否会改变其分化轨迹和独特的分子程序。
我们使用一种已建立的方法，即将小鼠胚胎干细胞（ESCs）分化为类上胚层细胞（EpiLCs），然后分化为类原始生殖细胞（PGCLCs），这两种细胞共同模拟了早期生殖细胞发育的特定阶段。通过细胞计数、固定和细胞内染色后用血细胞计数器检测，以及流式细胞术/荧光激活细胞分选（FACS）检测暴露于不同浓度（范围：）BPA 的细胞，来确定细胞的后续变化。为了探究和描述 BPA 暴露导致的基因表达差异，我们还从 FACS 纯化的 PGCLCs 中提取 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。
EpiLC 暴露于 BPA 会导致细胞数量增加，同时 S 期细胞比例升高，凋亡细胞比例降低；这种差异呈浓度依赖性。暴露还导致更多的 EpiLC 出现 DNA 损伤的迹象。值得注意的是，EpiLC 暴露不会对 PGC 特化产生负面影响，反而会导致 XX 细胞而非 XY 细胞中 PGCLC 增殖呈浓度依赖性。PGCLC 转录组分析显示，异常的基因表达程序与 X 连锁基因和逆转录转座子表达的显著失调有关。差异基因表达分析还揭示了与脂质代谢相关基因的失调，以及与配子发生后期阶段相关基因的失调表达。
据我们所知，本研究结果首次描述了早期 BPA 暴露对哺乳动物 PGC 发育模型的影响，突出了改变的细胞行为、潜在的改变途径以及改变的分子过程。
