Sex differences in embryonic gonad transcriptomes and benzo[a]pyrene metabolite levels after transplacental exposure. Sex differences in embryonic gonad transcriptomes and benzo[a]pyrene metabolite levels after transplacental exposure

Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are generated during incomplete combustion of organic materials. Prior research has demonstrated that BaP is a prenatal ovarian toxicant and carcinogen. However, the metabolic pathways active in the embryo and its developing gonads and the mechanisms by which prenatal exposure to BaP predisposes to ovarian tumors later in life remain to be fully elucidated. To address these data gaps, we orally dosed pregnant female mice with BaP from E6.5-11.5 (0, 0.2 or 2 mg/kg-day) for metabolite measurement or E9.5-11.5 (0 or 3.33 mg/kg-day) for embryonic gonad RNA-sequencing. Embryos were harvested at E13.5 for both experiments. The sum of BaP metabolite concentrations increased significantly with dose in the embryos and placentas, and concentrations were significantly higher in female than male embryos and in embryos than placentas. RNA sequencing revealed that enzymes involved in metabolic activation of BaP are expressed at moderate to high levels in embryonic gonads and that greater transcriptomic changes occurred in the ovaries in response to BaP than in the testes. We identified 490 differentially expressed genes (DEGs) with FDR p-values <0.05 when comparing BaP-exposed to control ovaries, but no statistically significant DEGs between BaP-exposed and control testes. Genes related to monocyte/macrophage recruitment and activity, prolactin family genes, and several keratin genes were among the most upregulated genes in the BaP-exposed ovaries. Results show that developing ovaries are more sensitive than testes to prenatal BaP exposure, which may be related to higher concentrations of BaP metabolites in female embryos. Overall design: Pregnant mice were exposed to benzo[a]pyrene, and gonads were collected from E13.5 embryos for RNA-seq.

多环芳烃（Polycyclic aromatic hydrocarbons）如苯并[a]芘（benzo[a]pyrene, BaP）可在有机物不完全燃烧过程中产生。既往研究已证实，BaP是一种孕期卵巢毒性物质与致癌物。然而，胚胎及其发育中性腺内的活性代谢通路，以及孕期BaP暴露何以增加个体后续生命中卵巢肿瘤易感性的具体机制，仍有待全面阐明。

为填补此类数据空白，本研究对孕鼠实施两种经口灌胃给药方案：于胚胎发育第6.5至11.5天（E6.5-11.5）给予0、0.2或2 mg/kg·天的BaP以开展代谢物检测；或于E9.5-11.5给予0或3.33 mg/kg·天的BaP以进行胚胎性腺RNA测序（RNA-sequencing）。两项实验均于胚胎发育第13.5天（E13.5）采集胚胎。

胚胎与胎盘中的BaP代谢物总浓度随给药剂量升高而显著增加，且雌性胚胎的代谢物浓度显著高于雄性胚胎，胚胎内的代谢物浓度也显著高于胎盘。

RNA测序结果显示，参与BaP代谢活化的酶在胚胎性腺中呈中等到高水平表达，且卵巢在BaP暴露下发生的转录组变化程度显著高于睾丸。在对比BaP暴露组与对照组卵巢的分析中，我们共鉴定出490个错误发现率（False Discovery Rate, FDR）校正后p值<0.05的差异表达基因（differentially expressed genes, DEGs）；但在对比BaP暴露组与对照组睾丸的分析中，未发现具有统计学意义的差异表达基因。

BaP暴露组卵巢中上调幅度最显著的基因包括与单核细胞/巨噬细胞招募及活化相关的基因、催乳素家族基因，以及多个角蛋白基因。

研究结果表明，发育中的卵巢较睾丸对孕期BaP暴露更为敏感，这一现象可能与雌性胚胎内BaP代谢物浓度更高有关。

实验整体设计：对孕鼠实施苯并[a]芘暴露，于胚胎发育第13.5天（E13.5）的胚胎中收集性腺进行RNA测序。