PDF(Pubmed)
Abstract：
To improve the mechanistic screening of reproductive toxicants in chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood-testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.
摘要：
为了改善化学风险评估和药物开发中对生殖毒物的机械筛选，我们从新生小鼠中建立了三维（3D）异质睾丸共培养模型。邻苯二甲酸二正丁酯(DBP),会对生殖健康产生负面影响的环境污染物，用作模型化合物以说明体外模型的实用性。用DBP(1nM至100μM)处理细胞7天。自动化高含量成像证实存在Leydig细胞的细胞特异性标志物（CYP11A1），支持细胞（SOX9+），和生殖细胞（DAZL+）。通过分析培养基中的睾酮水平来证明Leydig细胞的类固醇生成活性。与媒介物对照相比,DBP诱导睾酮水平的浓度依赖性降低并减少Leydig细胞的数量。在最低DBP浓度（1nM）下，类固醇生成调节剂StAR和类固醇生成酶CYP11A1的水平已经降低，证明了睾酮生物合成途径的上游效应。此外，暴露于10nMDBP降低生殖细胞特异性RNA结合蛋白DAZL的水平，精子发生的中心。3D模型还捕获了支持细胞连接蛋白的发育，N-钙黏着蛋白和包合Zonula蛋白1（ZO-1），对血液-睾丸屏障至关重要。然而,DBP暴露并未显着改变钙黏着蛋白和ZO-1的水平。总之,这个3D体外系统模拟睾丸细胞信号和功能，使其成为发育性睾丸毒性机制筛查的有力工具。这可以为在敏感的发育阶段高通量筛选化学诱导的生殖毒性开辟新的途径。
