Characterization of primary canine Sertoli cells as a model to test male reproductive toxicant.
Characterization of primary canine Sertoli cells as a model to test male reproductive toxicant.
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DOI：
10.1016/j.tiv.2022.105452
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发表时间：
2022-10
影响因子：
3.2
通讯作者：
Yin, Lei
中科院分区：
文献类型：
--
作者：
Hsiao, Zoey Hsuan;Li, Lu;Yu (John), Xiaozhong;Yin, Lei
支持细胞通过提供结构和营养支持在调节精子发生和睾丸发育中起关键作用。本研究旨在建立犬睾丸支持细胞分离培养的标准方案，并对其生物学特性、功能及在化合物毒性试验中的应用进行表征。从中性诊所获得犬睾丸，应用三步酶消化法分离支持细胞。我们的特点是生长和纯度的支持细胞与表达的SOX 9，GATA 4，和aperterin。此外，我们选择镉作为模型毒物，以评估在新建立的支持细胞的毒性反应，使用高含量分析（HCA）。利用我们优化的方案，分离的支持细胞的纯度高于95%，如用支持细胞特异性蛋白标记物SOX 9和GATA 4所确定的。更重要的是，原代支持细胞群可以在体外快速扩增、传代（最多7代）和冷冻保存。基于HCA的测定显示，1 μM的镉可诱导细胞骨架破坏和DNA损伤反应。此外，我们建立了一个新的分离和优化培养的犬支持细胞的HCA检测，以评估组蛋白修饰的表观遗传标记。我们发现镉诱导的组蛋白修饰H3Me3K9，H3Me3K36，H4Me3K20和H4acK5的差异变化。综上所述，我们已经建立了标准化的方案，以生产支持细胞特异性表型的犬支持细胞。大量分离和扩增的犬支持细胞将为研究男性不育、生殖毒理学、睾丸癌和细胞治疗提供广泛的应用。
Sertoli cells play critical roles in regulating spermatogenesis and testis development by providing structural and nutritional support. This study aimed to develop a standard protocol for canine Sertoli cell isolation and culture; and characterize its biological features, functionality, and application of compound toxicity testing. Canine testicles were received from the neuter clinic, and three-step of enzymatic digestion was applied to isolate Sertoli cells. We characterized the growth and purity of Sertoli cells with the expression of SOX9, GATA4, and Clusterin. In addition, we selected cadmium as a model toxicant to evaluate the toxic responses in the newly established Sertoli cells using High-content Analysis (HCA). With our optimized protocol, the purity of isolated Sertoli cells was above 95%, as determined with Sertoli cell-specific protein markers of SOX9 and GATA4. More importantly, primary Sertoli cell populations could be expanded rapidly in vitro, passaged (up to seven), and cryopreserved. The HCA-based assay revealed that cadmium at 1 μM induced both disruptions of cytoskeletal and DNA damage responses. Furthermore, we established an HCA assay with the newly isolated and optimized culture of canine Sertoli cells to evaluate the epigenetic markers of histone modification. We found cadmium-induced differential changes in histone modifications H3Me3K9, H3Me3K36, H4Me3K20, and H4acK5. In summary, we have established the standardized protocol to produce canine Sertoli cells with Sertoli cell-specific phenotype. The isolation and expansion of large quantities of canine Sertoli cells will provide broad applications in studying male infertility, reproductive toxicology, testicular cancer, and cell therapy.
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影响因子：
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作者：
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通讯作者：
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作者：
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通讯作者：
Legube G