Gene expression profiling of cultured mouse testis fragments treated with ethinylestradiol

Abstract

The assessment of xenobiotic-induced testicular toxicity is important in drug development. Nonetheless, in vitro models to test drugs and chemicals that may cause testicular toxicity are lacking, requiring the continued use of animal models for those studies. We previously evaluated an in vitro mouse testis organ culture system using ethinylestradiol (EE), a well-studied testicular toxicant, and demonstrated a dose-dependent relationship between adverse effects to germ cell differentiation and increasing EE concentrations. However, we terminated that study after 20 days of culture due to oxygen deficiency during germ cell differentiation. Therefore, in the current study, we aimed to identify gene(s) with potential for supporting the histopathological evaluations of testicular toxicity using in vitro testis organ culture system. We cultured testis fragments obtained from mice at postnatal day (PND) 5 in α-Minimal Essential Medium containing 40 mg/mL AlbuMAX ™ I and treated them with 0.01 or 1 nM EE on day 1 of culture. On day 20, we collected testis fragments for RNA sequencing analysis and quantitative polymerase chain reaction (qPCR). We found that phospholipase C, zeta 1 and testis-specific serine kinase 4 genes, that are involved in spermatogenesis and predominantly expressed in the testis, were significantly reduced in testis fragments treated with the highest concentration of EE. Also, cytochrome P450, family 26, subfamily b, polypeptide 1 (Cyp26b1) and interleukin 16 (Il16) were up-regulated in the highest EE-treated groups. Further studies are needed to confirm the variations of these gene expression using other testicular toxicants.

Cocktails of NSAIDs and 17α Ethinylestradiol at Environmentally Relevant Doses in Drinking Water Alter Puberty Onset in Mice Intergenerationally

“…Comparison of these DEGs with those identified after exposure to other EDCs indicated that 1 to 17 DEGs detected in F1 testes were also deregulated after exposure to the xenoestrogens bisphenol A (BPA) [ 86 ] and EE2 [ 87 ] and to the anti-androgenic EDCs di-2-ethylhexyl phthalate (DEHP) [ 88 ], DDT, and vinclozolin [ 89 ] ( Supplementary Table S9 ). Some genes that were also differentially expressed in F2 testes might be biomarkers of exposure to estrogenic ( Actn3 , Cntnap5b, Kcnk2 , Sall1 ) or anti-androgenic ( Dhrs4 , Fabp3 ) compounds.…”