Two-generational impacts of airway exposure to silver nanoparticles in utero on male reproductive function in mice

This study investigated the effects of airway exposure to silver nanoparticles (AgNPs) in utero on male reproductive function. Exposure of pregnant ICR mice to AgNPs (low dose: 5 μg/injection; high dose: 50 μg/injection) at embryonic day 7.5 and 14.5, by means of intratracheal administration, resulted in dose-dependent exacerbation of sperm parameters in both F1 and F2 males. Microarray analysis revealed that the high-dose group displayed significant upregulation of four microRNAs (miRNAs) and downregulation of seven miRNAs, as compared to the control group, results that were further verified using quantitative real-time PCR. Interestingly, in the mature sperm as well, the high-dose group displayed significant upregulation of one, and downregulation of three of the miRNAs found to be dysregulated in the testes. Notably, in silico analyses predicted that the target genes of the differentially expressed miRNAs in the sperm were significantly associated with neuronal development. Neuronal migration in the cerebral cortex was dysregulated in the F2 males, accompanied by depression-like behavior. These observations demonstrated for the first time that prenatal exposure to AgNPs can induce germ cell development dysfunction in F1 males, which is involved in developmental toxicity in F2 males born to those F1 males. Effects of prenatal exposure to AgNPs on microRNA (miRNA) expression in the testes.Using total RNA extracted from the testes of 12-week-old mice, the expression of 1,908 mature miRNAs (1,908 probe sets) and 1,176 mouse pre-miRNAs (1,255 probe sets) was profiled using the GeneChip® miRNA Array service, according to the protocol of Filgen (Aichi, Japan). The RNA samples were labeled with biotin using the FlashTag™ Biotin HSR RNA Labeling Kit (Applied Biosystems, Foster City, Union City, USA), according to GeneChip® miRNA Arrays User Manual. Briefly, the labeled RNA was hybridized using GeneChip® Hybridization Oven 645 (Applied Biosystems); the plate used in the microarray was miRNA 4.0 Array (Applied Biosystems). After hybridization, the microarray was washed using GeneChip® Fluidics Station 450 (Applied Biosystems), the slides scanned using GeneChip® Scanner 3000 7G system (Applied Biosystems), and the images digitized using Array-Pro Analyzer Ver. 4.5 (Media Cybernetics, Silver Spring, MD, USA). Finally, the data were normalized and expressed as fold increase in comparison to the control, using the Expression Console™ Software (Applied Biosystems). We identified differentially expressed miRNAs with >1.2-fold upregulation or downregulation, along with a false discovery rate of <0.1.