Nicotine Inhibits Murine Leydig Cell Differentiation And Maturation Via Regulating Hedgehog Signal Pathway

Nicotine,the main toxic substance in cigarette smoke and e-cigarette,inhibits male reproductive function by inducing autophagy.In this study,nicotine significantly reduced the differentiation and maturation ratio of Leydig cell in murine testes.As the main cell type which can realize synthesis and secretion of androgen,Leydig cell plays an important role in male reproduction.To investigate the underlying mechanism,C57BL/6J mice were divided into control(CT)and nicotine treated(NT)groups.Next generation RNA sequencing and bio-informatics analysis were carried out to analysis the effects of nicotine on the RNA profile of Leydig cells.There were 6377 extensive existing mRNAs discovered in our sequencing.Among these,there were 3670 up-regulated genes and 2706 down-regulated genes.By calculating the value of NT-RPKM/CT-RPKM,561 mRNAs show the fold change over 1000.According to the analysis via DAVID,expression level of 7 pathways remarkably changed after nicotine treatment: metabolic pathways,intestinal immune network for IgA production,pantothenate and CoA biosynthesis,Influenza A,Hedgehog signaling pathway,Hippo signaling pathway and circadian rhythm.As the positive regulating pathway of Leydig cell differentiation,Hedgehog signaling pathway was found among these pathways.PTCH1 and β-TrCP were down-regulated in nicotine treated mice Leydig cells,while GSK3β,GLI2 and GLI2 fragments increased significantly.Nicotine stimulated the destabilization of GLI2 via β-TrCP induced ubiquitination and degradation.GLI2 was phosphorylated by up-expressed GSK3β during this process.Destabilization of GLI2 reduced the activation rate of target genes of Hedgehog signaling pathway such as Ptch1.The differentiation of Leydig cell positively regulated by Hh pathway was thus inhibited by nicotine exposure.Consequently,the male reproduction process powered by Leydic cell-mediated androgen secretion was thus influenced.In conclusion,we find that nicotine inhibits murine Leydig cell differentiation and maturation via regulating Hedgehog signal pathway.