Nicotine Decreases Serum Testosterone Via Autophagy In TM3 Cells

Many reports have shown that nicotine exposure have hazardous effects on male fertility.Our previous study found that the mouse’s testis,sperm and epididymis changed after nicotine treatment,and nicotine also had an adverse effect on reproduction.Some studies have pointed out that nicotine can reduce the synthesis of male hormones,but its mechanism is not yet clear.The purpose of this study is to clarify the reasons for the decrease of male hormone synthesis caused by nicotine.We selected forty 6-week-old C57BL/6J male mice and randomly divided them into control and nicotine-treated groups.To simulate the dose of nicotine in severe smokers,mice in the treatment group were injected intraperitoneally with nicotine at a dose of 0.2 mg/100 g/day for 5 weeks.Analysis of serum testosterone of nicotine treated mice was performed by the electrochemiluminescence method.Our results confirmed that the concentration of testosterone in the sera of nicotine-treated mice statistically decreased(NT vs CT = 1.10 ± 0.095 vs 1.66 ± 0.060,P <0.05).Meanwhile,the levels of other hypothalamic-pituitary-gonadal(HPG)axis-related hormones were slightly altered.Furthermore,the data of single cell transcriptome indicated that the expression of autophagy-related genes was increased after nicotine exposure.Likewise,chemical and immune-histological staining demonstrated that autophagy of the TM3 cells increased after nicotine treatment.Apoptosis mainly exists in spermatids.Further,the expression of autophagy-related genes,such as Beclin1 and LC3,were up-regulated after nicotine exposure.Additionally,the data of transmission electron microscopy showed more autophagosomes in the TM3 cells of the nicotine-exposed groups than the cells of the control groups.Lysosomes also were aggregated and increased.Moreover,immunofluorescent staining of Beclin1 in the TM3 cell line in vitro indicated that rapamycin and nicotine exposure up-regulates the autophagy process and down-regulates their testosterone synthesis.Our data also proved that the decrease of testosterone synthesis is caused by the decrease of StAR,the key enzyme in testosterone synthesis.The reason is that compared with the control group,after the nicotine treatment,the methylation level of the promoter region of the TCL1 gene in the mesenchymal cell increased,thereby reducing the expression of TCL1.Mechanistically,the autophagy in TM3 cells induced by nicotine,which is set by the hyper-methylation of the TCL1 promoter region via the TCL1-mTOR-autophagy signaling pathway.This mechanism provides a new avenue in understanding the process of decreased serum testosterone after nicotine exposure.