Effects Of Nicotine On Sperm Acrosome Enzyme Activity And Acrosome Reaction In Mice

In recent years,infertility and abnormal embryonic development have become more and more seriously in the worldwide.It has become an indisputable fact that male fertility declines significantly.Clinically,patients with azoospermia and oligozoospermia are increasing.Quality problems such as sperm number,density,deformity,and motility have attracted attention.There are many factors that affect sperm quality,such as physical and chemical factors.Our previous research confirmed that when exposed to cigarette smoke,the testicular germ cells will have a corresponding stress response,and it will have the effect on spermatogenic cells,cell division,and serum hormone levels during sperm production.Our laboratory further confirmed that nicotine in cigarette smoke causes apoptosis in germ cells in the testis and it will increase the formation of autophagosomes in the cytoplasm of testicular stromal cells.In addition,nicotine also affects telomerase activity of spermatogenic cells,shortens telomere length,and causes structural damage of chromosomes.It is worth noting that after nicotine treatment,the expression of proteins involved in cytoskeleton regulation and energy metabolism in mouse testis was significantly up-regulated,and the sperm motility of mice was improved after nicotine treatment.However,it is unclear whether the sperm acrosome reaction and acrosome function will be abnormal after exposure to nicotine.Therefore,this study intends to observe the effects of nicotine on sperm acrosome and provide new ideas for smoking to affect male reproduction.First,we randomly divided male mice of C57BL/6J into nicotine group(n=20)and control group(n=20).Nicotine group was intraperitoneally injected with 0.2mg/100 g nicotine per day in 5weeks,and the control group was injected with equal volume of normal saline.RNA-Seq was then used to detect sperm RNA transcriptome expression levels before and after nicotine treatment.The results of RNA-Seq showed that the number of reads in the control group was 2089729,and the number of reads in the nicotine-treated group was 1739093,and a total of 8699 transcripts were obtained.Subsequently,the RPKM values of the control and nicotine RNA were corrected according to GAPDH.Based on the calibration results,we built a mathematical model.The model shows that RNA with RPKM values > 6 is constantly present insperm.Subsequently,we used this as a benchmark to screen for differential genes,and obtained 803 differential genes.The selected genes in the control group and the nicotine group were used to GO functional cluster analysis,which aim to obtain the concentrated distribution of the genes with different expression after nicotine treatment in the molecular function,biological process and cell component level.At the same time,the differentially expressed genes were screened for KEGG signaling pathway analysis.KEGG signaling pathway analysis indicated that genes differentially expressed after nicotine treatment mainly involved energy metabolism and protein metabolism signaling pathways.In the energy metabolism pathway,differential genes are mainly concentrated in the glycolytic signaling pathway,while in the protein metabolic pathway,they are mainly concentrated in the acrosomal(lysosomal enzyme)signaling pathway.Subsequent results showed that the acrosin activity and acrosome reaction rate were significantly increased by 1.48 times and 1.32 times respectively,after nicotine treatment.This result visually shows the effect of nicotine on the acrosome.To further reveal the molecular mechanism of this effect,we used Western blot to detect protein changes in the acrosomal enzyme(lysosomal enzyme)pathway.Western blot results showed that the expression of CLTC protein in the acrosomal(lysosomal)pathway was decreased by 1.42 times in the nicotine-treated group,which was significantly lower than that in the control group.This suggests that nicotine may increase the synthesis of acrosomal enzymes.In summary,this experimental study indicates that nicotine affects the expression of sperm genes at the transcriptome level during spermatogenesis.Through differential gene analysis,differential genes related to sperm acrosome function were discovered,and subsequent experiments demonstrated the inference results: nicotine down-regulates the reverse transport associated with acrosin(lysosomal enzyme)pathway during spermatogenesis The expression of CLTC protein up-regulates acrosomal enzyme activity and acrosome reaction rate,thus affecting the function of sperm acrosome.It is worth noting that the conclusions of this experimental study are helpful to understand the mechanism of the effect of nicotine on sperm acrosome function,and also provide insights for clinical male infertility and offspring embryo development.