Effects Of Cigarette Smoke On Reproductive Capacity In Male Mice And Bioinformatics Analysis Of Male Infertility

Objective: Infertility affects nearly 10 %-15 % of the couples,which has become a global public health problem.Among them,the male factor accounts for about 40 %-50 %,and this number is still increasing.Cigarette smoke-induced male infertility has been widely concerned by researchers over the past decades.However,the exact mechanism needs further exploration.Our previous study identified a differential methylated outer dense fibrous 1(ODF1)gene,and pyrosequencing in human semen samples further confirmed that ODF1 was hypermethylated in smokers.In the following study,the smoking mouse model was constructed by simulating a passive smoking environment in vitro.This study aims to investigate the effects of cigarette smoke on male reproductive system including testicular morphological changes,sperm motility and expressions of outer dense fibrous 1(ODF1)in smoking mouse model,and further elucidate the underlying the mechanism of cigarette smoke on the male reproductive health.Additionally,the expressions of ODF1 and other differential genes in male infertility were studied by bioinformatics analysis to further explore the mechanism of male infertility.Methods: 1.A smoking mouse model: six to eight weeks old C57BL/6J healthy male mice were selected to be treated with cigarette smoke for two weeks,four or six weeks by simulating passive smoking environment in vitro as described in previous studies,serum cotinine was detected by the ELISA kits.2.Experimental verification:(1)the histological changes in testicular tissues induced by cigarette smoke were observed by H&E staining;(2)sperm motility was counted by the hemocytometer;(3)the immunohistochemistry was used to detect the ODF1 protein in testicular tissue;(4)the expressions of ODF1 mRNA in testicular tissue were assayed by real-time quantitative PCR.3.Bioinformatics analysis:(1)the expressions of ODF1 gene in male infertility were studied by GEO2R;(2)the differential genes based on R / Bioconductor /DAVID in male infertility were analyzed.Results: 1.Serum cotinine was significantly higher in adult male mice exposed with cigarette smoke for two weeks,four or six weeks than the control group(all P < 0.05).Serum cotinine was up to(10.76 ± 2.19)ng/ml after six weeks of tobacco smoke exposure,which indicated the successful construction of smoking mouse model.2.The seminiferous epithelium in mouse testicular tissue was damaged by cigarette smoke,including spermatogenic cell deletion and germ cell apoptosis.3.The expressions of ODF1 mRNA in testicular tissue were significantly lower in six weeks of tobacco smoke exposure than in the control group(P < 0.05).4.Sperm motility was not significantly different among these groups after treatment with cigarette smoke.5.The expressions of ODF1 gene were statistically significant in patients with teratozoospermia or non-obstructive azoospermia compared with the control groups in the datasets of GSE6872 and GSE45885.Conclusion: 1.This study has successfully established the smoking mousemodel and may provide the theory and the experimental basis for subsequent studies and other diseases.2.Cigarette smoke affects the spermatogenesis by inducing germ cell apoptosis.3.Cigarette smoke may modulate the methylation levels of ODF1 and affect the expression of ODF1 mRNA and protein,which induces male infertility.4.The ODF1 gene is expected to be the screening index of male infertility.