| Background: The composition of cigarette smoke is very complex. Manycomponents of cigarette smoke are hazardous to health. Epidemiologicalresearch showed that there was a high correlation between smoking andmale fertility. Smoking could reduce sperm density in the semen, reducesperm motility, cause abnormal sperm morphology and decrease thepregnancy rate of IVF, which resulted in the depression of male fertility.Besides, smoking could even affect the health of the offspring. Manyresearchers tried to find the mechanism through which smoking affectmale fertility. Using methods include comet, TUNEL and SCSA, it wasproved that smoking could cause sperm DNA fragmentation,chromosome structural abnormalities and chromosome aneuploidy. It wasalso reported that smoking could change the hormone level in the serum,which would affect spermatogenesis. However, rare research has beendone on the molecular mechanism through which smoking affact semenquality using proteomic method.To the male reproductive system, testis and epididymis were the key place where spermatogenesis and sperm maturation occurred. Using thepre-established mice smoking model, this study aim to find out theproteins whose expressions were changed significantly after cigarettesmoke exposure in testis and epididymis, analysis their functions andtheir relationship with male fertility and figure out the molecularmechanisms through which cigarette smoke impact on male fertility.Research method: C57/BL6J mice at the age of7weeks were treatedwith cigarette smoke twice a day,1hour for each time while the mice ofcontrol group were treated without cigarette smoke. After being treatedfor2weeks, the testis and epididymis of these mice were collected andwhole proteins were extracted to perform2-DE. The results of2-DE werecompared and protein spots whose expressions were changedsignificantly were selected to be analyzed using MALDI-TOF-MS toidentify the proteins which could be represented by these protein spots.Several identified differential expressed proteins were selected, andWestern-blotting was used to detect their expression in the correspondingtissues, verifying the results of2-DE and MS. The transcript level of theirgenes in corresponding tissues was also detected through Real-time PCRto compare the differences between the two groups.Blast2GO was used to annotate the functions of these differentialexpressed proteins identified by2-DE/MS and classify them into groupsaccording to the Gene Ontology functional annotation. KEGG and IPA were used to analyze the pathways in which these proteins were involved,and the main pathways which were impacted by cigarette smoke exposurewere figured out. Immunohistochemistry were used to detect thedownstream markers of these pathways, verifying the results of thepathway analysis.Results: Over1000protein spots were found to be differential expressedfrom the testis and epididymis of the mice treated by cigarette smoke. Theexpressions of27protein spots from the testis changed over1.5foldwhile that of52protein spots from the epididymis changed over2fold,which was thought to have a significant change. These protein spots wereselected to be analyzed by MALDI-TOF-MS and27of them wereidentified as27proteins from the testis and the epididymis respectively.In the27proteins indentified from testis, the expressions of6proteinswere up-regulated and that of21proteins were down-regulated, while inthe27proteins indentified from epididymis, the expressions of12proteins were up-regulated and that of15proteins were down-regulated.Using western-blotting, the tendency of the expressions of these proteinswas proved to be identical to the results of2-DE/MS. So the results of2-DE/MS were thought to be reliable.IPA was used to perform the pathway analysis of the differentialexpressed proteins from the testis. Many differential expressed proteinswere found to have interaction with NF-κB, and the expression or activation of NF-κB could be suppressed by cigarette smoke exposureaccording to the pathway analysis. The subunit of NF-κB, p65, wasdetected on the testis sections to see whether the activation level ofNF-κB was changed after treatment and it was proved that the expressionor activation of NF-κB was inhibited in the germ cell in the testis of themice treated with cigarette smoke. The normal expression and activationof NF-κB was important for the proliferation and differentiation of thegerm cell in testis. Thus, the inhibition of the NF-κB expression oractivation caused by cigarette smoke exposure could affect the spermquality.KEGG was used to perform the pathway analysis of the differentialexpressed proteins from the epididymis. It was found that in theepididymis of the mice treated with cigarette smoke, differentialexpressed proteins were mainly enriched in glutathione metabolismpathway and ERAD, and many differential expressed proteins wererelated to the maintaining of redox homeostasis, which implied that theepididymis of the mice treated with cigarette smoke experiencedoxidative stress. The antibody of the specific marker of oxidative stress,8-OHdG, was used to detected8-OHdG on epididymis sections in situthrough immunohistochemistry, and it was proved that the epididymis ofthe mice treated with cigarette smoke experienced a serious oxidativestress. Oxidative stress could induce ERAD, altering the synthesis of the proteins, while the change tendency of transcript level of thecorresponding genes was not identical to that of the proteins according tothe results of real-time PCR, which suggested that the altering of proteinprofile in the epididymis of the mice treated with cigarette smoke wascaused by ERAD via oxidative stress.Besides, after being annotated and analysis, many other differentialexpressed proteins in the testis and epididymis truned out to have relationwith male fertility.Conclusion: cigarette smoke exposure could alter the protein profile inmice testis and epididymis. It could disturb normal proliferation anddifferentiation of germ cells via the inhibition of NF-κB pathway in tesitisand disturb the synthesis of proteins via ERAD induced by oxidativestress in epididymis, impairing the function of these organs. Otherdifferential expressed proteins also play roles in the control of spermquality. Through these ways, cigarette smoke exposure depressed spermquality and the fertility of male mice. |
PDF Full Text Request