香烟烟雾对雄性小鼠睾丸与附睾影响的蛋白质组学研究
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摘要
研究背景与目的：香烟烟雾成分复杂，许多成分能够危害人体健康。流行病学调查显示，吸烟行为与男性的生育能力之间具有较高的相关性，其主要表现为精液中精子密度降低、精子运动能力减弱、精子形态异常、体外受精受孕率降低。这一现象直接导致了吸烟男性生育能力的降低，甚至因而影响到后代的健康。很多研究者试图找出吸烟行为降低男性生育能力的原因，包括彗星电泳法（comet）、末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记法（TUNEL）与精子染色体结构分析（SCSA）在内的各种方法证实了吸烟会导致精子DNA的断裂、染色体的结构异常以及染色体非整倍性的发生，也有研究显示吸烟会改变血清中激素的水平，从而影响精子的生成。
雄性生殖系统中，睾丸是精子生成的主要场所，而附睾是精子成熟的主要场所，这两个器官对精子与精液的质量具有非常重要的作用。我们的研究使用实验室先期建立的小鼠吸烟模型，运用蛋白质组学的方法来观察香烟烟雾处理后小鼠睾丸与附睾中蛋白质总体的表达情况，找出表达水平较没有使用香烟烟雾处理的小鼠睾丸与附睾中的相应蛋白质发生显著差异的蛋白质，结合生物信息学的方法分析这些蛋白质的功能以及它们与生育能力之间的关系，试图找出香烟烟雾处理对雄性生育能力造成影响的分子机制。
研究方法：七周龄雄性C57/BL6J小鼠每日用香烟烟雾处理两次，每次一小时，持续处理两周，对照组小鼠不用香烟烟雾处理。处理期满后取小鼠睾丸与附睾，分别提取总蛋白进行双向电泳，比较处理组小鼠与对照组小鼠相关组织的双向电泳结果，挑选两者间表达水平发生显著差异的蛋白质点切胶进行基质辅助激光解吸/电离飞行时间质谱分析，以鉴定出这些蛋白质点所代表的蛋白质。从质谱分析鉴定得到的蛋白质中随机取数个蛋白质，使用Western-blotting的方法对这些蛋白质在处理组小鼠与对照组小鼠相关组织中表达水平差异的趋势进行验证，以确定双向电泳与质谱分析的结果是否可靠，同时利用Real-time PCR的方法对这些蛋白相应的基因在相应器官中的转录水平进行检测，以观察这些基因在处理组与对照组小鼠之间转录水平的差异。
将得到验证的由双向电泳与质谱分析得到的香烟烟雾处理后小鼠睾丸与附睾组织中表达水平发生变化的蛋白质使用Blast2GO进行功能注释，同时从生物学过程、分子功能两个层次上对这些蛋白质进行基于基因本体论的归类。使用通路分析工具KEGG与IPA对这些蛋白质所涉及的通路进行分析，找出受到影响的主要通路与生物学过程。对找出的通路的下游作用标志物使用免疫组织化学的方法在组织水平上对组织切片进行检测，以验证通路分析得到的结果与推测。
研究结果：通过双向电泳，我们发现香烟烟雾处理后的小鼠睾丸与附睾组织中各自有超过1000个蛋白点的表达水平与对照组小鼠存在差异，其中睾丸组织中有27个蛋白点的表达水平差异在1.5倍以上，附睾组织中有52个蛋白点的表达水平差异在2倍以上，可以认为差异水平显著。挑选这些在香烟烟雾处理后的小鼠睾丸与附睾组织中表达水平差异显著的蛋白点进行质谱分析，睾丸与附睾组织中分别有27个蛋白点成功地得到了鉴定，它们的表达水平各自可以用来代表27种相应蛋白质的表达水平。在小鼠睾丸中，得到鉴定的27种蛋白质中的6种蛋白质表达水平上调，21种蛋白质表达水平下调；在小鼠附睾中，得到鉴定的27种蛋白质中的12种蛋白质表达水平上调，15种蛋白质的表达水平下调。分别随机挑选睾丸组织中的5种蛋白质和附睾组织中的8种蛋白质，使用相应组织进行Western-blotting对双向电泳与质谱分析得到的结果进行验证，结果显示Western-blotting检测得到的处理组与对照组间表达差异的趋势与双向电泳和质谱分析得到的表达差异趋势基本一致，因此认为双向电泳与质谱分析的结果是可靠的。
将通过双向电泳与质谱分析得到的差异表达蛋白上传至IPA进行通路分析，发现在香烟烟雾处理后的小鼠睾丸中，表达水平与对照组小鼠具有显著差异的蛋白质中很多都能够直接或间接地与NF-κB发生作用，且根据通路分析它们的表达水平变化趋势能够抑制NF-κB的表达或激活。使用NF-κB复合体p65亚基的抗体在相应的睾丸组织切片上使用免疫组织化学的方法检测NF-κB的表达与激活情况，证实相较于对照组小鼠睾丸组织，香烟烟雾处理后的小鼠睾丸组织中生殖细胞内的NF-κB的表达或激活遭到了抑制。NF-κB在这些细胞中的正常水平的表达与激活对维持生殖细胞的正常增殖与精子的分化具有非常重要的意义，香烟烟雾抑制了这些细胞中的NF-κB的表达与激活，可能会通过影响到生殖细胞的正常增殖与精子的分化而影响到精液的质量。
同样的，将通过双向电泳与质谱分析得到的差异表达蛋白上传至KEGG进行通路分析后发现，在香烟烟雾处理后的小鼠附睾中，表达水平于对照组小鼠具有显著差异的蛋白质比较集中作用的通路有谷胱甘肽代谢通路与内质网相关降解途径（ERAD），且根据Blast2GO对这些蛋白质的注释，其中很多蛋白质均与维持机体正常的氧化还原稳态相关。这一结果暗示了香烟烟雾处理后小鼠附睾处于氧化应激状态。使用氧化应激的特异性标志物8-OHdG抗血清在相应的附睾组织切片上使用免疫组织化学的方法检测8-OHdG的存在，结果显示香烟烟雾处理后的小鼠附睾处于较为严重的氧化应激状态中。氧化应激能够引起ERAD从而影响蛋白质的正常合成，而Real-time PCR的结果显示处理组与对照组的小鼠附睾组织间差异表达蛋白在相应基因的转录水平上差异并不显著，且差异趋势与蛋白质水平的差异趋势不一致，这一点提示我们也许是氧化应激引起的ERAD导致了蛋白表达水平的差异。
此外，在对其他经过香烟烟雾处理后的小鼠睾丸与附睾组织中表达存在显著差异的蛋白经过注释与分析后我们发现，它们中的很多都能够通过不同途径影响到生育能力。
结论：香烟烟雾处理能够导致小鼠睾丸和附睾这两个重要的生殖器官中蛋白表达水平的变化，在睾丸中香烟烟雾能够通过对NF-κB途径的抑制干扰生殖细胞的正常增殖与精子的分化，在附睾中则能够通过造成氧化应激诱导ERAD的发生，从而影响蛋白质的正常合成，进一步损害附睾正常的功能。结合其他表达发生显著差异的蛋白对精子的影响，香烟烟雾最终通过这些途径降低精子的质量，从而使雄性小鼠的生育能力下降。
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.
雄性生殖系统中，睾丸是精子生成的主要场所，而附睾是精子成熟的主要场所，这两个器官对精子与精液的质量具有非常重要的作用。我们的研究使用实验室先期建立的小鼠吸烟模型，运用蛋白质组学的方法来观察香烟烟雾处理后小鼠睾丸与附睾中蛋白质总体的表达情况，找出表达水平较没有使用香烟烟雾处理的小鼠睾丸与附睾中的相应蛋白质发生显著差异的蛋白质，结合生物信息学的方法分析这些蛋白质的功能以及它们与生育能力之间的关系，试图找出香烟烟雾处理对雄性生育能力造成影响的分子机制。
研究方法：七周龄雄性C57/BL6J小鼠每日用香烟烟雾处理两次，每次一小时，持续处理两周，对照组小鼠不用香烟烟雾处理。处理期满后取小鼠睾丸与附睾，分别提取总蛋白进行双向电泳，比较处理组小鼠与对照组小鼠相关组织的双向电泳结果，挑选两者间表达水平发生显著差异的蛋白质点切胶进行基质辅助激光解吸/电离飞行时间质谱分析，以鉴定出这些蛋白质点所代表的蛋白质。从质谱分析鉴定得到的蛋白质中随机取数个蛋白质，使用Western-blotting的方法对这些蛋白质在处理组小鼠与对照组小鼠相关组织中表达水平差异的趋势进行验证，以确定双向电泳与质谱分析的结果是否可靠，同时利用Real-time PCR的方法对这些蛋白相应的基因在相应器官中的转录水平进行检测，以观察这些基因在处理组与对照组小鼠之间转录水平的差异。
将得到验证的由双向电泳与质谱分析得到的香烟烟雾处理后小鼠睾丸与附睾组织中表达水平发生变化的蛋白质使用Blast2GO进行功能注释，同时从生物学过程、分子功能两个层次上对这些蛋白质进行基于基因本体论的归类。使用通路分析工具KEGG与IPA对这些蛋白质所涉及的通路进行分析，找出受到影响的主要通路与生物学过程。对找出的通路的下游作用标志物使用免疫组织化学的方法在组织水平上对组织切片进行检测，以验证通路分析得到的结果与推测。
研究结果：通过双向电泳，我们发现香烟烟雾处理后的小鼠睾丸与附睾组织中各自有超过1000个蛋白点的表达水平与对照组小鼠存在差异，其中睾丸组织中有27个蛋白点的表达水平差异在1.5倍以上，附睾组织中有52个蛋白点的表达水平差异在2倍以上，可以认为差异水平显著。挑选这些在香烟烟雾处理后的小鼠睾丸与附睾组织中表达水平差异显著的蛋白点进行质谱分析，睾丸与附睾组织中分别有27个蛋白点成功地得到了鉴定，它们的表达水平各自可以用来代表27种相应蛋白质的表达水平。在小鼠睾丸中，得到鉴定的27种蛋白质中的6种蛋白质表达水平上调，21种蛋白质表达水平下调；在小鼠附睾中，得到鉴定的27种蛋白质中的12种蛋白质表达水平上调，15种蛋白质的表达水平下调。分别随机挑选睾丸组织中的5种蛋白质和附睾组织中的8种蛋白质，使用相应组织进行Western-blotting对双向电泳与质谱分析得到的结果进行验证，结果显示Western-blotting检测得到的处理组与对照组间表达差异的趋势与双向电泳和质谱分析得到的表达差异趋势基本一致，因此认为双向电泳与质谱分析的结果是可靠的。
将通过双向电泳与质谱分析得到的差异表达蛋白上传至IPA进行通路分析，发现在香烟烟雾处理后的小鼠睾丸中，表达水平与对照组小鼠具有显著差异的蛋白质中很多都能够直接或间接地与NF-κB发生作用，且根据通路分析它们的表达水平变化趋势能够抑制NF-κB的表达或激活。使用NF-κB复合体p65亚基的抗体在相应的睾丸组织切片上使用免疫组织化学的方法检测NF-κB的表达与激活情况，证实相较于对照组小鼠睾丸组织，香烟烟雾处理后的小鼠睾丸组织中生殖细胞内的NF-κB的表达或激活遭到了抑制。NF-κB在这些细胞中的正常水平的表达与激活对维持生殖细胞的正常增殖与精子的分化具有非常重要的意义，香烟烟雾抑制了这些细胞中的NF-κB的表达与激活，可能会通过影响到生殖细胞的正常增殖与精子的分化而影响到精液的质量。
同样的，将通过双向电泳与质谱分析得到的差异表达蛋白上传至KEGG进行通路分析后发现，在香烟烟雾处理后的小鼠附睾中，表达水平于对照组小鼠具有显著差异的蛋白质比较集中作用的通路有谷胱甘肽代谢通路与内质网相关降解途径（ERAD），且根据Blast2GO对这些蛋白质的注释，其中很多蛋白质均与维持机体正常的氧化还原稳态相关。这一结果暗示了香烟烟雾处理后小鼠附睾处于氧化应激状态。使用氧化应激的特异性标志物8-OHdG抗血清在相应的附睾组织切片上使用免疫组织化学的方法检测8-OHdG的存在，结果显示香烟烟雾处理后的小鼠附睾处于较为严重的氧化应激状态中。氧化应激能够引起ERAD从而影响蛋白质的正常合成，而Real-time PCR的结果显示处理组与对照组的小鼠附睾组织间差异表达蛋白在相应基因的转录水平上差异并不显著，且差异趋势与蛋白质水平的差异趋势不一致，这一点提示我们也许是氧化应激引起的ERAD导致了蛋白表达水平的差异。
此外，在对其他经过香烟烟雾处理后的小鼠睾丸与附睾组织中表达存在显著差异的蛋白经过注释与分析后我们发现，它们中的很多都能够通过不同途径影响到生育能力。
结论：香烟烟雾处理能够导致小鼠睾丸和附睾这两个重要的生殖器官中蛋白表达水平的变化，在睾丸中香烟烟雾能够通过对NF-κB途径的抑制干扰生殖细胞的正常增殖与精子的分化，在附睾中则能够通过造成氧化应激诱导ERAD的发生，从而影响蛋白质的正常合成，进一步损害附睾正常的功能。结合其他表达发生显著差异的蛋白对精子的影响，香烟烟雾最终通过这些途径降低精子的质量，从而使雄性小鼠的生育能力下降。
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.
引文
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