锌缺乏影响小鼠睾丸TGF-β1和FAK的表达并致圆形精子细胞脱落
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摘要
目的:探讨锌缺乏致小鼠精子发生障碍的机制。方法:4周龄CD-1雄性小鼠随机分为实验组(n=20)和对照组(n=20),实验组喂食低锌饲料(锌离子浓度为0. 85 ppm),对照组喂食锌正常饲料(锌离子浓度为30 ppm)。喂养5周后取睾丸和附睾标本,原子吸收光谱法检测睾丸组织中锌离子浓度,HE染色观察睾丸和附睾组织病理改变;采用H1T2和TRA54抗体进行免疫荧光双重染色鉴定脱落细胞的性质,Western印迹检测睾丸中ZO-1、FAK、TGF-β1、TGF-β2、TNF-α、Par6的表达。结果:实验组锌离子浓度显著低于对照组[(140. 59±16. 22)μg/gvs(218. 44±31. 92)μg/g,P <0. 05]。HE染色见对照组睾丸组织结构正常,生精小管密集,生精上皮完整,各级生精细胞层次清楚、排列有序,异常的生精小管占全部生精小管比例为0. 01±0. 01;实验组睾丸生精上皮退化,精子细胞数量减少,支持细胞胞质空泡化,生精小管闭塞,异常的生精小管数(0. 75±0. 04)显著多于对照组(P <0. 01),并在异常的生精小管和附睾的头、体、尾部发现脱落的细胞。H1T2和TRA54免疫荧光双重染色结果显示实验组附睾中脱落的细胞可能是圆形精子细胞。Western印迹结果显示对照组和实验组的蛋白表达水平分别为:ZO-1(1. 130±0. 054、0. 904±0. 052),FAK(0. 219±0. 048、0. 144±0. 047),Par6(0. 145±0. 047、0. 129±0. 049),TGF-β1(0. 586±0. 048、0. 024±0. 058),TGF-β2(0. 142±0. 048、0. 116±0. 047),TNF-α(0. 458±0. 023、0. 469±0. 022),其中实验组FAK和TGF-β1蛋白表达水平显著低于对照组(P <0. 05、<0. 01)。结论:锌缺乏能够引起小鼠睾丸组织病理改变,导致睾丸圆形精子细胞脱落,FAK和TGF-β1蛋白可能在锌缺乏引起小鼠睾丸病理改变过程中有重要的作用。
Objective:To investigate the mechanisms of zinc deficiency inducing spermatogenic disorders.Methods:Forty4-week-old CD-1 male mice were randomly divided into two groups of equal number:experimental and control,the former fed on a lowzinc diet and the latter on a normal diet,both for 5 weeks.Then all the mice were sacrificed and their testes and epididymides harvested for detection of the concentration of zinc ion in the testis by atomic absorption spectrophotometry,observation of the histopathological changes in the testis and epididymis by HE staining,examination of the properties of the exfoliated cells by dual immunofluorescence staining and determination of the expressions of ZO-1,FAK,TGF-β1,TGF-β2,TNF-α and Par6 proteins in the testicular tissue by Western blot.Results:The concentration of zinc ion in the testis was significantly lower in the experimental than in the control group([140.59 ± 16.22] vs [218.44 ± 31.29] μg/g,P < 0.05).HE staining showed normal testicular tissue structure,dense seminiferous tubules and intact seminiferous epithelium,with clear and orderly arrangement of spermatogenic cells at all levels in the control group.The ratio of the abnormal seminiferous tubules to the total number was 0.01 ± 0.01.The mice in the experimental group,however,exhibited degeneration of seminiferous epithelium,reduced number of spermatids,vacuolated cytoplasm of Sertoli cells,occluded seminiferous tubules,and a remarkably larger number of abnormal seminiferous tubules than that in the control(0.75 ± 0.04 vs 0.25 ± 0.04,P < 0.01).Exfoliated cells were observed in the abnormal tubules and the caput,corpus and cauda of the epididymis in the experimental group,which were shown to be immature round spermatids in H1T2 and TRA54 dual-immunofluore-scence staining.Western blot manifested that the protein expression of ZO-1 was 0.904 ± 0.052 vs 1.130 ± 0.054 in the experimental and control groups,that of Par6 was 0.129 ± 0.049 vs 0.145 ± 0.047,that of TGF-β2 was 0.116 ± 0.047 vs 0.142 ±0.048,and that of TNF-α was 0.469 ± 0.022 vs 0.458 ± 0.023,with significant decreases in the former group as compared with the latter in the levels of FAK(0.144 ± 0.047 vs 0.219 ± 0.048,P < 0.05) and TGF-β1(0.024 ± 0.058 vs 0.586 ± 0.048,P < 0.01).Conclusion:Zinc deficiency can induce histopathological changes in the testis of the mouse,leading to exfoliation of round spermatids,in which FAK and TGF-β1 may play an essential contributive role.
Objective:To investigate the mechanisms of zinc deficiency inducing spermatogenic disorders.Methods:Forty4-week-old CD-1 male mice were randomly divided into two groups of equal number:experimental and control,the former fed on a lowzinc diet and the latter on a normal diet,both for 5 weeks.Then all the mice were sacrificed and their testes and epididymides harvested for detection of the concentration of zinc ion in the testis by atomic absorption spectrophotometry,observation of the histopathological changes in the testis and epididymis by HE staining,examination of the properties of the exfoliated cells by dual immunofluorescence staining and determination of the expressions of ZO-1,FAK,TGF-β1,TGF-β2,TNF-α and Par6 proteins in the testicular tissue by Western blot.Results:The concentration of zinc ion in the testis was significantly lower in the experimental than in the control group([140.59 ± 16.22] vs [218.44 ± 31.29] μg/g,P < 0.05).HE staining showed normal testicular tissue structure,dense seminiferous tubules and intact seminiferous epithelium,with clear and orderly arrangement of spermatogenic cells at all levels in the control group.The ratio of the abnormal seminiferous tubules to the total number was 0.01 ± 0.01.The mice in the experimental group,however,exhibited degeneration of seminiferous epithelium,reduced number of spermatids,vacuolated cytoplasm of Sertoli cells,occluded seminiferous tubules,and a remarkably larger number of abnormal seminiferous tubules than that in the control(0.75 ± 0.04 vs 0.25 ± 0.04,P < 0.01).Exfoliated cells were observed in the abnormal tubules and the caput,corpus and cauda of the epididymis in the experimental group,which were shown to be immature round spermatids in H1T2 and TRA54 dual-immunofluore-scence staining.Western blot manifested that the protein expression of ZO-1 was 0.904 ± 0.052 vs 1.130 ± 0.054 in the experimental and control groups,that of Par6 was 0.129 ± 0.049 vs 0.145 ± 0.047,that of TGF-β2 was 0.116 ± 0.047 vs 0.142 ±0.048,and that of TNF-α was 0.469 ± 0.022 vs 0.458 ± 0.023,with significant decreases in the former group as compared with the latter in the levels of FAK(0.144 ± 0.047 vs 0.219 ± 0.048,P < 0.05) and TGF-β1(0.024 ± 0.058 vs 0.586 ± 0.048,P < 0.01).Conclusion:Zinc deficiency can induce histopathological changes in the testis of the mouse,leading to exfoliation of round spermatids,in which FAK and TGF-β1 may play an essential contributive role.
引文
[1]徐晨,宋宁.精子发生过程中的表观遗传学调控.中华男科学杂志,2014,20(5):387-391.
[2]顾娟,王一波,韩从辉.睾丸生精细胞体外培养的研究进展.中华男科学杂志,2009,15(8):742-745.
[3]Fanning AS,Anderson JM.Zonula occludens-1 and-2 are cytosolic scaffolds that regulate the assembly of cellular junctions.Ann N Y Acad Sci,2009,1165:113-120.
[4]Gungor-Ordueri NE,Mruk DD,Wan HT,et al.New insights into FAK function and regulation during spermatogenesis.Histol Histopathol,2014,29(8):977-989.
[5]Wan HT,Mruk DD,Tang El,et al.Role of non-receptor protein tyrosine kinases in spermatid transport during spermatogenesis.Semin Cell Dev Biol,2014,30:65-74.
[6]朱伟杰,黄瑞.睾丸支持细胞连接结构在精子发生过程的作用.生殖与避孕,2013,33(3):199-204.
[7]Li MW,Mruk DD,Lee WM,et al.Cytokines and junction restructuring events during spermatogenesis in the testis:An emerging concept of regulation.Cytokine Growth Factor Rev,2009,20(4):329-338.
[8]Suominen JS,Wang Y,Kaipia A,et al.Tumor necrosis factoralpha(TNF-alpha)promotes cell survival during spermatogenesis,and this affect can be blocked by infliximab,a TNF-alpha antagonist.Eur J Endocrinol,2004,151(5):629-640.
[9]Lydka M,Bilinska B,Cheng CY,et al.Tumor necrosis factorα-mediated restructuring of the sertoli cell barrier in vitro involves matrix metalloprotease 9(MMP9),membrane-bound intercellular adhesion molecule-1(ICAM-1)and the actin cytoskeleton.Spermatogenesis,2012,2(4):294-303.
[10]Gao Y,Lui WY.Transforming growth factor-β1(TGF-β1)regulates cell junction restructuring via Smad-msdiated repression and clathrin-mediated endocytosis of nectin-like molecule 2(Necl-2).PLo S One,2013,8(5):e64316.
[11]Wang Y,Lui WY.Opposite effects of interleukin-1alpha and transforming growth factor-beta2 induce stage-specific regulation of junctional adhesion molecule-B gene in sertoli cells.Endocrinology,2009,150(5):2404-2412.
[12]Li L,Gao Y,Chen H,et al.Cell polarity,cell adhesion,and spermatogenesis:Role of cytoskeletons.F1000Res,2017,6:1565.
[13]Gao Y,Cheng CY.Does cell polarity matter during spermatogenesis?Spermatogenesis,2016,6(2):e1218408.
[14]Fallah A,Mohammad-Hasani A,Colaqar AH.Zinc is an essential element for male fertility:A review of Zn roles in men's health,germination,sperm quality,and fertilization.J Reprod Infertil,2018,19(2):69-81.
[15]Yamaguchi S,Miura C,Kikuchi K,et al.Zinc is an essential trace element for spermatogenesis.Proc Natl Acad Sci USA,2009,106(26):10859-10864.
[16]Kumari D,Nair N,Bedwal RS.Effect of dietary zinc deficiency on testes of Wistar rats:Morphometric and cell quantification studies.J Trace Elem Med Biol,2011,25(1):47-53.
[17]Croxford TP,Mc Cormick NH,Kelleher SL.Moderate zinc deficiency reduces testicular Zip6 and Zip 10 abundance and impairs spermatogenesis in mice.J Nutr,2011,141(3):359-365.
[18]赵荣坡,熊承良.弱精子症、少弱精子症患者血清、精浆和精子锌含量分析.中华男科学杂志,2005,11(9):680-682.
[19]Martianov L,Brancorsini S,Catena R,et al.Polar nuclear localization of H1T2,a histone H1 variant,required for spermatid elongation and DNA condensation during spermiogenesis.Proc Natl Acad Sci U S A,2005,102(8):2808-2013.
[20]Ventela S,Toppari J,Parvinen M.Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat:Mechanisms of haploid gene product sharing.Mol Biol Cell,2003,14(7):2768-2780.
[21]Garcia PV,Arroteia KF,Joazeiro PP,et al.Orchidopexy restores morphometric-stereologic changes in the caput epididymis and daily sperm production in cryptorchidic mice,although sperm transit time and fertility parameters remain impaired.Fertil Steril,2011,96(3):739-744.
[22]Li SY,Mruk DD,Cheng CY.Focal adhesion kinase is a regulator of F-actin dynamics:New insights from studies in the testis.Spermatogenesis,2013,3(3):e25385.
[23]Ingman WV,Robertson SA.Defining the actions of transforming growth factor beta in reproduction.Bioessays,2002,24(10):904-914.
[24]Ingman WV,Robertson SA.The essential roles of TGF-β1 in reproduction.Cytokine Growth Factor Rev,2009,20(3):233-239.
[25]Teerds KJ,Dorrington JH.Localization of transforming growth factor beta 1 and beta 2 during testicular development in the rat.Biol Reprod,1993,48(1):40-45.
[26]Watrin F,Scotto L,Assoian RK,et al.Cell lineage specificity of expression of the murine transforming growth factor beta 3 and transforming growth factor beta 1 genes.Cell Growth Differ,1991,2(2):77-83.
[27]Ingman WV,Robertson SA.Transforming growth factor-beta1 null mutation causes infertility in male mice associated with testosterone deficiency and sexual dysfunction.Endocrinology,2007,148(8):4032-4043.
[2]顾娟,王一波,韩从辉.睾丸生精细胞体外培养的研究进展.中华男科学杂志,2009,15(8):742-745.
[3]Fanning AS,Anderson JM.Zonula occludens-1 and-2 are cytosolic scaffolds that regulate the assembly of cellular junctions.Ann N Y Acad Sci,2009,1165:113-120.
[4]Gungor-Ordueri NE,Mruk DD,Wan HT,et al.New insights into FAK function and regulation during spermatogenesis.Histol Histopathol,2014,29(8):977-989.
[5]Wan HT,Mruk DD,Tang El,et al.Role of non-receptor protein tyrosine kinases in spermatid transport during spermatogenesis.Semin Cell Dev Biol,2014,30:65-74.
[6]朱伟杰,黄瑞.睾丸支持细胞连接结构在精子发生过程的作用.生殖与避孕,2013,33(3):199-204.
[7]Li MW,Mruk DD,Lee WM,et al.Cytokines and junction restructuring events during spermatogenesis in the testis:An emerging concept of regulation.Cytokine Growth Factor Rev,2009,20(4):329-338.
[8]Suominen JS,Wang Y,Kaipia A,et al.Tumor necrosis factoralpha(TNF-alpha)promotes cell survival during spermatogenesis,and this affect can be blocked by infliximab,a TNF-alpha antagonist.Eur J Endocrinol,2004,151(5):629-640.
[9]Lydka M,Bilinska B,Cheng CY,et al.Tumor necrosis factorα-mediated restructuring of the sertoli cell barrier in vitro involves matrix metalloprotease 9(MMP9),membrane-bound intercellular adhesion molecule-1(ICAM-1)and the actin cytoskeleton.Spermatogenesis,2012,2(4):294-303.
[10]Gao Y,Lui WY.Transforming growth factor-β1(TGF-β1)regulates cell junction restructuring via Smad-msdiated repression and clathrin-mediated endocytosis of nectin-like molecule 2(Necl-2).PLo S One,2013,8(5):e64316.
[11]Wang Y,Lui WY.Opposite effects of interleukin-1alpha and transforming growth factor-beta2 induce stage-specific regulation of junctional adhesion molecule-B gene in sertoli cells.Endocrinology,2009,150(5):2404-2412.
[12]Li L,Gao Y,Chen H,et al.Cell polarity,cell adhesion,and spermatogenesis:Role of cytoskeletons.F1000Res,2017,6:1565.
[13]Gao Y,Cheng CY.Does cell polarity matter during spermatogenesis?Spermatogenesis,2016,6(2):e1218408.
[14]Fallah A,Mohammad-Hasani A,Colaqar AH.Zinc is an essential element for male fertility:A review of Zn roles in men's health,germination,sperm quality,and fertilization.J Reprod Infertil,2018,19(2):69-81.
[15]Yamaguchi S,Miura C,Kikuchi K,et al.Zinc is an essential trace element for spermatogenesis.Proc Natl Acad Sci USA,2009,106(26):10859-10864.
[16]Kumari D,Nair N,Bedwal RS.Effect of dietary zinc deficiency on testes of Wistar rats:Morphometric and cell quantification studies.J Trace Elem Med Biol,2011,25(1):47-53.
[17]Croxford TP,Mc Cormick NH,Kelleher SL.Moderate zinc deficiency reduces testicular Zip6 and Zip 10 abundance and impairs spermatogenesis in mice.J Nutr,2011,141(3):359-365.
[18]赵荣坡,熊承良.弱精子症、少弱精子症患者血清、精浆和精子锌含量分析.中华男科学杂志,2005,11(9):680-682.
[19]Martianov L,Brancorsini S,Catena R,et al.Polar nuclear localization of H1T2,a histone H1 variant,required for spermatid elongation and DNA condensation during spermiogenesis.Proc Natl Acad Sci U S A,2005,102(8):2808-2013.
[20]Ventela S,Toppari J,Parvinen M.Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat:Mechanisms of haploid gene product sharing.Mol Biol Cell,2003,14(7):2768-2780.
[21]Garcia PV,Arroteia KF,Joazeiro PP,et al.Orchidopexy restores morphometric-stereologic changes in the caput epididymis and daily sperm production in cryptorchidic mice,although sperm transit time and fertility parameters remain impaired.Fertil Steril,2011,96(3):739-744.
[22]Li SY,Mruk DD,Cheng CY.Focal adhesion kinase is a regulator of F-actin dynamics:New insights from studies in the testis.Spermatogenesis,2013,3(3):e25385.
[23]Ingman WV,Robertson SA.Defining the actions of transforming growth factor beta in reproduction.Bioessays,2002,24(10):904-914.
[24]Ingman WV,Robertson SA.The essential roles of TGF-β1 in reproduction.Cytokine Growth Factor Rev,2009,20(3):233-239.
[25]Teerds KJ,Dorrington JH.Localization of transforming growth factor beta 1 and beta 2 during testicular development in the rat.Biol Reprod,1993,48(1):40-45.
[26]Watrin F,Scotto L,Assoian RK,et al.Cell lineage specificity of expression of the murine transforming growth factor beta 3 and transforming growth factor beta 1 genes.Cell Growth Differ,1991,2(2):77-83.
[27]Ingman WV,Robertson SA.Transforming growth factor-beta1 null mutation causes infertility in male mice associated with testosterone deficiency and sexual dysfunction.Endocrinology,2007,148(8):4032-4043.