Ubi-p63E基因在果蝇睾丸生殖干细胞微环境中的功能研究
|
摘要
目的 Ubi-p63E基因是否调控精子发生和肿瘤形成过程还尚不清楚。文章讨论Ubi-p63E基因在果蝇睾丸生殖干细胞(GSCs)微环境中对GSCs的影响。方法利用UAS-Gal4系统实现果蝇睾丸细胞特异性敲减。实验分为对照组(W1118品系果蝇),nos>Ubi-p63E RNAi组(早期生殖细胞中敲减Ubi-p63E基因)和tj>Ubi-p63E RNAi组(包囊细胞中敲减Ubi-p63E基因),利用生育率测试观察雄性果蝇的生育能力。采用免疫荧光染色观察对照组和实验组雄性果蝇睾丸在GSCs微环境中的作用。结果生育率测试结果表明,与对照组生育率(97.26%)相比,nos>Ubi-p63E RNAi组(0)和tj>Ubi-p63E RNAi组生育能力(4.12%)明显降低(P<0.01)。光镜下观察,与对照组相比,nos>Ubi-p63E RNAi组和tj>Ubi-p63E RNAi组睾丸形态明显异常,长度分别为对照组的22.77%、18.86%,均出现小睾丸的表型。免疫荧光结果显示,与对照组睾丸比较,tj>Ubi-p63E RNAi组正常睾丸形态消失,且出现较多异常的细胞团块,这些细胞团大多为Vasa阳性细胞。结论 Ubi-p63E基因在果蝇睾丸GSCs微环境中影响GSCs的维持,并可以通过包囊细胞影响GSCs的分化过程,最终导致生殖细胞肿瘤的形成。
Objective Whether the Ubi-p63E gene regulates spermatogenesis and tumorigenesis remains unclear. This study aimed to explore the regulatory effect of Ubi-p63E on germline stem cells(GSC) in the GSC niche of the Drosophila testis.Methods We used the UAS-Gal4 system for knockdown of the Ubi-p63E gene in the specific GSCs of the Drosophila testis and divided the male flies for this experiment into three groups:control(wild-type W1118 flies),nos>Ubi-p63E RNAi(knockdown of the Ubip63E gene in the early germ cells),and tj>Ubi-p63E RNAi(knockdown of the Ubi-p63E gene in the cystoblasts). We determined the fertility rate of the flies by fertility tests and examined the effect of Ubi-p63E on the Drosophila testis in the GSC niche by immunofluorescence staining.Results Fertility tests manifested a significantly lower rate of fertility in the nos>Ubi-p63E RNAi and tj>Ubi-p63E RNAi groups than in the control(0.00% and 4.12% vs 97.26%,P <0.01). Morphologically abnormal testes were observed in the nos>Ubip63E RNAi and tj>Ubi-p63E RNAi groups,only 22.77% and 18.86% as long as the testes of the control flies. Immunofluorescence staining revealed no morphologically normal testes in the tj>Ubip63E RNAi group,but quite a few masses of abnormal cells,and mostly Vasa-positive cells.Conclusion The Ubi-p63E gene affects the self-renewal ability of GSCs in the GSC niche of the Drosophila testis as well as the differentiation of GSCs via cystoblasts,and consequently leads to the formation of germ cell tumors.
Objective Whether the Ubi-p63E gene regulates spermatogenesis and tumorigenesis remains unclear. This study aimed to explore the regulatory effect of Ubi-p63E on germline stem cells(GSC) in the GSC niche of the Drosophila testis.Methods We used the UAS-Gal4 system for knockdown of the Ubi-p63E gene in the specific GSCs of the Drosophila testis and divided the male flies for this experiment into three groups:control(wild-type W1118 flies),nos>Ubi-p63E RNAi(knockdown of the Ubip63E gene in the early germ cells),and tj>Ubi-p63E RNAi(knockdown of the Ubi-p63E gene in the cystoblasts). We determined the fertility rate of the flies by fertility tests and examined the effect of Ubi-p63E on the Drosophila testis in the GSC niche by immunofluorescence staining.Results Fertility tests manifested a significantly lower rate of fertility in the nos>Ubi-p63E RNAi and tj>Ubi-p63E RNAi groups than in the control(0.00% and 4.12% vs 97.26%,P <0.01). Morphologically abnormal testes were observed in the nos>Ubip63E RNAi and tj>Ubi-p63E RNAi groups,only 22.77% and 18.86% as long as the testes of the control flies. Immunofluorescence staining revealed no morphologically normal testes in the tj>Ubip63E RNAi group,but quite a few masses of abnormal cells,and mostly Vasa-positive cells.Conclusion The Ubi-p63E gene affects the self-renewal ability of GSCs in the GSC niche of the Drosophila testis as well as the differentiation of GSCs via cystoblasts,and consequently leads to the formation of germ cell tumors.
引文
[1]de Cuevas M,Matunis EL.The stem cell niche:lessons from the Drosophila testis[J].Development,2011,138(14):2861-2869.
[2]Fuller MT,Spradling AC.Male and female Drosophila germline stem cells:two versions of immortality[J].Science,2007,316(5823):402-404.
[3]Spradling A,Fuller MT,Braun RE,et al.Germline stem cells[J].Cold Spring Harb Perspect Biol,2011,3(11):a002642.
[4]Roessler E,Ouspenskaia MV,Karkera JD,et al.Reduced NODAL signaling strength via mutation of several pathway members including FOXH1 is linked to human heart defects and holoprosencephaly[J].Am J Hum Genet,2008,83(1):18-29.
[5]Evans-Anderson HJ,Alfieri CM,Yutzey KE.Regulation of cardiomyocyte proliferation and myocardial growth during development by FOXO transcription factors[J].Circ Res,2008,102(6):686-694.
[6]Yu J,Lan X,Chen X,et al.Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes[J].Development,2016,143(16):2930-2945.
[7]Chen D,Wu C,Zhao S,et al.Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila[J].PLoS Genet,2014,10(11):e1004797.
[8]Eun SH,Stoiber PM,Wright HJ,et al.MicroRNAs downregulate Bag of marbles to ensure proper terminal differentiation in the Drosophila male germline[J].Development,2013,140(1):23-30.
[9]Insco ML,Bailey AS,Kim J,et al.A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage[J].Cell Stem Cell,2012,11(5):689-700.
[10]Archibald JM,Teh EM,Keeling PJ.Novel ubiquitin fusion proteins:ribosomal protein P1 and actin[J].J Mol Biol,2003,328(4):771-778.
[11]Swatek KN,Komander D.Ubiquitin modifications[J].Cell Res,2016,26(4):399-422.
[12]Chang TK,Shravage BV,Hayes SD,et al.Uba1 functions in Atg7-and Atg3-independent autophagy[J].Nat Cell Biol,2013,15(9):1067-1078.
[13]White-Cooper H.Tissue,cell type and stage-specific ectopic gene expression and RNAi induction in the Drosophila testis[J].Spermatogenesis,2012,2(1):11-22.
[14]Ni JQ,Zhou R,Czech B,et al.A genome-scale shRNA resource for transgenic RNAi in Drosophila[J].Nat Methods,2011,8(5):405-407.
[15]Yan D,Neumu?ller RA,Buckner M,et al.A regulatory network of Drosophila germline stem cell self-renewal[J].Dev Cell,2014,28(4),459-473.
[16]Zeng X,Han L,Singh SR,et al.Genome-wide RNAi screen identifies networks involved in intestinal stem cell regulation in Drosophila[J].Cell Rep,2015,10(7):1226-1238.
[17]崔英霞,夏欣一,卢洪涌,等.45,X,der(Y)t(Y;13)不育男性患者的临床细胞和分子遗传学研究[J].医学研究生学报,2008,21(11):1168-1171.
[18]魏莉,史轶超,范晓博,等.泛素特异蛋白酶26基因序列改变与精子发生相关性的研究[J].医学研究生学报,2010,23(10):1020-1024.
[19]Lu C,Kim J,Fuller MT.The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila[J].Development,2013,140(17):3522-3531.
[20]王少冰,王芳.泛素结合酶E2C与肿瘤的关系研究进展[J].医学研究生学报,2015,28(5):543-548.
[21]Rajpert-De Meyts E,McGlynn KA,Okamoto K,et al.Testicular germ cell tumours[J].Lancet,2016,387(10029):1762-1774.
[22]Walsh TJ,Croughan MS,Schembri M,et al.Increased risk of testicular germ cell cancer among infertile men[J].Arch Intern Med,2009,169(4):351-356.
[23]Hime GR,Loveland KL,Abud HE.Drosophila spermatogenesis:insights into testicular cancer[J].Int J Androl,2007,30(4),265-274.
[24]Lim JG,Fuller MT.Somatic cell lineage is required for differentiation and not maintenance of germline stem cells in Drosophila testes[J].Proc Natl Acad Sci USA,2012,109(45):18477-18481.
[2]Fuller MT,Spradling AC.Male and female Drosophila germline stem cells:two versions of immortality[J].Science,2007,316(5823):402-404.
[3]Spradling A,Fuller MT,Braun RE,et al.Germline stem cells[J].Cold Spring Harb Perspect Biol,2011,3(11):a002642.
[4]Roessler E,Ouspenskaia MV,Karkera JD,et al.Reduced NODAL signaling strength via mutation of several pathway members including FOXH1 is linked to human heart defects and holoprosencephaly[J].Am J Hum Genet,2008,83(1):18-29.
[5]Evans-Anderson HJ,Alfieri CM,Yutzey KE.Regulation of cardiomyocyte proliferation and myocardial growth during development by FOXO transcription factors[J].Circ Res,2008,102(6):686-694.
[6]Yu J,Lan X,Chen X,et al.Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes[J].Development,2016,143(16):2930-2945.
[7]Chen D,Wu C,Zhao S,et al.Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila[J].PLoS Genet,2014,10(11):e1004797.
[8]Eun SH,Stoiber PM,Wright HJ,et al.MicroRNAs downregulate Bag of marbles to ensure proper terminal differentiation in the Drosophila male germline[J].Development,2013,140(1):23-30.
[9]Insco ML,Bailey AS,Kim J,et al.A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage[J].Cell Stem Cell,2012,11(5):689-700.
[10]Archibald JM,Teh EM,Keeling PJ.Novel ubiquitin fusion proteins:ribosomal protein P1 and actin[J].J Mol Biol,2003,328(4):771-778.
[11]Swatek KN,Komander D.Ubiquitin modifications[J].Cell Res,2016,26(4):399-422.
[12]Chang TK,Shravage BV,Hayes SD,et al.Uba1 functions in Atg7-and Atg3-independent autophagy[J].Nat Cell Biol,2013,15(9):1067-1078.
[13]White-Cooper H.Tissue,cell type and stage-specific ectopic gene expression and RNAi induction in the Drosophila testis[J].Spermatogenesis,2012,2(1):11-22.
[14]Ni JQ,Zhou R,Czech B,et al.A genome-scale shRNA resource for transgenic RNAi in Drosophila[J].Nat Methods,2011,8(5):405-407.
[15]Yan D,Neumu?ller RA,Buckner M,et al.A regulatory network of Drosophila germline stem cell self-renewal[J].Dev Cell,2014,28(4),459-473.
[16]Zeng X,Han L,Singh SR,et al.Genome-wide RNAi screen identifies networks involved in intestinal stem cell regulation in Drosophila[J].Cell Rep,2015,10(7):1226-1238.
[17]崔英霞,夏欣一,卢洪涌,等.45,X,der(Y)t(Y;13)不育男性患者的临床细胞和分子遗传学研究[J].医学研究生学报,2008,21(11):1168-1171.
[18]魏莉,史轶超,范晓博,等.泛素特异蛋白酶26基因序列改变与精子发生相关性的研究[J].医学研究生学报,2010,23(10):1020-1024.
[19]Lu C,Kim J,Fuller MT.The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila[J].Development,2013,140(17):3522-3531.
[20]王少冰,王芳.泛素结合酶E2C与肿瘤的关系研究进展[J].医学研究生学报,2015,28(5):543-548.
[21]Rajpert-De Meyts E,McGlynn KA,Okamoto K,et al.Testicular germ cell tumours[J].Lancet,2016,387(10029):1762-1774.
[22]Walsh TJ,Croughan MS,Schembri M,et al.Increased risk of testicular germ cell cancer among infertile men[J].Arch Intern Med,2009,169(4):351-356.
[23]Hime GR,Loveland KL,Abud HE.Drosophila spermatogenesis:insights into testicular cancer[J].Int J Androl,2007,30(4),265-274.
[24]Lim JG,Fuller MT.Somatic cell lineage is required for differentiation and not maintenance of germline stem cells in Drosophila testes[J].Proc Natl Acad Sci USA,2012,109(45):18477-18481.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |