淇河鲫R-spondin1基因cDNA克隆和表达
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摘要
为探索R-spondin1(Rspo1)基因在雌核发育三倍体鱼淇河鲫性别决定与分化过程中的作用,本研究利用RACE技术克隆Rspo1基因cDNA序列,同时分析它的时空表达模式,并检测芳香化酶抑制剂Letrozole及高温养殖诱导性逆转下它在性腺中的表达情况。将扩增的PCR产物经亚克隆测序、拼接后获得淇河鲫Rspo1 cDNA序列,长1 243 bp(MH243761),包含5′非编码区318 bp,3′非编码区127 bp和开放读码框798 bp,共编码265个氨基酸残基。氨基酸序列比对及系统进化分析结果显示,淇河鲫Rspo1与其他鲤科鱼类同源性较高,与哺乳类、爬行类等脊椎动物同源性相对较低;组织分布检测结果显示,Rspo1基因在淇河鲫各个组织中均有表达,其中肌肉组织表达量最高,卵巢次之,而在脾脏、肾脏、心脏组织中表达量较低;在胚胎发育过程中Rspo1的表达量在未受精卵与受精卵中无差异,随着胚胎发育而降低,神经胚期最低,从尾芽期至出膜期又逐步升高。胚后阶段,Rspo1在性腺中的表达量从淇河鲫性别决定与分化关键时期(孵化后20 d)开始上调。Letrozole处理或高温养殖引起的性逆转中,Rspo1在性腺中表达量升高。研究表明,淇河鲫Rspo1作为一个母源性因子可能在卵巢分化与维持中起到一定作用,同时它可能也参与精子发生过程。
In order to study the roles of R-spondin1(Rspo1) gene in the sex determination and differentiation of the Qihe crucian carp(Carassius auratus), a natural gynogenesis triploid fish, we cloned its cDNA by using reverse transcription-polymerase chain reaction(RT-PCR) and rapid amplification of cDNA 5′ and 3′ ends(RACE). We also examined Rspo1 spatio-temporal expression profiles by real-time PCR, at the same time, expression level of Rspo1 in the testis induced by Letrozole or high temperature treatment was also examined. The results showed that the cDNA of Rspo1 was 1243 bp including a 318 bp 5′-untranslated region(UTR), a 127 bp 3′UTR, and a 798 bp open reading frame(ORF) encoding a polypeptide of 265 amino acids. Sequence analysis revealed that Rspo1 from Qihe crucian carp showed the highest similarity with Cyprinidae fish, while had low similarity with other vertebrates including mammalians and reptiles. Tissue distribution by real-time PCR revealed that Rspo1 was abundantly expressed in the muscle, and then in the ovary, gill, intestine, and was barely detectable in other tissues.The expression of Rspo1 was gradually downregulated from fertilization stage, and then upregulated from the tailbud stage, so Rspo1 might be a maternal factor. Ontogenic analysis demonstrated that Rspo1 was expressed abundantly from 20 days after hatching(dah) in the gonad. However, Rspo1 was upregulated in the gonad of sexreversed males by Letrozole or high temperature treatment, indicating that Rspo1 exhibited a sexual dimophic expression pattern. These results suggested that, as a maternal factor, Rspo1 might be involved in both the ovarian differentiation and spermatogenesis in the testis of C. auratus.
In order to study the roles of R-spondin1(Rspo1) gene in the sex determination and differentiation of the Qihe crucian carp(Carassius auratus), a natural gynogenesis triploid fish, we cloned its cDNA by using reverse transcription-polymerase chain reaction(RT-PCR) and rapid amplification of cDNA 5′ and 3′ ends(RACE). We also examined Rspo1 spatio-temporal expression profiles by real-time PCR, at the same time, expression level of Rspo1 in the testis induced by Letrozole or high temperature treatment was also examined. The results showed that the cDNA of Rspo1 was 1243 bp including a 318 bp 5′-untranslated region(UTR), a 127 bp 3′UTR, and a 798 bp open reading frame(ORF) encoding a polypeptide of 265 amino acids. Sequence analysis revealed that Rspo1 from Qihe crucian carp showed the highest similarity with Cyprinidae fish, while had low similarity with other vertebrates including mammalians and reptiles. Tissue distribution by real-time PCR revealed that Rspo1 was abundantly expressed in the muscle, and then in the ovary, gill, intestine, and was barely detectable in other tissues.The expression of Rspo1 was gradually downregulated from fertilization stage, and then upregulated from the tailbud stage, so Rspo1 might be a maternal factor. Ontogenic analysis demonstrated that Rspo1 was expressed abundantly from 20 days after hatching(dah) in the gonad. However, Rspo1 was upregulated in the gonad of sexreversed males by Letrozole or high temperature treatment, indicating that Rspo1 exhibited a sexual dimophic expression pattern. These results suggested that, as a maternal factor, Rspo1 might be involved in both the ovarian differentiation and spermatogenesis in the testis of C. auratus.
引文
[1]Chassot A A,Gillot I,Chaboissier M C.R-spondin1,WNT4,and the CTNNB1 signaling pathway:strict control over ovarian differentiation[J].Reproduction,2014,148(6):R97-R110.
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[11]Chassot A A,Gregoire E P,Lavery R,et al.RSPO1/β-Catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary[J].PLoS One,2011,6(10):e25641.
[12]Chassot A A,Gregoire E P,Magliano M,et al.Genetics of ovarian differentiation:Rspo1,a major player[J].Sexual Development,2008,2(4-5):219-227.
[13]Jeays-Ward K,Hoyle C,Brennan J,et al.Endothelial and steroidogenic cell migration are regulated by WNT4in the developing mammalian gonad[J].Development,2003,130(16):3663-3670.
[14]Chassot A A,Bradford S T,Auguste A,et al.WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad[J].Development,2012,139(23):4461-4472.
[15]Nam J S,Turcotte T J,Smith P F,et al.Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activateβ-catenin-dependent gene expression[J].Journal of Biological Chemistry,2006,281(19):13247-13257.
[16]Kim K A,Wagle M,Tran K,et al.R-Spondin family members regulate the Wnt pathway by a common mechanism[J].Molecular Biology of the Cell,2008,19(6):2588-2596.
[17]Nicol B,Guiguen Y.Expression profiling of Wnt signaling genes during gonadal differentiation and gametogenesis in rainbow trout[J].Sexual Development,2011,5(6):318-329.
[18]Wu G C,Chang C F.wnt4 is associated with the development of ovarian tissue in the protandrous black porgy,Acanthopagrus schlegeli[J].Biology of Reproduction,2009,81(6):1073-1082.
[19]Zhang Y M,Li F,Sun D C,et al.Molecular analysis shows differential expression of R-spondin1 in zebrafish (Danio rerio)gonads[J].Molecular Biology Reports,2011,38(1):275-282.
[20]Zhou L Y,Charkraborty T,Yu X G,et al.R-spondins are involved in the ovarian differentiation in a teleost,medaka(Oryzias latipes)[J].BMC Developmental Biology,2012,12(1):36.
[21]Zhou L Y,Charkraborty T,Zhou Q,et al.Rspo1-activated signalling molecules are sufficient to induce ovarian differentiation in XY medaka(Oryzias latipes)[J].Scientific Reports,2016,6(1):19543.
[22]Wu L M,Yang P,Luo F,et al.R-spondin1 signaling pathway is required for both the ovarian and testicular development in a teleosts,Nile tilapia(Oreochromis niloticus)[J].General and Comparative Endocrinology,2016,230-231:177-185.
[23]Zhang J,Sun M,Zhou L,et al.Meiosis completion and various sperm responses lead to unisexual and sexual reproduction modes in one clone of polyploid Carassius gibelio[J].Scientific Reports,2015,5:10898.
[24]Kamata T,Katsube K I,Michikawa M,et al.R-spondin,a novel gene with thrombospondin type 1 domain,was expressed in the dorsal neural tube and affected in Wnts mutants[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,2004,1676(1):51-62.
[25]De Lau W B,Snel B,Clevers H C.The R-spondin protein family[J].Genome Biology,2012,13(3):242.
[26]Vainio S,Heikkil?M,Kispert A,et al.Female development in mammals is regulated by Wnt-4 signalling[J].Nature,1999,397(6718):405-409.
[27]Nam J S,Turcotte T J,Yoon J K.Dynamic expression of R-spondin family genes in mouse development[J].Gene Expression Patterns,2007,7(3):306-312.
[28]Díaz N,Piferrer F.Lasting effects of early exposure to temperature on the gonadal transcriptome at the time of sex differentiation in the European sea bass,a fish with mixed genetic and environmental sex determination[J].BMC Genomics,2015,16(1):679.
[29]Valenzuela N,Neuwald J L,Literman R.Transcriptional evolution underlying vertebrate sexual development[J].Developmental Dynamics,2013,242(4):307-319.
[30]Navarro-Martín L,Vi?as J,Ribas L,et al.DNAmethylation of the gonadal aromatase(cyp19a)Promoter is involved in temperature-dependent sex ratio shifts in the European Sea bass[J].PLoS Genetics,2011,7(12):e1002447.
[31]Yamaguchi T,Yamaguchi S,Hirai T,et al.Folliclestimulating hormone signaling and Foxl2 are involved in transcriptional regulation of aromatase gene during gonadal sex differentiation in Japanese flounder,Paralichthys olivaceus[J].Biochemical and Biophysical Research Communications,2007,359(4):935-940.
[32]Fernandino J I,Hattori R S,Kimura H,et al.Expression profile and estrogenic regulation of anti-Müllerian hormone during gonadal development in pejerrey Odontesthes bonariensis,a teleost fish with strong temperature-dependent sex determination[J].Developmental Dynamics,2008,237(11):3192-3199.
[33]D'Cotta H,Fostier A,Guiguen Y,et al.Search for genes involved in the temperature-induced gonadal sex differentiation in the tilapia,Oreochromis niloticus[J].Journal of Experimental Zoology,2001,290(6):574-585.
[34]Poonlaphdecha S,Pepey E,Canonne M,et al.Temperature induced-masculinisation in the Nile tilapia causes rapid up-regulation of both dmrt1 and amh expressions[J].General and Comparative Endocrinology,2013,193:234-242.
[35]Li X Y,Liu X L,Zhu Y J,et al.Origin and transition of sex determination mechanisms in a gynogenetic hexaploid fish[J].Heredity,2018,121(1):64-74.
[36]Li H L,Xu W T,Zhu Y,et al.Characterization and expression pattern of r‐spondin1 in Cynoglossus semilaevis[J].Journal of Experimental Zoology Part B:Molecular and Developmental Evolution,2017,328(8):772-780.
[2]Kim K A,Zhao J S,Andarmani S,et al.R-Spondin proteins:a novel link toβ-catenin activation[J].Cell Cycle,2006,5(1):23-26.
[3]Wilhelm D.R-spondin1-discovery of the long-missing,mammalian female-determining gene?[J].Bioessays,2007,29(4):314-318.
[4]Tomizuka K,Horikoshi K,Kitada R,et al.R-spondin1plays an essential role in ovarian development through positively regulating Wnt-4 signaling[J].Human Molecular Genetics,2008,17(9):1278-1291.
[5]Kim Y,Kobayashi A,Sekido R,et al.Fgf 9 and Wnt4act as antagonistic signals to regulate mammalian sex determination[J].PLoS Biology,2006,4(6):e187.
[6]Kocer A,Pinheiro I,Pannetier M,et al.R-spondin1 and FOXL2 act into two distinct cellular types during goat ovarian differentiation[J].BMC Developmental Biology,2008,8(1):36.
[7]Nef S,Vassalli J D.Complementary pathways in mammalian female sex determination[J].Journal of Biology,2009,8(8):74.
[8]Parma P,Radi O,Vidal V,et al.R-spondin1 is essential in sex determination,skin differentiation and malignancy[J].Nature Genetics,2006,38(11):1304-1309.
[9]Smith C A,Shoemaker C M,Roeszler K N,et al.Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development[J].BMC Developmental Biology,2008,8(1):72.
[10]Lavery R,Chassot A A,Pauper E,et al.Testicular differentiation occurs in absence of R-spondin1 and Sox9 in mouse sex reversals[J].PLoS Genetics,2012,8(12):e1003170.
[11]Chassot A A,Gregoire E P,Lavery R,et al.RSPO1/β-Catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary[J].PLoS One,2011,6(10):e25641.
[12]Chassot A A,Gregoire E P,Magliano M,et al.Genetics of ovarian differentiation:Rspo1,a major player[J].Sexual Development,2008,2(4-5):219-227.
[13]Jeays-Ward K,Hoyle C,Brennan J,et al.Endothelial and steroidogenic cell migration are regulated by WNT4in the developing mammalian gonad[J].Development,2003,130(16):3663-3670.
[14]Chassot A A,Bradford S T,Auguste A,et al.WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad[J].Development,2012,139(23):4461-4472.
[15]Nam J S,Turcotte T J,Smith P F,et al.Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activateβ-catenin-dependent gene expression[J].Journal of Biological Chemistry,2006,281(19):13247-13257.
[16]Kim K A,Wagle M,Tran K,et al.R-Spondin family members regulate the Wnt pathway by a common mechanism[J].Molecular Biology of the Cell,2008,19(6):2588-2596.
[17]Nicol B,Guiguen Y.Expression profiling of Wnt signaling genes during gonadal differentiation and gametogenesis in rainbow trout[J].Sexual Development,2011,5(6):318-329.
[18]Wu G C,Chang C F.wnt4 is associated with the development of ovarian tissue in the protandrous black porgy,Acanthopagrus schlegeli[J].Biology of Reproduction,2009,81(6):1073-1082.
[19]Zhang Y M,Li F,Sun D C,et al.Molecular analysis shows differential expression of R-spondin1 in zebrafish (Danio rerio)gonads[J].Molecular Biology Reports,2011,38(1):275-282.
[20]Zhou L Y,Charkraborty T,Yu X G,et al.R-spondins are involved in the ovarian differentiation in a teleost,medaka(Oryzias latipes)[J].BMC Developmental Biology,2012,12(1):36.
[21]Zhou L Y,Charkraborty T,Zhou Q,et al.Rspo1-activated signalling molecules are sufficient to induce ovarian differentiation in XY medaka(Oryzias latipes)[J].Scientific Reports,2016,6(1):19543.
[22]Wu L M,Yang P,Luo F,et al.R-spondin1 signaling pathway is required for both the ovarian and testicular development in a teleosts,Nile tilapia(Oreochromis niloticus)[J].General and Comparative Endocrinology,2016,230-231:177-185.
[23]Zhang J,Sun M,Zhou L,et al.Meiosis completion and various sperm responses lead to unisexual and sexual reproduction modes in one clone of polyploid Carassius gibelio[J].Scientific Reports,2015,5:10898.
[24]Kamata T,Katsube K I,Michikawa M,et al.R-spondin,a novel gene with thrombospondin type 1 domain,was expressed in the dorsal neural tube and affected in Wnts mutants[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,2004,1676(1):51-62.
[25]De Lau W B,Snel B,Clevers H C.The R-spondin protein family[J].Genome Biology,2012,13(3):242.
[26]Vainio S,Heikkil?M,Kispert A,et al.Female development in mammals is regulated by Wnt-4 signalling[J].Nature,1999,397(6718):405-409.
[27]Nam J S,Turcotte T J,Yoon J K.Dynamic expression of R-spondin family genes in mouse development[J].Gene Expression Patterns,2007,7(3):306-312.
[28]Díaz N,Piferrer F.Lasting effects of early exposure to temperature on the gonadal transcriptome at the time of sex differentiation in the European sea bass,a fish with mixed genetic and environmental sex determination[J].BMC Genomics,2015,16(1):679.
[29]Valenzuela N,Neuwald J L,Literman R.Transcriptional evolution underlying vertebrate sexual development[J].Developmental Dynamics,2013,242(4):307-319.
[30]Navarro-Martín L,Vi?as J,Ribas L,et al.DNAmethylation of the gonadal aromatase(cyp19a)Promoter is involved in temperature-dependent sex ratio shifts in the European Sea bass[J].PLoS Genetics,2011,7(12):e1002447.
[31]Yamaguchi T,Yamaguchi S,Hirai T,et al.Folliclestimulating hormone signaling and Foxl2 are involved in transcriptional regulation of aromatase gene during gonadal sex differentiation in Japanese flounder,Paralichthys olivaceus[J].Biochemical and Biophysical Research Communications,2007,359(4):935-940.
[32]Fernandino J I,Hattori R S,Kimura H,et al.Expression profile and estrogenic regulation of anti-Müllerian hormone during gonadal development in pejerrey Odontesthes bonariensis,a teleost fish with strong temperature-dependent sex determination[J].Developmental Dynamics,2008,237(11):3192-3199.
[33]D'Cotta H,Fostier A,Guiguen Y,et al.Search for genes involved in the temperature-induced gonadal sex differentiation in the tilapia,Oreochromis niloticus[J].Journal of Experimental Zoology,2001,290(6):574-585.
[34]Poonlaphdecha S,Pepey E,Canonne M,et al.Temperature induced-masculinisation in the Nile tilapia causes rapid up-regulation of both dmrt1 and amh expressions[J].General and Comparative Endocrinology,2013,193:234-242.
[35]Li X Y,Liu X L,Zhu Y J,et al.Origin and transition of sex determination mechanisms in a gynogenetic hexaploid fish[J].Heredity,2018,121(1):64-74.
[36]Li H L,Xu W T,Zhu Y,et al.Characterization and expression pattern of r‐spondin1 in Cynoglossus semilaevis[J].Journal of Experimental Zoology Part B:Molecular and Developmental Evolution,2017,328(8):772-780.