CYP11A1、CYP17A1、CYP19A1基因在小尾寒羊下丘脑-垂体-卵巢轴的表达
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摘要
为揭示类固醇生成相关基因CYP11A1、CYP17A1、CYP19A1在绵羊多羔繁殖中的作用,以不同繁殖力小尾寒羊为研究对象,采用实时荧光定量PCR技术检测CYP11A1、CYP17A1、CYP19A1基因在小尾寒羊大脑、小脑、下丘脑、垂体、子宫、卵巢、输卵管中的表达。结果表明:CYP11A1基因主要在小尾寒羊卵巢、输卵管、子宫内表达;CYP11A1基因在单羔小尾寒羊卵巢的表达极显著低于多羔群体(P<0.01),在子宫的表达量极显著高于多羔群体(P<0.01);CYP17A1基因主要在小尾寒羊卵巢、输卵管、下丘脑表达;CYP17A1基因在多羔小尾寒羊群体下丘脑、子宫的表达量显著低于单羔群体(P<0.05)。CYP19A1基因主要在小尾寒羊卵巢、下丘脑表达。本究结果提示CYP11A1、CYP17A1基因可能参与小尾寒羊多羔性状的调控。
To reveal the roles of CYP11A1,CYP17A1 and CYP19A1 genes in the high fecundity of sheep,Real-time fluorescence quantitative PCR(RT-qPCR)was performed to detect CYP11A1,CYP17A1 and CYP19A1 mRNA expressions in seven different tissues(brain,cerebellum,hypothalamus,pituitary,uterus,ovary and oviduct)of multiparous and uniparous Small Tail Han sheep.The results showed that CYP11A1 was expressed in ovary,oviduct and uterus of Small Tail Han sheep.The expression of CYP11A1 in ovary of multiparous Small Tail Han sheep was extremely significantly higher than that of uniparous Small Tail Han sheep(P<0.01).However,the expression of CYP11A1 in the uterus of multiparous Small Tail Han sheep was extremely significantly lower than that of uniparous Small Tail Han sheep(P<0.01).The CYP17A1 gene was mainly expressed in ovary,oviduct and hypothalamus of Small Tail Han sheep.The expression of CYP17A1 in hypothalamus,uterus of multiparous Small Tail Han sheep was significantly lower than that of uniparous Small Tail Han sheep(P<0.05).The CYP19A1 gene was mainly expressed in ovary and hypothalamus of Small Tail Han sheep.In conclusion,the results suggested that CYP11A1 and CYP17A1 might be involved in the regulation of multiple births in Small Tail Han sheep.
To reveal the roles of CYP11A1,CYP17A1 and CYP19A1 genes in the high fecundity of sheep,Real-time fluorescence quantitative PCR(RT-qPCR)was performed to detect CYP11A1,CYP17A1 and CYP19A1 mRNA expressions in seven different tissues(brain,cerebellum,hypothalamus,pituitary,uterus,ovary and oviduct)of multiparous and uniparous Small Tail Han sheep.The results showed that CYP11A1 was expressed in ovary,oviduct and uterus of Small Tail Han sheep.The expression of CYP11A1 in ovary of multiparous Small Tail Han sheep was extremely significantly higher than that of uniparous Small Tail Han sheep(P<0.01).However,the expression of CYP11A1 in the uterus of multiparous Small Tail Han sheep was extremely significantly lower than that of uniparous Small Tail Han sheep(P<0.01).The CYP17A1 gene was mainly expressed in ovary,oviduct and hypothalamus of Small Tail Han sheep.The expression of CYP17A1 in hypothalamus,uterus of multiparous Small Tail Han sheep was significantly lower than that of uniparous Small Tail Han sheep(P<0.05).The CYP19A1 gene was mainly expressed in ovary and hypothalamus of Small Tail Han sheep.In conclusion,the results suggested that CYP11A1 and CYP17A1 might be involved in the regulation of multiple births in Small Tail Han sheep.
引文
[1]Hashem N M,El-Azrak K M,Nour El-Din A N M,Taha T A,Salem M H.Effect of GnRH treatment on ovarian activity and reproductive performance of low-prolific Rahmani ewes[J].Theriogenology,2015,83(2):192-198
[2]Mcnatty K P,Fidler A E,Juengel J L,Quirke L D,Smith P R,Heath D A,Lundy T,O’connell A,Tisdall D J.Growth and paracrine factors regulating follicular formation and cellular function[J].Molecular&Cellular Endocrinology,2000,163(1-2):11-20
[3]Lebbe M,Taylor A E,Visser J A,Kirkman-Brown J C,Woodruff T K,Arlt W.The steroid metabolome in the isolated ovarian follicle and its response to androgen exposure and antagonism[J].Endocrinology,2017,158(5):1474-1485
[4]Stocco D M,Zhao A H,Tu L N,Morohaku K,Selvaraj V.Abrief history of the search for the protein(s)involved in the acute regulation of steroidogenesis[J].Molecular and Cellular Endocrinology,2017,441(2):7-16
[5]O’hara L,York J P,Zhang P M,Smith L B.Targeting of GFP-Cre to the mouse Cyp11a1locus both drives cre recombinase expression in steroidogenic cells and permits generation of Cyp11a1knock out mice[J].PloS ONE,2014,9(1):e84541
[6]Johnson A L,Woods D C.Dynamics of avian ovarian follicle development:Cellular mechanisms of granulosa cell differentiation[J].General and Comparative Endocrinology,2009,163(1):12-17
[7]Rangel P L,Rodríguez A,Gutierrez C G.Testosterone directly induces progesterone production and interacts with physiological concentrations of LH to increase granulosa cell progesterone production in laying hens(Gallus domesticus)[J].Animal Reproduction Science,2007,102(1):56-65
[8]Bao B,Garverick H A.Expression of steroidogenic enzyme and gonadotropin receptor genes in bovine follicles during ovarian follicular waves:A review[J].Journal of Animal Science,1998,76(7):1903-1921
[9]Kwon J Y,Mcandrew B J,Penman D J.Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia,Oreochromis niloticus[J].Molecular Reproduction and Development,2001,59(4):359-370
[10]Juengel J L,Hudson N L,Heath D A,Smith P,Reader K L,Lawrence S B,O’connell A R,Laitinen M P,Cranfield M,Groome N P,Ritvos O,McNatty K P.Growth differentiation factor 9and bone morphogenetic protein 15are essential for ovarian follicular development in sheep[J].Biology of Reproduction,2002,67(6):1777-1789
[11]Hess M F,Roser J F.Immunocytochemical localization of cytochrome P450 aromatase in the testis of prepubertal,pubertal,and postpubertal horses[J].Theriogenology,2004,61(2-3):293-299
[12]Albrecht B A,Daels P F.Immunolocalization of 3β-hydroxysteroid dehydrogenase,cytochrome P450 17α-hydroxylase/17,20-lyase and cytochrome P450aromatase in the equine corpus luteum of dioestrus and early pregnancy[J].Journal of Reproduction and Fertility,1997,111(1):127-133
[13]Davis G H,Galloway S M,Ross I K,Gregan S M,Ward J,Nimbkar B V,Ghalsasi P M,Nimbkar C,Gray G D,Inounu I.DNA tests in prolific sheep from eight countries provide new evidence on origin of the Booroola(FecB)mutation[J].Biology of Reproduction,2002,66(6):1869-1874
[14]Souza C,Macdougall C,Campbell B,Mcneilly A,Baird D.The Booroola(FecB)phenotype is associated with a mutation in the bone morphogenetic receptor type 1B(BMPR1B)gene[J].Journal of Endocrinology,2001,169(2):R1-R6
[15]Mulsant P,Lecerf F,Fabre S,Schibler L,Monget P,Lanneluc I,Pisselet C,Riquet J,Monniaux D,Callebaut I,Cribiu E,Thimonier J,Teyssier J,Bodin L,CogniéY,Chitour N,Elsen J-M.Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes[J].Proceedings of the National Academy of Sciences,2001,98(9):5104-5109
[16]Drouilhet L,Taragnat C,Fontaine J,Duittoz A,Mulsant P,Bodin L,Fabre S.Endocrine characterization of the reproductive axis in highly prolific lacaune sheep homozygous for the FecLL mutation[J].Biology of Reproduction,2010,82(5):815-824
[17]Schmittgen T D,Livak K J.Analyzing real-time PCR data by the comparative CT method[J].Nature Protocols,2008,3(6):1101-1108
[18]曹素梅,万雪萍,严美姣,李昂,吴旭.miRNAs介导下丘脑-垂体-性腺轴调控动物生殖的研究进展[J].中国畜牧杂志,2017,53(1):1-6Cao S M,Wan X P,Yan M J,Li A,Wu X.Research progress on miRNAs-mediated HPG axis in regulating animal reproduction[J].Chinese Journal of Animal Science,2017,53(1):11-6(in Chinese)
[19]Herndon M K,Nilson J H.Maximal expression of Foxl2in pituitary gonadotropes requires ovarian hormones[J].PLoSOne,2015,10(5):e0126527
[20]Hashem N M,El-Azrak K M,Nour El-Din A N M,Taha T A,Salem M H.Effect of GnRH treatment on ovarian activity and reproductive performance of low-prolific Rahmani ewes[J].Theriogenology,2015,83(2):192-198
[21]Sechman A,Pawlowska K,Hrabia A.Effect of 3,3′,5-triiodothyronine and 3,5-diiodothyronine on progesterone production,cAMPsynthesis,and mRNA expression of STAR,CYP11A1,and HSD3B genes in granulosa layer of chicken preovulatory follicles[J].Domestic Animal Endocrinology,2011,41(3):137-149
[22]李娟.豁眼鹅卵巢组织产蛋性能相关基因的筛选及表达分析[D].沈阳:沈阳农业大学,2016Li J.Screening and expression analysis of genes related to laying production in huoyan geese ovarian tissus[D].Shenyang:Shenyang Agricultural University,2016(in Chinese)
[23]刘锐,李江凌,陈晓晖,龚建军,杨跃奎,吕学斌.CYP11A1基因在藏猪和藏梅猪卵巢,子宫和输卵管组织表达差异[J].中国猪业,2016,11(12):59-62Li R,Li J L,Chen X H,Fan J J,Yang Y K,Lv X B.Differential expression of CYP11A1 gene in ovary,uterus,and oviduct of Tibetan and Tibetan mutators[J].China Swine Industry,2016,11(12):59-62(in Chinese)
[24]Iwade R,Maruo K,Okada G,Nakamura M.Elevated expression of P450c17(CYP17)during testicular formation in the frog[J].General and Comparative Endocrinology,2008,155(1):79-87
[25]Gervsio C G,Bernuci M P,Silva-De-SM F,Rosa-E-Silva AC.The role of androgen hormones in early follicular development[J].Isrn Obstetrics&Gynecology,2014,(18):e818010
[26]Nakamura K,Shibuya K,Saito N,Shimada K,Ohshima A,Hirai T,Nunoya T.mRNA expression of cytochrome P45017αhydroxylase,cytochrome P450 aromatase,anti-müllerian hormone,estrogen receptorα,and androgen receptor in developing gonads of Japanese quail[J].The Journal of Poultry Science,2008,45(4):298-302
[27]王伟伟.尼罗罗非鱼cyp17a2生物信息学和雌雄性腺表达差异分析[J].安徽农业科学,2016,44(7):103-106Wang W W.Bioinformatics analysis of cyp17a2in Nile Tilapia and differences of expression in gonads[J].Journal of Anhui Agricultural Sciences,2016,44(9):103-106(in Chinese)
[28]温海深,任源远,张冬茜,赵梅琳.绿鳍马面鲀CYP17-I基因克隆及其在繁殖周期中的表达[J].水产学报,2014,(12):1945-1955Wen H S,Ren Y Y,Zhang D Q,Zhao M L.Molecular cloning and expression patterns of the cytochrome CYP17-I gene during the reproductive cycle in Navodon septentrionalis[J].Journal of Fisheries of China,2014,(12):1945-1955(in Chinese)
[29]王彩云,潘登,张召议,范奎奎,李婷,杜晨光.类固醇激素合成调节因子在小鼠下丘脑和卵巢上的分布[J].中国兽医杂志,2017,53(2):29-32Wang C Y,Pan D,Zhang Z Y,Fan K K,Li T,Du C G.Expression of steroidogenic factor in mice ovary and brain[J].Chinese Journal of Veterinary Medicine,2017,53(2):29-32(in Chinese)
[30]Bershtein L.Molecular-genetic aspects of estrogen production:Aromatase gene[J].Molecular Biology,1997,31(5):655-659
[31]苏节,刘庆友,朱鹏,沈开元,石德顺,崔奎青.沼泽型水牛CYP19A1基因克隆,序列分析及组织表达研究[J].畜牧兽医学报,2013,44(4):514-521Su J,Liu Q Y,Zhu P,Sen K Y,Shi D S,Cui K Q.Cloning and sequence analysis of swamp buffalo CYP19A1 gene and determination of its expression pattern in different tissus[J].Chinese Journal of Animal and Veterinary Sciences,2013,44(4):514-521(in Chinese)
[32]包海声,蔡晗,韩伟,张海艳,孙伟,葛楚天,钱国英.Cyp19a1基因在中华鳖早期卵巢分化中的功能研究[J].中国科学:生命科学,2017,47(6):640-649Bao H S,Cai H,Han W,Zang H Y,Sun W,Ge C T,Qian G Y.Functional characterization of Cyp19a1 in female sexual differentiation in Pelodiscus sinensis[J].Scientia Sinica(Vitae),2017,47(6):640-649(in Chinese)
[33]孙甜甜,杨祝良,邓继贤,曾令湖,蒋和生,杨秀荣.广西麻鸡CYP19A1基因的克隆、序列分析和组织表达谱研究[J].中国家禽,2017,39(18):60-62Sun T T,Yang Z L,Deng J X,Zeng L H,Jiang H S,Yang XR.Cloning,sequence analysis and tissue expression of CYP19A1 gene in Guangxi chicken[J].China Poultry,2017,39(18):60-62(in Chinese)
[2]Mcnatty K P,Fidler A E,Juengel J L,Quirke L D,Smith P R,Heath D A,Lundy T,O’connell A,Tisdall D J.Growth and paracrine factors regulating follicular formation and cellular function[J].Molecular&Cellular Endocrinology,2000,163(1-2):11-20
[3]Lebbe M,Taylor A E,Visser J A,Kirkman-Brown J C,Woodruff T K,Arlt W.The steroid metabolome in the isolated ovarian follicle and its response to androgen exposure and antagonism[J].Endocrinology,2017,158(5):1474-1485
[4]Stocco D M,Zhao A H,Tu L N,Morohaku K,Selvaraj V.Abrief history of the search for the protein(s)involved in the acute regulation of steroidogenesis[J].Molecular and Cellular Endocrinology,2017,441(2):7-16
[5]O’hara L,York J P,Zhang P M,Smith L B.Targeting of GFP-Cre to the mouse Cyp11a1locus both drives cre recombinase expression in steroidogenic cells and permits generation of Cyp11a1knock out mice[J].PloS ONE,2014,9(1):e84541
[6]Johnson A L,Woods D C.Dynamics of avian ovarian follicle development:Cellular mechanisms of granulosa cell differentiation[J].General and Comparative Endocrinology,2009,163(1):12-17
[7]Rangel P L,Rodríguez A,Gutierrez C G.Testosterone directly induces progesterone production and interacts with physiological concentrations of LH to increase granulosa cell progesterone production in laying hens(Gallus domesticus)[J].Animal Reproduction Science,2007,102(1):56-65
[8]Bao B,Garverick H A.Expression of steroidogenic enzyme and gonadotropin receptor genes in bovine follicles during ovarian follicular waves:A review[J].Journal of Animal Science,1998,76(7):1903-1921
[9]Kwon J Y,Mcandrew B J,Penman D J.Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia,Oreochromis niloticus[J].Molecular Reproduction and Development,2001,59(4):359-370
[10]Juengel J L,Hudson N L,Heath D A,Smith P,Reader K L,Lawrence S B,O’connell A R,Laitinen M P,Cranfield M,Groome N P,Ritvos O,McNatty K P.Growth differentiation factor 9and bone morphogenetic protein 15are essential for ovarian follicular development in sheep[J].Biology of Reproduction,2002,67(6):1777-1789
[11]Hess M F,Roser J F.Immunocytochemical localization of cytochrome P450 aromatase in the testis of prepubertal,pubertal,and postpubertal horses[J].Theriogenology,2004,61(2-3):293-299
[12]Albrecht B A,Daels P F.Immunolocalization of 3β-hydroxysteroid dehydrogenase,cytochrome P450 17α-hydroxylase/17,20-lyase and cytochrome P450aromatase in the equine corpus luteum of dioestrus and early pregnancy[J].Journal of Reproduction and Fertility,1997,111(1):127-133
[13]Davis G H,Galloway S M,Ross I K,Gregan S M,Ward J,Nimbkar B V,Ghalsasi P M,Nimbkar C,Gray G D,Inounu I.DNA tests in prolific sheep from eight countries provide new evidence on origin of the Booroola(FecB)mutation[J].Biology of Reproduction,2002,66(6):1869-1874
[14]Souza C,Macdougall C,Campbell B,Mcneilly A,Baird D.The Booroola(FecB)phenotype is associated with a mutation in the bone morphogenetic receptor type 1B(BMPR1B)gene[J].Journal of Endocrinology,2001,169(2):R1-R6
[15]Mulsant P,Lecerf F,Fabre S,Schibler L,Monget P,Lanneluc I,Pisselet C,Riquet J,Monniaux D,Callebaut I,Cribiu E,Thimonier J,Teyssier J,Bodin L,CogniéY,Chitour N,Elsen J-M.Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes[J].Proceedings of the National Academy of Sciences,2001,98(9):5104-5109
[16]Drouilhet L,Taragnat C,Fontaine J,Duittoz A,Mulsant P,Bodin L,Fabre S.Endocrine characterization of the reproductive axis in highly prolific lacaune sheep homozygous for the FecLL mutation[J].Biology of Reproduction,2010,82(5):815-824
[17]Schmittgen T D,Livak K J.Analyzing real-time PCR data by the comparative CT method[J].Nature Protocols,2008,3(6):1101-1108
[18]曹素梅,万雪萍,严美姣,李昂,吴旭.miRNAs介导下丘脑-垂体-性腺轴调控动物生殖的研究进展[J].中国畜牧杂志,2017,53(1):1-6Cao S M,Wan X P,Yan M J,Li A,Wu X.Research progress on miRNAs-mediated HPG axis in regulating animal reproduction[J].Chinese Journal of Animal Science,2017,53(1):11-6(in Chinese)
[19]Herndon M K,Nilson J H.Maximal expression of Foxl2in pituitary gonadotropes requires ovarian hormones[J].PLoSOne,2015,10(5):e0126527
[20]Hashem N M,El-Azrak K M,Nour El-Din A N M,Taha T A,Salem M H.Effect of GnRH treatment on ovarian activity and reproductive performance of low-prolific Rahmani ewes[J].Theriogenology,2015,83(2):192-198
[21]Sechman A,Pawlowska K,Hrabia A.Effect of 3,3′,5-triiodothyronine and 3,5-diiodothyronine on progesterone production,cAMPsynthesis,and mRNA expression of STAR,CYP11A1,and HSD3B genes in granulosa layer of chicken preovulatory follicles[J].Domestic Animal Endocrinology,2011,41(3):137-149
[22]李娟.豁眼鹅卵巢组织产蛋性能相关基因的筛选及表达分析[D].沈阳:沈阳农业大学,2016Li J.Screening and expression analysis of genes related to laying production in huoyan geese ovarian tissus[D].Shenyang:Shenyang Agricultural University,2016(in Chinese)
[23]刘锐,李江凌,陈晓晖,龚建军,杨跃奎,吕学斌.CYP11A1基因在藏猪和藏梅猪卵巢,子宫和输卵管组织表达差异[J].中国猪业,2016,11(12):59-62Li R,Li J L,Chen X H,Fan J J,Yang Y K,Lv X B.Differential expression of CYP11A1 gene in ovary,uterus,and oviduct of Tibetan and Tibetan mutators[J].China Swine Industry,2016,11(12):59-62(in Chinese)
[24]Iwade R,Maruo K,Okada G,Nakamura M.Elevated expression of P450c17(CYP17)during testicular formation in the frog[J].General and Comparative Endocrinology,2008,155(1):79-87
[25]Gervsio C G,Bernuci M P,Silva-De-SM F,Rosa-E-Silva AC.The role of androgen hormones in early follicular development[J].Isrn Obstetrics&Gynecology,2014,(18):e818010
[26]Nakamura K,Shibuya K,Saito N,Shimada K,Ohshima A,Hirai T,Nunoya T.mRNA expression of cytochrome P45017αhydroxylase,cytochrome P450 aromatase,anti-müllerian hormone,estrogen receptorα,and androgen receptor in developing gonads of Japanese quail[J].The Journal of Poultry Science,2008,45(4):298-302
[27]王伟伟.尼罗罗非鱼cyp17a2生物信息学和雌雄性腺表达差异分析[J].安徽农业科学,2016,44(7):103-106Wang W W.Bioinformatics analysis of cyp17a2in Nile Tilapia and differences of expression in gonads[J].Journal of Anhui Agricultural Sciences,2016,44(9):103-106(in Chinese)
[28]温海深,任源远,张冬茜,赵梅琳.绿鳍马面鲀CYP17-I基因克隆及其在繁殖周期中的表达[J].水产学报,2014,(12):1945-1955Wen H S,Ren Y Y,Zhang D Q,Zhao M L.Molecular cloning and expression patterns of the cytochrome CYP17-I gene during the reproductive cycle in Navodon septentrionalis[J].Journal of Fisheries of China,2014,(12):1945-1955(in Chinese)
[29]王彩云,潘登,张召议,范奎奎,李婷,杜晨光.类固醇激素合成调节因子在小鼠下丘脑和卵巢上的分布[J].中国兽医杂志,2017,53(2):29-32Wang C Y,Pan D,Zhang Z Y,Fan K K,Li T,Du C G.Expression of steroidogenic factor in mice ovary and brain[J].Chinese Journal of Veterinary Medicine,2017,53(2):29-32(in Chinese)
[30]Bershtein L.Molecular-genetic aspects of estrogen production:Aromatase gene[J].Molecular Biology,1997,31(5):655-659
[31]苏节,刘庆友,朱鹏,沈开元,石德顺,崔奎青.沼泽型水牛CYP19A1基因克隆,序列分析及组织表达研究[J].畜牧兽医学报,2013,44(4):514-521Su J,Liu Q Y,Zhu P,Sen K Y,Shi D S,Cui K Q.Cloning and sequence analysis of swamp buffalo CYP19A1 gene and determination of its expression pattern in different tissus[J].Chinese Journal of Animal and Veterinary Sciences,2013,44(4):514-521(in Chinese)
[32]包海声,蔡晗,韩伟,张海艳,孙伟,葛楚天,钱国英.Cyp19a1基因在中华鳖早期卵巢分化中的功能研究[J].中国科学:生命科学,2017,47(6):640-649Bao H S,Cai H,Han W,Zang H Y,Sun W,Ge C T,Qian G Y.Functional characterization of Cyp19a1 in female sexual differentiation in Pelodiscus sinensis[J].Scientia Sinica(Vitae),2017,47(6):640-649(in Chinese)
[33]孙甜甜,杨祝良,邓继贤,曾令湖,蒋和生,杨秀荣.广西麻鸡CYP19A1基因的克隆、序列分析和组织表达谱研究[J].中国家禽,2017,39(18):60-62Sun T T,Yang Z L,Deng J X,Zeng L H,Jiang H S,Yang XR.Cloning,sequence analysis and tissue expression of CYP19A1 gene in Guangxi chicken[J].China Poultry,2017,39(18):60-62(in Chinese)
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