FecB突变对小尾寒羊卵泡发育及AMH和AMHR2基因表达的影响
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摘要
小尾寒羊(Ovis aries)是我国拥有高繁殖力的优良品种,其骨形态发生蛋白受体1B(bone morphogenetic protein receptor 1B, BMPR1B)基因发生FecB突变能提高排卵数和产羔数,但具体机制尚不清楚。本研究旨在探讨小尾寒羊不同FecB基因型(突变纯合型BB和野生型++)对卵泡数量和直径以及抗苗勒氏激素基因(anti-Müllerian hormone, AMH)和抗苗勒氏激素Ⅱ型受体基因(anti-Müllerian hormone receptorⅡ, AMHR2)表达模式的影响。基于TaqMan探针分型技术,运用同期发情方法,统计卵泡期++型(n=21)和BB型(n=16)直径在3 mm以上的卵泡数量和直径。利用qRT-PCR技术检测BB型小尾寒羊(n=3)卵泡期14种组织(下丘脑,垂体,卵巢,心脏,肝脏,脾脏,肺脏,肾脏,大脑,肾上腺,小肠,输卵管,子宫,甲状腺)以及BB型和++型小尾寒羊黄体期和卵泡期(n=3, 3, 3, 3)下丘脑-垂体-卵巢性腺轴中AMH和AMHR2的表达量。结果显示,FecB突变极显著增加了卵泡数量(P<0.01)和降低了卵泡直径(P<0.01)。AMH和AMHR2在BB型小尾寒羊卵泡期各组织中均有表达,其中下丘脑-垂体-卵巢性腺轴中两者均高表达,卵巢中AMH表达量最高,肾上腺中AMHR2表达量最高。AMH在不同发育阶段不同FecB基因型小尾寒羊下丘脑-垂体-卵巢性腺轴的表达差异不显著(P>0.05)。AMHR2在卵巢中表达量无显著差异(P>0.05),在垂体中表达量BB型极显著高于++型(P<0.01),卵泡发育阶段和基因型对下丘脑AMHR2的表达量存在极显著影响(P<0.01)。综上表明,FecB突变可以引起小尾寒羊卵泡发育和AMHR2表达时空的改变,AMHR2在下丘脑-垂体-卵巢性腺轴系统的特定表达可能参与FecB突变调节卵泡发育,这为我国高繁殖力肉羊育种提供了新的理论依据。
As an excellent domestic breed with fecundity, Small Tail Han sheep(Ovis aries) could get high ovulation rate and little size as a result of A476 G mutation in bone morphogenetic protein receptor 1 B(BMPR1 B) gene(FecB mutation), under which the mechanism remains to poorly understand. The objective of this study was to investigate the effects on the number and diameter of follicle and the expression patterns of anti-Müllerian hormone(AMH) gene and its receptor Ⅱ(anti-Müllerian hormone receptor Ⅱ, AMHR2) gene in Small Tail Han sheep with or without FecB mutation(mutant homozygous BB and wide type ++, respectively).Based on TaqMan probe genotyping and estrus synchronization, the number and diameter of follicle over 3 mm from ++(n=21) and BB ewes(n=16) were recorded. qRT-PCR technology was applied to detect the expression of AMH and AMHR2 genes in 14 tissues(hypothalamus, pituitary, ovary, heart, liver, spleen, lung,kidney, brain, adrenal gland, small intestine, oviduct, uterus, thyroid) from 3 BB ewes during follicular phase and 3 tissues(hypothalamus, pituitary and ovary) from BB and ++ ewes during luteal phase and follicular phase(n=3, 3, 3, 3, respectively). The results revealed that FecB mutation had a highly significant increase in number of follicle(P<0.01) and a highly remarkable decrease in follicular diameter(P<0.01). Two genes were expressed in 14 tissues of BB ewes during follicular phase, wherein both genes expressed highly in hypothalamus-pituitary-ovary gonad axis as well as AMH gene expressed highest in ovary and AMHR2 gene in adrenal gland. The expression of AMH gene in hypothalamus-pituitary-ovary gonad axis between BB and ++animals during different phases of follicular development were not significantly different(P>0.05). There was no significant difference for the expression of AMHR2 gene in ovary between 2 categories of animals during different phases of follicular development(P>0.05). The expression of AMHR2 gene in pituitary of BB ewes was higher than ++ contemporaries(P<0.01). Stage of follicular development and FecB genotype had a highly significant effect on expression of AMHR2 gene in hypothalamus(P<0.01). A conclusion can be drew that FecB mutation caused the change of follicular development and expression of AMHR2 gene in time and space in Small Tail Han sheep and the special expression of AMHR2 gene in hypothalamus-pituitary-ovary gonad axis system may be involved in the regulation that FecB mutation alters follicular development, which would give novel theoretical basis to multiparous meat sheep breeding in our country.
As an excellent domestic breed with fecundity, Small Tail Han sheep(Ovis aries) could get high ovulation rate and little size as a result of A476 G mutation in bone morphogenetic protein receptor 1 B(BMPR1 B) gene(FecB mutation), under which the mechanism remains to poorly understand. The objective of this study was to investigate the effects on the number and diameter of follicle and the expression patterns of anti-Müllerian hormone(AMH) gene and its receptor Ⅱ(anti-Müllerian hormone receptor Ⅱ, AMHR2) gene in Small Tail Han sheep with or without FecB mutation(mutant homozygous BB and wide type ++, respectively).Based on TaqMan probe genotyping and estrus synchronization, the number and diameter of follicle over 3 mm from ++(n=21) and BB ewes(n=16) were recorded. qRT-PCR technology was applied to detect the expression of AMH and AMHR2 genes in 14 tissues(hypothalamus, pituitary, ovary, heart, liver, spleen, lung,kidney, brain, adrenal gland, small intestine, oviduct, uterus, thyroid) from 3 BB ewes during follicular phase and 3 tissues(hypothalamus, pituitary and ovary) from BB and ++ ewes during luteal phase and follicular phase(n=3, 3, 3, 3, respectively). The results revealed that FecB mutation had a highly significant increase in number of follicle(P<0.01) and a highly remarkable decrease in follicular diameter(P<0.01). Two genes were expressed in 14 tissues of BB ewes during follicular phase, wherein both genes expressed highly in hypothalamus-pituitary-ovary gonad axis as well as AMH gene expressed highest in ovary and AMHR2 gene in adrenal gland. The expression of AMH gene in hypothalamus-pituitary-ovary gonad axis between BB and ++animals during different phases of follicular development were not significantly different(P>0.05). There was no significant difference for the expression of AMHR2 gene in ovary between 2 categories of animals during different phases of follicular development(P>0.05). The expression of AMHR2 gene in pituitary of BB ewes was higher than ++ contemporaries(P<0.01). Stage of follicular development and FecB genotype had a highly significant effect on expression of AMHR2 gene in hypothalamus(P<0.01). A conclusion can be drew that FecB mutation caused the change of follicular development and expression of AMHR2 gene in time and space in Small Tail Han sheep and the special expression of AMHR2 gene in hypothalamus-pituitary-ovary gonad axis system may be involved in the regulation that FecB mutation alters follicular development, which would give novel theoretical basis to multiparous meat sheep breeding in our country.
引文
海米提·阿布都力木,李艳,郭琼,等.2016.OrexinA、OrexinB和AMH受体在性腺轴的分布及其生殖生物学意义的研究[J].中国男科学杂志,30(11):3-8.(Hai M T·A B D L M,Li Y,Guo Q,et al.2016.Study on the distribution of orexin and anti-mullerian hormone receptors in the hypothalamic-pituitary-testicular axis and their reproductive biological significance[J].Chinese Journal of Andrology,30(11):3-8.)
胡姗.2017.BMP15基因对绒山羊mGSCs增殖分化的影响[D].硕士学位论文,西北农林科技大学,导师:胡建宏.pp.11.(Hu S,2017.The effects of BMP15 gene on proliferation and differentiation of cashmere goats mG-SCS[D].Thesis for M.S.,North West Agriculture and Forestry University,Supervisor:Hu J H.pp.11.)
赖海燕,桂文武.2018.抗苗勒管激素在辅助生殖领域的临床研究进展[J].国际生殖健康/计划生育杂志,37(01):71-75.(Lai H Y,Gui W W.2018.Study progress of antiMullerian hormone in assisted reproduction[J].Journal of International Reproductive Health/Family Planning,37(01):71-75.)
李运生.2015.抗缪勒氏管激素在猪卵母细胞和颗粒细胞体外发育中的作用及其机制研究[D].博士学位论文,安徽农业大学,导师:章孝荣.pp.6-7;21-24.(Li Y S.2015.Effects and mechanisms of anti-Müllerian hormone on in vitro development of porcine oocytes and granulosa cells[D].Thesis for Ph.D.,Anhui Agricultural University,Supervisor:Zhang X R.pp.6-7;21-24.)
刘秋月,胡文萍,贺小云,等.2017.绵羊多羔主效基因FecB高通量检测方法的建立及应用[J].畜牧兽医学报,48(01):39-51.(Liu Q Y,Hu W P,He X Y,et al.2017.Establishment of high-throughput molecular detection methods for ovine high fecundity major gene FecB and their application[J].Chinese Journal of Animal and Veterinary Sciences,48(01):39-51.)
刘世佳.2014.绵羊产羔数相关候选基因联合效应的初步分析[D].硕士学位论文,甘肃农业大学,导师:李发弟.pp.2.(Liu S J.2014.Preliminary analysis of number of sheep lambing combined effects of candidate gene[D].Thesis for M.S.,Gansu Agricultural University,Supervisor:Li F D.pp.2.)
罗庆苗,秦晓玉,苗向阳.2012.利用数字基因表达谱对小尾寒羊高繁性状相关基因的筛选研究[J].中国农学通报,28(23):8-25.(Luo Q M,Qing X Y,Miao X Y.2012.The study on related genes of high fecundity using digital gene expression tag profiling in Small-Tail-Han-Sheep[J].Chinese Agricultural Science Bulletin,28(23):8-25.)
潘章源,狄冉,刘秋月,等.2015a.绵羊BMPR1B基因生物信息学分析[J].中国草食动物科学,35(06):1-5.(Pan Z Y,Di R,Liu Q Y,et al.2015a.Bioinformatics analysis on BMPR1B gene of sheep[J].China Herbivore Science,35(06):1-5.)
潘章源,狄冉,刘秋月,等.2015b.绵羊多羔主效基因BM?PR1B的研究进展[J].家畜生态学报,36(05):1-6.(Pan Z Y,Di R,Liu Q Y,et al.2015b.Advance in ovine prolificacy gene BMPR1B[J].Journal of Domestic Animal Ecology,36(05):1-6.)
孙晓卫.2017.山羊卵巢中BMP15通过p38 MAPK通路调控AMH的表达[D].硕士学位论文,西南大学,导师:赵中权.pp.43.(Sun X W.2017.BMP15 regulates AMH expression by p38 MAPK passway in the goat ovary[D].Thesis for M.S.,Southwest University,Supervisor:Zhao Z Q.pp.43.)
杨利国.2010.动物繁殖学.第2版[M].北京:中国农业出版社,pp.30.(Yang L G.2010.Animal Reproduction.Second Edition[M].China Agriculture Press,Beijing.pp.30.)
于嘉瑞,王翔宇,郭晓飞,等.2018.LEPR及LEP基因表达量对FecB不同基因型小尾寒羊产羔数的影响[J].畜牧兽医学报,49(05):954-961.(Yu J R,Wang X Y,Guo X F,et al.2018.Effects of expression of LEPR and LEPgenes on litter size of Small Tail Han Sheep with different FecB genotypes[J].Chinese Journal of Animal and Veterinary Sciences,49(05):954-961.)
Bédécarrats G Y,O'Neill F H,Norwitz E R,et al.2003.Regulation of gonadotropin gene expression by Müllerian inhibiting iubstance[J].Proceedings of the National Academy of Sciences of the USA,100(16):9348-9353.
Cai J,Liu L,Zheng J,et al.2018.Differential response of AMH to GnRH agonist among individuals:The effect on ovarian stimulation outcomes[J].Journal of Assisted Reproduction and Genetics,35(3):467-473.
Campbell B K,Clinton M,Webb R.2012.The role of antiMüllerian hormone(AMH)during follicle development in a monovulatory species(sheep)[J].Endocrinology,153(9):4533-4543.
Center K,Dixon D,Lonney C,et al.2018.Anti-Mullerian hormone and follicle counts as predictors of superovulatory response and embryo production in beef cattle[J].Advances in Reproductive Sciences,06(1):22-33.
Cimino I,Casoni F,Liu X,et al.2016.Novel role for antiMüllerian hormone in the regulation of GnRH neuron excitability and hormone secretion[J].Nature Communications,7(1):10055.
Cognie Y,Benoit F,Poulin N,et al.1998.Effect of follicle size and of the FecB Booroola gene on oocyte function in sheep[J].Journal of Reproduction and Fertility,112:379-386.
Cutting A D,Ayers K,Davidson N,et al.2014.Identification,expression,and regulation of anti-Müllerian hormone type-II receptor in the embryonic chicken gonad1[J].Biology of Reproduction,90(5):106.
Durlinger A L,Visser J A,Themmen A P N.2002.Regulation of ovarian function:The role of anti-Müllerian hormone[J].Reproduction,124(5):601-609.
Estienne A,Pierre A,di Clemente N,et al.2015.Anti-Müllerian hormone regulation by the bone morphogenetic proteins in the sheep ovary:Deciphering a direct regulatory pathway[J].Endocrinology,156(1):301-313.
Guo X F,Wang X Y,Di R,et al.2018.Metabolic effects of FecB gene on follicular fluid and ovarian vein serum in sheep(Ovis aries)[J].International Journal of Molecular Sciences,19(2):539.
Josso N,Clemente N D.2003.Transduction pathway of antiMüllerian hormone,a sex-specific member of the TGF-βfamily[J].Trends in Endocrinology&Metabolism,14(2):91-97.
Juengel J L,Davis G H,Mcnatty K P.2013.Using sheep lines with mutations in single genes to better understand ovarian function[J].Reproduction,146(4):111-123.
Juengel J L,French M C,Quirke L D,et al.2017.Differential expression of CART in ewes with differing ovulation rates[J].Reproduction,153(4):471-479.
Lahoz B,Alabart J L,Monniaux D,et al.2012.Anti-Müllerian hormone plasma concentration in prepubertal ewe lambs as a predictor of their fertility at a young age[J].BMC Veterinary Research,118(8):1-9.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,25(4):402-408.
Oliveira M E F,Ayres H,Oliveira L G,et al.2016.Effects of season and ovarian status on the outcome of long-term progesterone-based estrus synchronization protocols and ovulatory follicle development in Santa Inês ewes under subtropical conditions[J].Theriogenology,85(3):452-460.
Poole D H,Ocón-Grove O M,Johnson A L.2016.Anti-Müllerian hormone(AMH)receptor typeⅡexpression and AMH activity in bovine granulosa cells[J].Theriogenology,86(5):1353-1360.
Roy S,Gandra D,Seger C,et al.2018.Oocyte-derived factors(GDF9 and BMP15)and FSH regulate AMH expression via modulation of H3K27AC in granulosa cells[J].Endocrinology,2018,159(9):3433-3445.
Tata B,Mimouni N E H,Barbotin A,et al.2018.Elevated prenatal anti-Müllerian hormone reprograms the fetus and induces polycystic ovary syndrome in adulthood[J].Nature Medicine,24(6):834-846.
van Houten E L A F,Themmen A P N,Visser J A.2010.AntiMüllerian hormone(AMH):Regulator and marker of ovarian function[J].Annales Dendocrinologie,71(3):191-197.
胡姗.2017.BMP15基因对绒山羊mGSCs增殖分化的影响[D].硕士学位论文,西北农林科技大学,导师:胡建宏.pp.11.(Hu S,2017.The effects of BMP15 gene on proliferation and differentiation of cashmere goats mG-SCS[D].Thesis for M.S.,North West Agriculture and Forestry University,Supervisor:Hu J H.pp.11.)
赖海燕,桂文武.2018.抗苗勒管激素在辅助生殖领域的临床研究进展[J].国际生殖健康/计划生育杂志,37(01):71-75.(Lai H Y,Gui W W.2018.Study progress of antiMullerian hormone in assisted reproduction[J].Journal of International Reproductive Health/Family Planning,37(01):71-75.)
李运生.2015.抗缪勒氏管激素在猪卵母细胞和颗粒细胞体外发育中的作用及其机制研究[D].博士学位论文,安徽农业大学,导师:章孝荣.pp.6-7;21-24.(Li Y S.2015.Effects and mechanisms of anti-Müllerian hormone on in vitro development of porcine oocytes and granulosa cells[D].Thesis for Ph.D.,Anhui Agricultural University,Supervisor:Zhang X R.pp.6-7;21-24.)
刘秋月,胡文萍,贺小云,等.2017.绵羊多羔主效基因FecB高通量检测方法的建立及应用[J].畜牧兽医学报,48(01):39-51.(Liu Q Y,Hu W P,He X Y,et al.2017.Establishment of high-throughput molecular detection methods for ovine high fecundity major gene FecB and their application[J].Chinese Journal of Animal and Veterinary Sciences,48(01):39-51.)
刘世佳.2014.绵羊产羔数相关候选基因联合效应的初步分析[D].硕士学位论文,甘肃农业大学,导师:李发弟.pp.2.(Liu S J.2014.Preliminary analysis of number of sheep lambing combined effects of candidate gene[D].Thesis for M.S.,Gansu Agricultural University,Supervisor:Li F D.pp.2.)
罗庆苗,秦晓玉,苗向阳.2012.利用数字基因表达谱对小尾寒羊高繁性状相关基因的筛选研究[J].中国农学通报,28(23):8-25.(Luo Q M,Qing X Y,Miao X Y.2012.The study on related genes of high fecundity using digital gene expression tag profiling in Small-Tail-Han-Sheep[J].Chinese Agricultural Science Bulletin,28(23):8-25.)
潘章源,狄冉,刘秋月,等.2015a.绵羊BMPR1B基因生物信息学分析[J].中国草食动物科学,35(06):1-5.(Pan Z Y,Di R,Liu Q Y,et al.2015a.Bioinformatics analysis on BMPR1B gene of sheep[J].China Herbivore Science,35(06):1-5.)
潘章源,狄冉,刘秋月,等.2015b.绵羊多羔主效基因BM?PR1B的研究进展[J].家畜生态学报,36(05):1-6.(Pan Z Y,Di R,Liu Q Y,et al.2015b.Advance in ovine prolificacy gene BMPR1B[J].Journal of Domestic Animal Ecology,36(05):1-6.)
孙晓卫.2017.山羊卵巢中BMP15通过p38 MAPK通路调控AMH的表达[D].硕士学位论文,西南大学,导师:赵中权.pp.43.(Sun X W.2017.BMP15 regulates AMH expression by p38 MAPK passway in the goat ovary[D].Thesis for M.S.,Southwest University,Supervisor:Zhao Z Q.pp.43.)
杨利国.2010.动物繁殖学.第2版[M].北京:中国农业出版社,pp.30.(Yang L G.2010.Animal Reproduction.Second Edition[M].China Agriculture Press,Beijing.pp.30.)
于嘉瑞,王翔宇,郭晓飞,等.2018.LEPR及LEP基因表达量对FecB不同基因型小尾寒羊产羔数的影响[J].畜牧兽医学报,49(05):954-961.(Yu J R,Wang X Y,Guo X F,et al.2018.Effects of expression of LEPR and LEPgenes on litter size of Small Tail Han Sheep with different FecB genotypes[J].Chinese Journal of Animal and Veterinary Sciences,49(05):954-961.)
Bédécarrats G Y,O'Neill F H,Norwitz E R,et al.2003.Regulation of gonadotropin gene expression by Müllerian inhibiting iubstance[J].Proceedings of the National Academy of Sciences of the USA,100(16):9348-9353.
Cai J,Liu L,Zheng J,et al.2018.Differential response of AMH to GnRH agonist among individuals:The effect on ovarian stimulation outcomes[J].Journal of Assisted Reproduction and Genetics,35(3):467-473.
Campbell B K,Clinton M,Webb R.2012.The role of antiMüllerian hormone(AMH)during follicle development in a monovulatory species(sheep)[J].Endocrinology,153(9):4533-4543.
Center K,Dixon D,Lonney C,et al.2018.Anti-Mullerian hormone and follicle counts as predictors of superovulatory response and embryo production in beef cattle[J].Advances in Reproductive Sciences,06(1):22-33.
Cimino I,Casoni F,Liu X,et al.2016.Novel role for antiMüllerian hormone in the regulation of GnRH neuron excitability and hormone secretion[J].Nature Communications,7(1):10055.
Cognie Y,Benoit F,Poulin N,et al.1998.Effect of follicle size and of the FecB Booroola gene on oocyte function in sheep[J].Journal of Reproduction and Fertility,112:379-386.
Cutting A D,Ayers K,Davidson N,et al.2014.Identification,expression,and regulation of anti-Müllerian hormone type-II receptor in the embryonic chicken gonad1[J].Biology of Reproduction,90(5):106.
Durlinger A L,Visser J A,Themmen A P N.2002.Regulation of ovarian function:The role of anti-Müllerian hormone[J].Reproduction,124(5):601-609.
Estienne A,Pierre A,di Clemente N,et al.2015.Anti-Müllerian hormone regulation by the bone morphogenetic proteins in the sheep ovary:Deciphering a direct regulatory pathway[J].Endocrinology,156(1):301-313.
Guo X F,Wang X Y,Di R,et al.2018.Metabolic effects of FecB gene on follicular fluid and ovarian vein serum in sheep(Ovis aries)[J].International Journal of Molecular Sciences,19(2):539.
Josso N,Clemente N D.2003.Transduction pathway of antiMüllerian hormone,a sex-specific member of the TGF-βfamily[J].Trends in Endocrinology&Metabolism,14(2):91-97.
Juengel J L,Davis G H,Mcnatty K P.2013.Using sheep lines with mutations in single genes to better understand ovarian function[J].Reproduction,146(4):111-123.
Juengel J L,French M C,Quirke L D,et al.2017.Differential expression of CART in ewes with differing ovulation rates[J].Reproduction,153(4):471-479.
Lahoz B,Alabart J L,Monniaux D,et al.2012.Anti-Müllerian hormone plasma concentration in prepubertal ewe lambs as a predictor of their fertility at a young age[J].BMC Veterinary Research,118(8):1-9.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,25(4):402-408.
Oliveira M E F,Ayres H,Oliveira L G,et al.2016.Effects of season and ovarian status on the outcome of long-term progesterone-based estrus synchronization protocols and ovulatory follicle development in Santa Inês ewes under subtropical conditions[J].Theriogenology,85(3):452-460.
Poole D H,Ocón-Grove O M,Johnson A L.2016.Anti-Müllerian hormone(AMH)receptor typeⅡexpression and AMH activity in bovine granulosa cells[J].Theriogenology,86(5):1353-1360.
Roy S,Gandra D,Seger C,et al.2018.Oocyte-derived factors(GDF9 and BMP15)and FSH regulate AMH expression via modulation of H3K27AC in granulosa cells[J].Endocrinology,2018,159(9):3433-3445.
Tata B,Mimouni N E H,Barbotin A,et al.2018.Elevated prenatal anti-Müllerian hormone reprograms the fetus and induces polycystic ovary syndrome in adulthood[J].Nature Medicine,24(6):834-846.
van Houten E L A F,Themmen A P N,Visser J A.2010.AntiMüllerian hormone(AMH):Regulator and marker of ovarian function[J].Annales Dendocrinologie,71(3):191-197.
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