甘加藏羊发情周期下丘脑-垂体-卵巢轴中MTR1a基因的表达分析
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摘要
褪黑激素(melatonin, MT)是松果体分泌的一种吲哚类激素,与其受体结合可作用于雌性动物下丘脑-垂体-卵巢轴(hypothalamic-pituitary-ovarian, HPO),对动物的季节性繁殖起一定的调控作用。为阐明高原甘加藏羊(Ovis aries)发情周期HPO中褪黑素1a受体基因(melatonin receptor 1a, MTR1a)的表达规律,本实验选取健康空怀且处于发情周期的甘加藏羊32只,应用qRT-PCR的方法,检测其发情周期下丘脑、垂体、卵巢组织中MTR1a基因表达差异。结果显示,甘加藏羊整个发情周期MTR1a在HPO轴组织中均有表达。在甘加藏羊发情前期、发情期、发情后期、间情期下丘脑组织中MTR1a基因的相对表达量发情前期最高,极显著高于发情期和发情后期(P<0.01);在垂体组织中MTR1a基因的相对表达量间情期最高,与发情期差异显著(P<0.05);在卵巢组织中MTR1a基因的相对表达量发情前期最高,其中发情前期MTR1a基因的相对表达量极显著高于其他三个时期(P<0.01);甘加藏羊发情周期HPO轴组织中均有MTR1a的表达,且表达量具有动态变化差异性,表明MT在甘加藏羊生殖活动中具有调节作用,这一研究结果在mRNA水平上为进一步揭示MT对甘加藏羊繁殖性能调控提供了科学依据。
Melatonin is a steroid hormone secreted by the pineal gland. Its receptor binds to the hypothalamicpituitary-ovarian axis(HPO) of female animals and plays a role in regulating the seasonal reproduction of animals. To describe the expression pattern of the HPO axis of the estrus cycle melatonin receptor 1a(MTR1a)in Ganjia Tibetan sheep(Ovis aries), 32 healthy Ganjia Tibetan sheep were selected in the erotic cycles, and detected the differential expression of melatonin receptor 1a(MTR1a) in Ganjia Tibetan sheep hypothalamus,pituitary and ovarian tissues of plateau in estrous cycle by using qRT-PCR assays. The results showed that MTR1 a was expressed in HPO tissues with entire estrous cycles. In the pre-estrus, estrus, late estrus, and inter-temporal hypothalamic tissue of Ganjia Tibetan sheep, the relative expression of MTR1 a gene in hypothalamic tissue was the highest in proestrus, which was significantly higher than that in estrus and late estrus(P<0.01).At the same period, the relative expression of MTR1 a gene in pituitary tissue was the highest in diestrus,which was significantly different from estrus(P<0.05); The relative expression of MTR1 a in ovarian tissue was the highest in proestrus, which was significantly higher than the other three periods(P<0.01). Taken together, this study found that MTR1 a in different stages with estrous cycle was expressed in HPO tissues, and the expression levels had dynamic variation. These results indicated that MT had a regulatory role for Tibetan sheep reproductive activities. This study provides a scientific basis for MT regulation of sheep reproductive performance in molecular level.
Melatonin is a steroid hormone secreted by the pineal gland. Its receptor binds to the hypothalamicpituitary-ovarian axis(HPO) of female animals and plays a role in regulating the seasonal reproduction of animals. To describe the expression pattern of the HPO axis of the estrus cycle melatonin receptor 1a(MTR1a)in Ganjia Tibetan sheep(Ovis aries), 32 healthy Ganjia Tibetan sheep were selected in the erotic cycles, and detected the differential expression of melatonin receptor 1a(MTR1a) in Ganjia Tibetan sheep hypothalamus,pituitary and ovarian tissues of plateau in estrous cycle by using qRT-PCR assays. The results showed that MTR1 a was expressed in HPO tissues with entire estrous cycles. In the pre-estrus, estrus, late estrus, and inter-temporal hypothalamic tissue of Ganjia Tibetan sheep, the relative expression of MTR1 a gene in hypothalamic tissue was the highest in proestrus, which was significantly higher than that in estrus and late estrus(P<0.01).At the same period, the relative expression of MTR1 a gene in pituitary tissue was the highest in diestrus,which was significantly different from estrus(P<0.05); The relative expression of MTR1 a in ovarian tissue was the highest in proestrus, which was significantly higher than the other three periods(P<0.01). Taken together, this study found that MTR1 a in different stages with estrous cycle was expressed in HPO tissues, and the expression levels had dynamic variation. These results indicated that MT had a regulatory role for Tibetan sheep reproductive activities. This study provides a scientific basis for MT regulation of sheep reproductive performance in molecular level.
引文
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史军红,何玉琴,葛文博,等.2016.高原甘加藏羊生殖周期垂体FSHβ和LHβmRNA表达差异的研究[J].中国兽医科学,46(11):1444-1449.(Shi J H,He Y Q,Ge W B,et al.2016.Study on the difference of FSHβand LHβmRNA expression in pituitary gland of Ganjia Tibetan sheep in the plateau[J].Chinese Veterinary Science,46(11):1444-1449.)
宋倩,王慧,康丽,等.2013.寿光鸡褪黑素受体基因(MT-NR1A)5'控区多态性与产蛋性状的关联分析[J].农业生物技术学报,21(05):579-587.(Song Q,Wang H,Kang L,et al.2013.Association analysis between 5'control region polymorphism and egg production traits of melatonin receptor gene(MTNR1A)in Shouguang chicken[J].Journal of Agricultural Biotechnology,21(05):579-587.)
王淑娟,刘文举,刘晓丽,等.2016.褪黑素受体Mel1a在鸭不同组织中的表达研究[J].浙江农业学报,44(6):15-20.(Wang S J,Liu W J,Liu X L,et al.2016.Expression of melatonin receptor Mel1a in different tissues of duck[J].Acta Agricultural Zhejiangensis,44(6):15-20.)
汪运舟,曾启繁,陈维云,等.2011.多胎与单胎山羊发情期卵巢褪黑素受体基因的差异表达研究[J].中国农学通报,27(03):262-267.(Wang Y Z,Zeng Q F,Chen W Y,et al.2011.Differential expression of ovarian melatonin receptor gene in multiple and single fetal goats during estrus[J].Chinese Agricultural Science Bulletin,27(03):262-267.)
杨颖,陈黎,卢立志.2017.松果体调控动物季节性繁殖概述[J].农业生物技术学报,25(07):1086-1101.(Yang Y,Chen L,Lu L Z.2017.Overview of pineal body regulating seasonal reproduction of animals[J].Journal of Agricultural Biotechnology,25(07):1086-1101)
朱根宝,于兰兰,刘亚.2016.褪黑素调控动物生殖内分泌的研究进展[J].动物医学进展,37(03):94-98.(Zhu G B,Yu L L,Liu Y.2016.Advances in research on melatonin regulating reproductive endocrine in animals[J].Progress in Animal Medicine,37(03):94-98.)
张勇,朱家桥,李发弟,等.2009.绵羊松果体及生殖轴系褪黑素受体MT1的克隆[J].中国兽医学报,29(11):1495-1499.(Zhang Y,Zhu J Q,Li F D,et al.2009.Cloning of melatonin receptor MT1 in pine pineal and reproductive axis[J].Chinese Veterinary Journal,29(11):1495-1499.)
赵艳红,王琳,张铁佳,等.MTNR1a基因在内蒙古绒山羊下丘脑、垂体及卵巢中的表达[J].东北农业大学学报,2014,45(5):92-96.ZHAO Y H,WANG L,ZHANG T Jet al.Expression of MTNR1a gene in hypothalamus,pituitary and ovary of Inner Mongolian Cashmere goats[J].Journal of northeast agricultural university,2014,45(5):92-96.
郑竹清,石玲,王梦一,等.2012.褪黑激素受体1a基因在内蒙古绒山羊垂体中的表达[J].中国畜牧兽医,39(04):39-42.(Zheng Z Q,Shi L,Wang M Y,et al.2012.Expression of melatonin receptor 1a gene in pituitary of Inner Mongolian cashmere goat[J].China Animal Husbandry&Veterinary Medicine,39(04):39-42.)
Clemens J W,Jarzynka M J.2001.Witt-Enderby PA:Downregulation of mt1 melatonin receptors in rat ovary following estrogen exposure[J].Life Sciences,69(1):27-35.
Chowdhuyv S,Ubuka T,Tsutsui K.2013.Review:Melatonin stimu-lates the synthesis and release of gonadotropin-inhibitory hormonein birds[J].General&Comparative Endocrinology,181(1):175-178.
Forcada F,Abecia J A,Casao A,et al.2007.Muino-Blanco T,Palacin I:Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in ewes during anestrus and the reproductive season[J].Theriogenology,67(4):855-862.
Jockers R,Maurics P,Boutin J A,et al.2008.Melatonin receptors,heterodimerization,signal transduction and binding sites:What's new[J].British Journal of Pharmacology,154(6):1182-1195.
Lee C K,Moon D H,Shin C S.2003.Circadian expression of Mel1a and PL-II genes in placenta:Effects of melatoninon the PL-II gene expression in the rat placenta[J].Molecular and Cellular Endocrinology,200(1-2):57-66.
Migaud M,Daveau A,Malpaux B.2005.MTR1a melatonin receptors in the ovine premammillary hypothalamus:Day-night variation in the expression of the transcripts[J].Biology of Reproduction,72(2):393-398
Nishiwaki-Ohkawa T,Yoshimura T.2016.Molecular basis for regulating seasonal reproduction in vertebrates[J].Journal of Endocrinology,229(3):R117-R127.
Sundaresan N R,Marcus Leo M D,Subramani J,et al.2009.Expression analysis of melatonin receptor subtypes in the ovary of domestic chicken[J].Veterinary Research Communicantications,33(1):49-56.
Tamura H,Nakamura Y,Korkmaz A,et al.2009.Melatonin and the ovary:Physiological and pathophysiological implications[J].Fertility and Sterility,92(1):328-343.
Vincent L,Cohen W,Delagrange P,et al.2010.Molecular and cellular pharmaacological properties of 5-methoxycarbonylamino-N-acetyltryptamine(MCA-NTA):A nonspecific MT3 ligand[J].Journal of Pineal Research,48(3):222-229.
冯涛,储明星,王西筠,等.2010.哺乳动物繁殖节律相关基因的研究进展[J].中国畜牧兽医,37(7):89-92.(Feng T,Chu M X,Wang X J,et al.2010.Advances in research on mammalian reproductive rhythm related genes[J].China Animal Husbandry&Veterinary M-edicine37(7):89-92.)
霍生东,袁其建,卢建雄,等.2015.褪黑素受体在未妊娠和妊娠牦牛子宫表达的研究[J].畜牧兽医学报,46(02)239-244.(Huo D S,Yuan Q J,Lu J X,et al.2015.Expression of melatonin receptor in unpregnant and pregnant yak uterus[J].Chinese Journal of Animal and Veterinary Sciences,46(02):239-244.)
葛闻博,何玉琴,张全伟,等.2018.高原甘加羊生殖周期GNRHR基因在垂体和卵巢表达规的研究[J].中国兽医科学,48(06):756-765.(Ge W B,He Y Q,Zhang Q Wet al.2018.Study on expression regulation of GDRHRgene in pituitary and ovary of plateau Ganjia Sheep reproductive cycle[J].Chinese Veterinary Science,48(06)756-765.)
雷梦媛,狄冉,刘秋月,等.2015.褪黑激素受体1A基因(MTR1a)的分子遗传学研究进展[J].农业生物技术学报,23(03):388-396.(Lei M Y,Di R,Liu Q Y,et al.2015.Progress in molecular genetics of melatonin receptor 1A gene(MTR1a)[J].Journal of Agricultural Biotechnology,23(03):388-396.)
史军红,何玉琴,葛文博,等.2016.高原甘加藏羊生殖周期垂体FSHβ和LHβmRNA表达差异的研究[J].中国兽医科学,46(11):1444-1449.(Shi J H,He Y Q,Ge W B,et al.2016.Study on the difference of FSHβand LHβmRNA expression in pituitary gland of Ganjia Tibetan sheep in the plateau[J].Chinese Veterinary Science,46(11):1444-1449.)
宋倩,王慧,康丽,等.2013.寿光鸡褪黑素受体基因(MT-NR1A)5'控区多态性与产蛋性状的关联分析[J].农业生物技术学报,21(05):579-587.(Song Q,Wang H,Kang L,et al.2013.Association analysis between 5'control region polymorphism and egg production traits of melatonin receptor gene(MTNR1A)in Shouguang chicken[J].Journal of Agricultural Biotechnology,21(05):579-587.)
王淑娟,刘文举,刘晓丽,等.2016.褪黑素受体Mel1a在鸭不同组织中的表达研究[J].浙江农业学报,44(6):15-20.(Wang S J,Liu W J,Liu X L,et al.2016.Expression of melatonin receptor Mel1a in different tissues of duck[J].Acta Agricultural Zhejiangensis,44(6):15-20.)
汪运舟,曾启繁,陈维云,等.2011.多胎与单胎山羊发情期卵巢褪黑素受体基因的差异表达研究[J].中国农学通报,27(03):262-267.(Wang Y Z,Zeng Q F,Chen W Y,et al.2011.Differential expression of ovarian melatonin receptor gene in multiple and single fetal goats during estrus[J].Chinese Agricultural Science Bulletin,27(03):262-267.)
杨颖,陈黎,卢立志.2017.松果体调控动物季节性繁殖概述[J].农业生物技术学报,25(07):1086-1101.(Yang Y,Chen L,Lu L Z.2017.Overview of pineal body regulating seasonal reproduction of animals[J].Journal of Agricultural Biotechnology,25(07):1086-1101)
朱根宝,于兰兰,刘亚.2016.褪黑素调控动物生殖内分泌的研究进展[J].动物医学进展,37(03):94-98.(Zhu G B,Yu L L,Liu Y.2016.Advances in research on melatonin regulating reproductive endocrine in animals[J].Progress in Animal Medicine,37(03):94-98.)
张勇,朱家桥,李发弟,等.2009.绵羊松果体及生殖轴系褪黑素受体MT1的克隆[J].中国兽医学报,29(11):1495-1499.(Zhang Y,Zhu J Q,Li F D,et al.2009.Cloning of melatonin receptor MT1 in pine pineal and reproductive axis[J].Chinese Veterinary Journal,29(11):1495-1499.)
赵艳红,王琳,张铁佳,等.MTNR1a基因在内蒙古绒山羊下丘脑、垂体及卵巢中的表达[J].东北农业大学学报,2014,45(5):92-96.ZHAO Y H,WANG L,ZHANG T Jet al.Expression of MTNR1a gene in hypothalamus,pituitary and ovary of Inner Mongolian Cashmere goats[J].Journal of northeast agricultural university,2014,45(5):92-96.
郑竹清,石玲,王梦一,等.2012.褪黑激素受体1a基因在内蒙古绒山羊垂体中的表达[J].中国畜牧兽医,39(04):39-42.(Zheng Z Q,Shi L,Wang M Y,et al.2012.Expression of melatonin receptor 1a gene in pituitary of Inner Mongolian cashmere goat[J].China Animal Husbandry&Veterinary Medicine,39(04):39-42.)
Clemens J W,Jarzynka M J.2001.Witt-Enderby PA:Downregulation of mt1 melatonin receptors in rat ovary following estrogen exposure[J].Life Sciences,69(1):27-35.
Chowdhuyv S,Ubuka T,Tsutsui K.2013.Review:Melatonin stimu-lates the synthesis and release of gonadotropin-inhibitory hormonein birds[J].General&Comparative Endocrinology,181(1):175-178.
Forcada F,Abecia J A,Casao A,et al.2007.Muino-Blanco T,Palacin I:Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in ewes during anestrus and the reproductive season[J].Theriogenology,67(4):855-862.
Jockers R,Maurics P,Boutin J A,et al.2008.Melatonin receptors,heterodimerization,signal transduction and binding sites:What's new[J].British Journal of Pharmacology,154(6):1182-1195.
Lee C K,Moon D H,Shin C S.2003.Circadian expression of Mel1a and PL-II genes in placenta:Effects of melatoninon the PL-II gene expression in the rat placenta[J].Molecular and Cellular Endocrinology,200(1-2):57-66.
Migaud M,Daveau A,Malpaux B.2005.MTR1a melatonin receptors in the ovine premammillary hypothalamus:Day-night variation in the expression of the transcripts[J].Biology of Reproduction,72(2):393-398
Nishiwaki-Ohkawa T,Yoshimura T.2016.Molecular basis for regulating seasonal reproduction in vertebrates[J].Journal of Endocrinology,229(3):R117-R127.
Sundaresan N R,Marcus Leo M D,Subramani J,et al.2009.Expression analysis of melatonin receptor subtypes in the ovary of domestic chicken[J].Veterinary Research Communicantications,33(1):49-56.
Tamura H,Nakamura Y,Korkmaz A,et al.2009.Melatonin and the ovary:Physiological and pathophysiological implications[J].Fertility and Sterility,92(1):328-343.
Vincent L,Cohen W,Delagrange P,et al.2010.Molecular and cellular pharmaacological properties of 5-methoxycarbonylamino-N-acetyltryptamine(MCA-NTA):A nonspecific MT3 ligand[J].Journal of Pineal Research,48(3):222-229.