绵羊繁殖轴相关组织RXRs基因的表达及RXRA基因多态性与季节性发情之间的关系
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摘要
大多数绵羊(Ovis aries)品种为现季节性发情品种,严重制约了绵羊的繁殖效率,而类视黄醇X受体(retinoid X receptors, RXRs)作为季节性发情候选基因受到广泛关注。为了探讨RXRs基因3个亚型RXRA、RXRB和RXRG在绵羊不同繁殖状态下的表达差异,本研究利用qRT-PCR检测3个基因分别在常年发情品种小尾寒羊母羊的卵泡期和黄体期以及季节性发情品种苏尼特羊母羊的长光照和短光照条件下的大脑、小脑、下丘脑、松果体、垂体、卵巢、输卵管、子宫、肾脏、肾上腺共10种组织中的相对表达情况。同时采用Sequenom MassARRAY?SNP技术检测RXRA基因2个SNPs在常年发情绵羊品种(小尾寒羊,湖羊和策勒黑羊)与季节性发情绵羊品种(滩羊,苏尼特羊和草原型藏羊)的多态性,并与季节性发情性状进行关联分析。结果表明,RXRA、RXRB和RXRG基因在苏尼特羊和小尾寒羊各组织中广泛表达,RXRA基因在苏尼特羊松果体中短光照下表达量显著高于长光照(P<0.05),在小尾寒羊卵泡期卵巢中的表达量极显著高于黄体期(P<0.01),在卵泡期子宫体中的表达量极显著低于黄体期(P<0.01);RXRB基因在苏尼特羊垂体中短光照下的表达量显著高于长光照(P<0.05),在小尾寒羊卵泡期卵巢中的表达量极显著高于黄体期(P<0.01);RXRG基因在苏尼特羊下丘脑、垂体和松果体中高表达,其中松果体中在短光照下的表达量极显著低于长光照(P<0.01),在小尾寒羊卵泡期子宫体中的表达量极显著低于黄体期(P<0.01)。通过MassARRAY飞行质谱技术分型发现RXRA基因2个SNP位点g.1699247 G>A和g. 1699276 G>A在小尾寒羊中分布符合哈代温伯格平衡,且2个SNP位点的基因型频率和等位基因频率在6个绵羊品种的2种不同发情模式间均存在显著差异(P<0.05)。以上结果提示,RXRs基因表达与绵羊的繁殖有关,可能参与绵羊季节性发情上游基因调控,本研究结果有助于理解RXRs基因在季节性发情及发情周期转换中的调控作用。
Most of the sheep(Ovis aries) are seasonal estrus animal, which severely restricts the reproductive efficiency of sheep, and retinoid X receptors(RXRs) have received attention as seasonal estrus candidate genes. In order to investigate the differences in the expression of seasonal estrous candidate genes retinoid X receptors(RXRA, RXRB and RXRG) in sheep under different reproductive conditions, q RT-PCR was used to detect these 3 genes for the relative expression of 10 tissues about fallopian tube, brain, cerebellum,hypothalamus, pineal body, pituitary, ovary, fallopian tube, uterus, kidney and adrenal gland, in the follicular and luteal phases of Small Tail Han sheep(perennial estrus) ewes and the long day and short day conditions of the Sunite sheep(seasonal estrus) ewes. The Sequenom MassARRAY?SNP technique was used to detect the 2 SNPs of RXRA gene in perennial estrus sheep breeds(Small Tail Han sheep, Hu sheep and Cele black sheep)and seasonal estrus sheep breeds(Tan sheep, Sunite sheep and Tibetan sheep). The results showed that RXRA,RXRB and RXRG genes were widely expressed in the tissues of the 2 sheep breeds. The expression of RXRA gene in the pineal body of Sunite sheep in short day was significantly higher than that in long day(P<0.05),and the expression of follicular phase in the ovary of Small Tail Han sheep was extremely significant higher than the luteal phase(P<0.01), but the expression of follicular phase in the uterus was extremely significant lower than that in the luteal phase(P<0.01). The expression of RXRB gene in the pituitary of Sunite sheep in short day was significantly higher than in long day(P<0.05). The expression of follicular phase was extremely significant higher than that of luteal phase in the ovary of Small Tail Han sheep(P<0.01). RXRG gene was highly expressed in the hypothalamus, pituitary and pineal body of Sunite, and the expression level in pineal body in long day was extremely significant higher than in short day(P<0.01). The expression level in the uterus of the Small Tail Han sheep was extremely significant lower in the follicular phase than in the luteal phase(P<0.01). From genotyping by MassARRAY time-of-flight mass spectrometry, the study found the genotype frequencies and allele frequencies of the RXRA gene g.1699247 G>A and g. 1699276 G>A were distributed in the Small Tail Han sheep in accordance with the Hardy-Weinberg equilibrium, and were significantly different between seasonal and perennial estrus sheep(P>0.05). The above results implied that RXRs genes expression might be related to reproduction of sheep. These genes might be involved in the regulation of upstream genes of sheep seasonal estrus. The results of this study helps to understand the regulatory role of RXRs in seasonal estrus and estrus cycle transition.
Most of the sheep(Ovis aries) are seasonal estrus animal, which severely restricts the reproductive efficiency of sheep, and retinoid X receptors(RXRs) have received attention as seasonal estrus candidate genes. In order to investigate the differences in the expression of seasonal estrous candidate genes retinoid X receptors(RXRA, RXRB and RXRG) in sheep under different reproductive conditions, q RT-PCR was used to detect these 3 genes for the relative expression of 10 tissues about fallopian tube, brain, cerebellum,hypothalamus, pineal body, pituitary, ovary, fallopian tube, uterus, kidney and adrenal gland, in the follicular and luteal phases of Small Tail Han sheep(perennial estrus) ewes and the long day and short day conditions of the Sunite sheep(seasonal estrus) ewes. The Sequenom MassARRAY?SNP technique was used to detect the 2 SNPs of RXRA gene in perennial estrus sheep breeds(Small Tail Han sheep, Hu sheep and Cele black sheep)and seasonal estrus sheep breeds(Tan sheep, Sunite sheep and Tibetan sheep). The results showed that RXRA,RXRB and RXRG genes were widely expressed in the tissues of the 2 sheep breeds. The expression of RXRA gene in the pineal body of Sunite sheep in short day was significantly higher than that in long day(P<0.05),and the expression of follicular phase in the ovary of Small Tail Han sheep was extremely significant higher than the luteal phase(P<0.01), but the expression of follicular phase in the uterus was extremely significant lower than that in the luteal phase(P<0.01). The expression of RXRB gene in the pituitary of Sunite sheep in short day was significantly higher than in long day(P<0.05). The expression of follicular phase was extremely significant higher than that of luteal phase in the ovary of Small Tail Han sheep(P<0.01). RXRG gene was highly expressed in the hypothalamus, pituitary and pineal body of Sunite, and the expression level in pineal body in long day was extremely significant higher than in short day(P<0.01). The expression level in the uterus of the Small Tail Han sheep was extremely significant lower in the follicular phase than in the luteal phase(P<0.01). From genotyping by MassARRAY time-of-flight mass spectrometry, the study found the genotype frequencies and allele frequencies of the RXRA gene g.1699247 G>A and g. 1699276 G>A were distributed in the Small Tail Han sheep in accordance with the Hardy-Weinberg equilibrium, and were significantly different between seasonal and perennial estrus sheep(P>0.05). The above results implied that RXRs genes expression might be related to reproduction of sheep. These genes might be involved in the regulation of upstream genes of sheep seasonal estrus. The results of this study helps to understand the regulatory role of RXRs in seasonal estrus and estrus cycle transition.
引文
文禹粱,王翔宇,郭晓飞,等.2019.不同产羔数小尾寒羊BMP4基因表达及其错义突变与产羔数关联分析[J].农业生物技术学报,27(01):80-88.(Wen Y L,Wang XY,Guo X F et al.2019.BMP4 gene expression in Small Tail Han Sheep(Ovis aries)with different litter size and association analysis between its missense mutation and litter size[J].Journal of Agricultural Biotechnology,27(1):80-88.)
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Dupont J,Reverchon M,Cloix L,et al.2012.Involvement of adipokines,AMPK,PI3K and the PPAR signaling pathways in ovarian follicle development and cancer[J].The International Journal of Developmental Biology,56(10-12):959-967.
Dupre S M,Miedzinska K,Duval C V,et al.2010.Identification of Eya3 and TAC1 as long-day signals in the sheep pituitary[J].Current Biology,20(9):829-835.
Froment P,Fabre S,Dupont J,et al.2003.Expression and functional role of peroxisome proliferator-activated receptor-gamma in ovarian folliculogenesis in the sheep[J].Biology of Reproduction,69(5):1665-1674.
Gampe R J,Montana V G,Lambert M H,et al.2000.Structural basis for autorepression of retinoid X receptor by tetramer formation and the AF-2 helix[J].Genes Development,14(17):2229-2241.
Girish B P,Swetha C,Reddy P S.2015.Induction of ecdysteroidogenesis,methyl farnesoate synthesis and expression of ecdysteroid receptor and retinoid X receptor in the hepatopancreas and ovary of the giant mud crab,Scylla serrata by melatonin[J].General and Comparative Endocrinology,217-218:37-42.
Gunin A G,Bitter A D,Demakov A B,et al.2004.Effects of peroxisome proliferator activated receptors-alpha andgamma agonists on estradiol-induced proliferation and hyperplasia formation in the mouse uterus[J].The Journal of Endocrinology,182(2):229-239.
Heyman R A,Mangelsdorf D J,Dyck J A,et al.1992.9-cis retinoic acid is a high affinity ligand for the retinoid Xreceptor[J].Cell,68(2):397-406.
Hill S M,Cheng C,Yuan L,et al.2013.Age-related decline in melatonin and its MT1 receptor are associated with decreased sensitivity to melatonin and enhanced mammary tumor growth[J].Current Aging Science,6(1):125-133.
Kersten S,Pan L,Chambon P,et al.1995.Role of ligand in retinoid signaling.9-cis-retinoic acid modulates the oligomeric state of the retinoid X receptor[J].Biochemistry,34(42):13717-13721.
Komar C M.2005.Peroxisome proliferator-activated receptors(PPARs)and ovarian function--implications for regulating steroidogenesis,differentiation,and tissue remodeling[J].Reproductive Biology and Endocrinology,3:41.
Meade E A,Mcintyre T M,Zimmerman G A,et al.1999.Peroxisome proliferators enhance cyclooxygenase-2 expression in epithelial cells[J].The Journal of Biological Chemistry,274(12):8328-8334.
Ortega M S,Denicol A C,Cole J B,et al.2016.Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows[J].Animal Genetics,47(3):288-297.
Pinto V,Minakhina S,Qiu S,et al.2017.Naturally occurring amino acids in helix 10 of the thyroid hormone receptor mediate isoform-specific TH gene regulation[J].Endocrinology,158(9):3067-3078.
Schmittgen T D,Livak K J.2016.Analyzing Real-time PCRdata by the comparative CT method[J].Nature Protocols,3(6):1101-1108.
Shiue Y L,Chen L R,Tsai C J,et al.2013.Emerging roles of peroxisome proliferator-activated receptors in the pituitary gland in female reproduction[J].Biomarkers&Genomic Medicine,5(1-2):1-11.
Singh B K,Sinha R A,Ohba K,et al.2017.Role of thyroid hormone in hepatic gene regulation,chromatin remodeling,and autophagy[J].Molecular&Cellular Endocrinology,458:160-168.
Wan X,Wang S,Xu J,et al.2017.Dietary protein-induced hepatic IGF-1 secretion mediated by PPAR gamma activation[J].PLOS ONE,12(3):e173174.
Willy P J,Mangelsdorf D J.1997.Unique requirements for retinoid-dependent transcriptional activation by the orphan receptor LXR[J].Genes development,11(3):289-298.
Ackerman W T,Zhang X L,Rovin B H,et al.2005.Modulation of cytokine-induced cyclooxygenase 2 expression by PPARG ligands through NFkappaB signal disruption in human WISH and amnion cells[J].Biology of Reproduction,73(3):527-535.
Bogacka I,Kurzynska A,Bogacki M,et al.2015.Peroxisome proliferator-activated receptors in the regulation of female reproductive functions[J].Folia Histochemica et Cytobiologica,53(3):189-200.
Darnerud P O,Risberg S.2006.Tissue localisation of tetraand pentabromodiphenyl ether congeners(BDE-47,-85and-99)in perinatal and adult C57BL mice[J].Chemosphere,62(3):485-493.
Dokmanovic M,Chang B D,Fang J,et al.2002.Retinoid-induced growth arrest of breast carcinoma cells involves co-activation of multiple growth-inhibitory genes[J].Cancer Biology&Therapy,1(1):24-27.
Dupont J,Chabrolle C,Rame C,et al.2008.Role of the peroxisome proliferator-activated receptors,adenosine monophosphate-activated kinase,and adiponectin in the ovary[J].PPAR Research,2008:176275.
Dupont J,Reverchon M,Cloix L,et al.2012.Involvement of adipokines,AMPK,PI3K and the PPAR signaling pathways in ovarian follicle development and cancer[J].The International Journal of Developmental Biology,56(10-12):959-967.
Dupre S M,Miedzinska K,Duval C V,et al.2010.Identification of Eya3 and TAC1 as long-day signals in the sheep pituitary[J].Current Biology,20(9):829-835.
Froment P,Fabre S,Dupont J,et al.2003.Expression and functional role of peroxisome proliferator-activated receptor-gamma in ovarian folliculogenesis in the sheep[J].Biology of Reproduction,69(5):1665-1674.
Gampe R J,Montana V G,Lambert M H,et al.2000.Structural basis for autorepression of retinoid X receptor by tetramer formation and the AF-2 helix[J].Genes Development,14(17):2229-2241.
Girish B P,Swetha C,Reddy P S.2015.Induction of ecdysteroidogenesis,methyl farnesoate synthesis and expression of ecdysteroid receptor and retinoid X receptor in the hepatopancreas and ovary of the giant mud crab,Scylla serrata by melatonin[J].General and Comparative Endocrinology,217-218:37-42.
Gunin A G,Bitter A D,Demakov A B,et al.2004.Effects of peroxisome proliferator activated receptors-alpha andgamma agonists on estradiol-induced proliferation and hyperplasia formation in the mouse uterus[J].The Journal of Endocrinology,182(2):229-239.
Heyman R A,Mangelsdorf D J,Dyck J A,et al.1992.9-cis retinoic acid is a high affinity ligand for the retinoid Xreceptor[J].Cell,68(2):397-406.
Hill S M,Cheng C,Yuan L,et al.2013.Age-related decline in melatonin and its MT1 receptor are associated with decreased sensitivity to melatonin and enhanced mammary tumor growth[J].Current Aging Science,6(1):125-133.
Kersten S,Pan L,Chambon P,et al.1995.Role of ligand in retinoid signaling.9-cis-retinoic acid modulates the oligomeric state of the retinoid X receptor[J].Biochemistry,34(42):13717-13721.
Komar C M.2005.Peroxisome proliferator-activated receptors(PPARs)and ovarian function--implications for regulating steroidogenesis,differentiation,and tissue remodeling[J].Reproductive Biology and Endocrinology,3:41.
Meade E A,Mcintyre T M,Zimmerman G A,et al.1999.Peroxisome proliferators enhance cyclooxygenase-2 expression in epithelial cells[J].The Journal of Biological Chemistry,274(12):8328-8334.
Ortega M S,Denicol A C,Cole J B,et al.2016.Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows[J].Animal Genetics,47(3):288-297.
Pinto V,Minakhina S,Qiu S,et al.2017.Naturally occurring amino acids in helix 10 of the thyroid hormone receptor mediate isoform-specific TH gene regulation[J].Endocrinology,158(9):3067-3078.
Schmittgen T D,Livak K J.2016.Analyzing Real-time PCRdata by the comparative CT method[J].Nature Protocols,3(6):1101-1108.
Shiue Y L,Chen L R,Tsai C J,et al.2013.Emerging roles of peroxisome proliferator-activated receptors in the pituitary gland in female reproduction[J].Biomarkers&Genomic Medicine,5(1-2):1-11.
Singh B K,Sinha R A,Ohba K,et al.2017.Role of thyroid hormone in hepatic gene regulation,chromatin remodeling,and autophagy[J].Molecular&Cellular Endocrinology,458:160-168.
Wan X,Wang S,Xu J,et al.2017.Dietary protein-induced hepatic IGF-1 secretion mediated by PPAR gamma activation[J].PLOS ONE,12(3):e173174.
Willy P J,Mangelsdorf D J.1997.Unique requirements for retinoid-dependent transcriptional activation by the orphan receptor LXR[J].Genes development,11(3):289-298.