NR4A1在猪卵巢黄体中的作用探讨
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摘要
通过分析NR4A1在猪卵巢黄体中的表达与定位,探讨其在猪黄体发育与退化中的潜在作用。获取猪发情周期不同阶段卵巢组织,分离黄体(早期、中期和晚期),应用放射免疫法检测孕酮和雌二醇浓度,应用蛋白免疫印迹法检测NR4A1、CYP17A1和cleaved-Caspase-3的表达,应用免疫组织化学方法检测NR4A1和cleaved-Caspase-3的定位。结果:晚期黄体(白体)的孕酮水平显著低于早期和中期,雌二醇水平显著高于早期和中期(P<0.05);早期黄体(红体)与中期黄体的NR4A1表达量无显著差异,而晚期黄体(白体)中NR4A1显著低于早期和中期(P<0.05);CYP17A1在早期黄体与中期黄体无显著差异,晚期黄体显著上升;cleaved-Caspase-3在中期黄体中表达最低,而在晚期黄体中最高(P<0.05)。此外,整个黄体发育与退化过程中,NR4A1主要在黄体类固醇生成细胞的细胞质中表达,而cleaved-Caspase-3在包括黄体类固醇生成细胞的所有细胞中均有表达。结果提示:猪卵巢黄体发育与退化过程中,NR4A1的蛋白表达趋势与孕酮水平一致,NR4A1可能参与调控猪黄体组织中孕酮的合成代谢,为深入研究NR4A1参与猪卵巢机能调控提供理论依据。
Based on the natural porcine ovarian corpus luteum,the expression pattern and localization of luteal NR4A1 were analyzed to investigate the potential role during the development and regression of the corpus luteum.Porcine ovaries at different estrous cycle were collected to isolate the luteal tissues(early phase,middle phase and late phase).Radioimmunoassay(RIA) was used to determine the progesterone and estrogen concentrations,western blot(WB) used to analyze the expression pattern of NR4A1,CYP17A1 and cleaved-Caspase-3and immunohistochemistry(IHC) applied to localize the NR4A1 and cleaved-Caspase-3.Results showed that the progesterone level of early and middle luteal tissue were lower than that in the late corpus luteum,while estrodiol were higher than that(P<0.05) in late corpus luteum(corpus albicans).In addition,the NR4A1 expression had no significant differences between early(corpus haemorrhagicum) and middle corpus luteum,which decreased significantly(P<0.05) in the late corpus luteum.However,CYP17A1 expression increased significantly in the late corpus luteum,and no changes were found between the early and middle corpus luteum.Cleaved-Caspase-3 expression level was the lowest in the middle corpus luteum,while it was the highest in the late corpus luteum(P<0.05).Furthermore,the NR4A1 mainly localized in the cytoplasm of luteal steroidogenic cells,while cleaved-Caspase-3 expressed in the cytoplasm of nearly all kinds of luteal cells including steroidogenic cells.Our finding suggested that NR4A1 might be involved in the regulation of progesterone metabolism in porcine corpus lutea,providing a theoretical basis for the study of NR4A1 in the regulation of porcine ovarian functions.
Based on the natural porcine ovarian corpus luteum,the expression pattern and localization of luteal NR4A1 were analyzed to investigate the potential role during the development and regression of the corpus luteum.Porcine ovaries at different estrous cycle were collected to isolate the luteal tissues(early phase,middle phase and late phase).Radioimmunoassay(RIA) was used to determine the progesterone and estrogen concentrations,western blot(WB) used to analyze the expression pattern of NR4A1,CYP17A1 and cleaved-Caspase-3and immunohistochemistry(IHC) applied to localize the NR4A1 and cleaved-Caspase-3.Results showed that the progesterone level of early and middle luteal tissue were lower than that in the late corpus luteum,while estrodiol were higher than that(P<0.05) in late corpus luteum(corpus albicans).In addition,the NR4A1 expression had no significant differences between early(corpus haemorrhagicum) and middle corpus luteum,which decreased significantly(P<0.05) in the late corpus luteum.However,CYP17A1 expression increased significantly in the late corpus luteum,and no changes were found between the early and middle corpus luteum.Cleaved-Caspase-3 expression level was the lowest in the middle corpus luteum,while it was the highest in the late corpus luteum(P<0.05).Furthermore,the NR4A1 mainly localized in the cytoplasm of luteal steroidogenic cells,while cleaved-Caspase-3 expressed in the cytoplasm of nearly all kinds of luteal cells including steroidogenic cells.Our finding suggested that NR4A1 might be involved in the regulation of progesterone metabolism in porcine corpus lutea,providing a theoretical basis for the study of NR4A1 in the regulation of porcine ovarian functions.
引文
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[23]Zhang X K.Targeting Nur77 translocation[J].Expert Opinion on Therapeutic Targets,2007,11:69-79.
[24]张灵枝,王磊磊,梁超,等.奶山羊妊娠黄体的组织形态观察[J].西北农林科技大学学报(自然科学版),2010(6):31-36.
[2]Winoto A,Dan R L.Nuclear hormone receptors in T lymphocytes[J].Cell,2002,109(Suppl):57-66.
[3]Li Q X,Ke N,Sundaram R,et al.NR4A1,2,3--an orphan nuclear hormone receptor family involved in cell apoptosis and carcinogenesis[J].Histology&Histopathology,2006,21:533-540.
[4]马红梅,刘嘉茵.核受体激素NR4A1在人卵巢周期中的表达[J].江苏医药,2007,33(5):439-440.
[5]Li M,Xue K,Ling J,et al.The orphan nuclear receptor NR4A1regulates transcription of key steroidogenic enzymes in ovarian theca cells[J].Molecular&Cellular Endocrinology,2010,319:39-46.
[6]Atli M O,Bender R W,Mehta V,et al.Patterns of gene expression in the bovine corpus luteum following repeated intrauterine infusions of low doses of prostaglandin F2alpha[J].Biology of Reproduction,2012,86:130-130.
[7]Stocco C O,Lau L F,Gibori G.A calcium/calmodulin-dependent activation of ERK1/2 mediates Jun D phosphorylation and induction of nur77 and 20 alpha-hsd genes by prostaglandin F2alpha in ovarian cells[J].Journal of Biological Chemistry,2002,277:3293-3302.
[8]Wu Y,Ghosh S,Nishi Y,et al.The orphan nuclear receptors NURR1 and NGFI-B modulate aromatase gene expression in ovarian granulosa cells:a possible mechanism for repression of aromatase expression upon luteinizing hormone surge[J].Endocrinology,2005,146:237-246.
[9]Liu L Q,Li F E,Deng C Y,et al.Molecular cloning,tissue expression and association of porcine NR4A1 gene with reproductive traits[J].Molecular Biology Reports,2011,38:103-114.
[10]Akins E L,Morrissette M C.Gross ovarian changes during estrous cycle of swine[J].American Journal of Veterinary Research,1968,29:1953-1957.
[11]Bai W J,Jin P J,Kuang M Q,et al.Temporal regulation of extracellular signal-regulated kinase 1/2 phosphorylation,heat shock protein 70 and activating transcription factor 3 during prostaglandin F-induced luteal regression in pseudopregnant rats following heat stress[J].Reproduction Fertility&Development,2016 May 12.doi:10.1071/RD15415.
[12]Kallen C B,Billheimer J T,Summers S A,et al.Steroidogenic acute regulatory protein(St AR)is a sterol transfer protein[J].Journal of Biological Chemistry,1998,273:26285-26288.
[13]Taniguchi K,Matsuoka A,Kizuka F,et al.Prostaglandin F2α(PGF2α)stimulates PTGS2 expression and PGF2αsynthesis through NFKB activation via reactive oxygen species in the corpus luteum of pseudopregnant rats[J].Reproduction,2010,140:885-892.
[14]张广智,胡长敏,陈颖钰,等.细胞凋亡的主要检测方法研究进展[J].动物医学进展,2011,32(8):89-92.
[15]Hong C Y,Park J H,Ahn R S,et al.Molecular mechanism of suppression of testicular steroidogenesis by proinflammatory cytokine tumor necrosis factor alpha[J].Molecular&Cellular Biology,2004,24:2593-2604.
[16]Martin L J,Tremblay J J.Glucocorticoids antagonize c AMP-induced Star transcription in Leydig cells through the orphan nuclear receptor NR4A1[J].Journal of Molecular Endocrinology,2008,41:165-175.
[17]Guo N,Meng C,Bai W,et al.Prostaglandin F2αinduces expression of activating transcription factor 3(ATF3)and activates MAPK signaling in the rat corpus luteum[J].Acta Histochemica,2015,117:211-218.
[18]Stocco C O,Zhong L,Sugimoto Y,et al.Prostaglandin F2α-induced expression of 20alpha-hydroxysteroid dehydrogenase involves the transcription factor NUR77[J].Journal of Biological Chemistry,2000,275:37202-37211.
[19]Sudeshna T.Analysis of 20α-hydroxysteroid dehydrogenase expression in the corpus luteum of the buffalo cow:effect of prostaglandin F2αtreatment on circulating 20α-hydroxyprogesterone levels[J].Reproductive Biology and Endocrinology,2013,11:1-10.
[20]屠迪,邬静,袁莉芸,等.软脂酸在体外激活Caspase 9和Caspase 3引起L02细胞凋亡[J].中国兽医杂志,2013,49(2):10-13.
[21]Zorrilla L M,D'Annibale M A,Swing S E,et al.Expression of genes associated with apoptosis in the porcine corpus luteum during the oestrous cycle[J].Reproduction in Domestic Animals,2013,48:755-761.
[22]杨晨.妊娠期山羊黄体中凋亡蛋白Bax/Bcl-2和Caspase-3/Caspase-9的表达[D].杨凌:西北农林科技大学,2011.
[23]Zhang X K.Targeting Nur77 translocation[J].Expert Opinion on Therapeutic Targets,2007,11:69-79.
[24]张灵枝,王磊磊,梁超,等.奶山羊妊娠黄体的组织形态观察[J].西北农林科技大学学报(自然科学版),2010(6):31-36.