热应激对黄体孕酮代谢通路的影响
|
摘要
[目的]以假孕大鼠为模型,探讨热应激对氯前列烯醇(PGF)诱导的黄体退化过程中孕酮水平及其代谢通路相关蛋白表达的影响。[方法]用PMSG/h CG(D0)联合处理50只大鼠,诱导假孕,然后随机分为非热应激(NHS)和热应激(HS)两组。每天在相同时间,将HS组置于40℃应激2 h。假孕第7天,热应激结束2 h后,各组大鼠以PGF分别处理0、1、2、8和24 h后屠宰,收集血清和卵巢组织。用酶联免疫法检测血清孕酮浓度,蛋白免疫印迹法和免疫组织化学法检测孕酮水平及其代谢通路相关蛋白的表达。[结果]热应激前、后假孕大鼠的直肠温度差异显著(P<0.05);PGF降低了两组大鼠血清孕酮浓度,热应激抑制了PGF诱导的孕酮浓度下降(P<0.05);PGF或热应激处理对PKA磷酸化底物、St AR、P450scc、3βHSD及NR4A1-F表达均无显著影响(P>0.05),但均诱导NR4A1-S表达上调(P<0.05),且热应激降低PGF诱导的NR4A1-S表达(P<0.05);PKA磷酸化底物主要在类固醇生成细胞的细胞核中表达,而St AR、P450scc、3βHSD和NR4A1-F均在类固醇生成细胞的细胞质表达。[结论]热应激降低了PGF诱导的假孕大鼠黄体退化过程中NR4A1-S的表达,提示NR4A1-S可能参与调控黄体退化过程中孕酮的代谢。
[Objectives]This study was designed to investigate the effect of heat stress on the pathway associated with the progesterone synthesis and catabolism during cloprostenol( prostaglandin F_(2α)analogue,PGF)-induced luteal regression in pseudopregnant rats.[Methods]A total of 50 rats were induced pseudopregnancy by sequential PMSG/h CG( D0) treatment paradigm and randomly distributed into non-heat stress( NHS) group and heat stress( HS) group. The rats in HS group were exposed to 40 ℃ at the same time every day for 2 h. On day 7 of pseudopregnancy,after 2 h post-stress recovery,rats were treated with PGF for 0,1,2,8 or 24 h and then were sampled. Blood were collected to determine serum concentrations of progesterone by enzyme-linked immunosorbent assay,and ovaries were collected to determine the proteins associated with the progesterone metablism by western blot and immunohistochemistry.[Results]The changes in the rectal temperature in pseudopregnant rats was significant before and after heat stress( P<0.05); PGF decreased progesterone concentrations in both HS and NHS groups,and heat stress blunted PGF-induced decline in the progesterone concentrations( P<0.05); PGF or heat treatment alone didn't alter the expressions of phospho-PKA substrates,St AR,P450 scc,3βHSD and NR4A1-F( P > 0. 05),but significantly increased NR4A1-S expression( P < 0. 05). Furthermore,heat stress reduced NR4A1-S expression in response to PGF( P < 0. 05); the phospho-PKA substrates expressed mainly in the nuclei of steroidogenic cells,while St AR,P450 scc,3βHSD and NR4A1-F uniformly expressed in the cytoplasm of steroidogenic cells. [Conclusions]Heat stress could reduce PGF-induced NR4A1-S expressions in pseudopregnant rats,which might be correlated with regulation of the progesterone catabolism during luteal regression.
[Objectives]This study was designed to investigate the effect of heat stress on the pathway associated with the progesterone synthesis and catabolism during cloprostenol( prostaglandin F_(2α)analogue,PGF)-induced luteal regression in pseudopregnant rats.[Methods]A total of 50 rats were induced pseudopregnancy by sequential PMSG/h CG( D0) treatment paradigm and randomly distributed into non-heat stress( NHS) group and heat stress( HS) group. The rats in HS group were exposed to 40 ℃ at the same time every day for 2 h. On day 7 of pseudopregnancy,after 2 h post-stress recovery,rats were treated with PGF for 0,1,2,8 or 24 h and then were sampled. Blood were collected to determine serum concentrations of progesterone by enzyme-linked immunosorbent assay,and ovaries were collected to determine the proteins associated with the progesterone metablism by western blot and immunohistochemistry.[Results]The changes in the rectal temperature in pseudopregnant rats was significant before and after heat stress( P<0.05); PGF decreased progesterone concentrations in both HS and NHS groups,and heat stress blunted PGF-induced decline in the progesterone concentrations( P<0.05); PGF or heat treatment alone didn't alter the expressions of phospho-PKA substrates,St AR,P450 scc,3βHSD and NR4A1-F( P > 0. 05),but significantly increased NR4A1-S expression( P < 0. 05). Furthermore,heat stress reduced NR4A1-S expression in response to PGF( P < 0. 05); the phospho-PKA substrates expressed mainly in the nuclei of steroidogenic cells,while St AR,P450 scc,3βHSD and NR4A1-F uniformly expressed in the cytoplasm of steroidogenic cells. [Conclusions]Heat stress could reduce PGF-induced NR4A1-S expressions in pseudopregnant rats,which might be correlated with regulation of the progesterone catabolism during luteal regression.
引文
[1]Hansen P J.Effects of heat stress on mammalian reproduction[J].Philosophical Transactions of the Royal Society London Series B:Biological Sciences,2009,364(1534):3341-3350.
[2]de S Torres-Júnior J R,de FA Pires M,de SaW F,et al.Effect of maternal heat-stress on follicular growth and oocyte competence in Bos indicus cattle[J].Theriogenology,2008,69(2):155-166.
[3]Hansen P J.Exploitation of genetic and physiological determinants of embryonic resistance to elevated temperature to improve embryonic survival in dairy cattle during heat stress[J].Theriogenology,2007,68(Suppl 1):S242-S249.
[4]Mizutani T,Ishikane S,Kawabe S,et al.Transcriptional regulation of genes related to progesterone production[J].Endocrine Journal,2015,62(9):757-763.
[5]Sudeshna T,Anand K,Medhamurthy R.Analysis of 20alpha-hydroxysteroid dehydrogenase expression in the corpus luteum of the buffalo cow:effect of prostaglandin F2-alpha treatment on circulating 20alpha-hydroxyprogesterone levels[J].Reproductive Biology and Endocrinology,2013,11(1):111-118.
[6]Guo N,Meng C,Bai W,et al.Prostaglandin F2alpha induces expression of activating transcription factor 3(ATF3)and activates MAPK signaling in the rat corpus luteum[J].Acta Histochem,2015,117(2):211-218.
[7]Macias-Cruz U,Gastelum M A,Alvarez F D,et al.Effects of summer heat stress on physiological variables,ovulation and progesterone secretion in Pelibuey ewes under natural outdoor conditions in an arid region[J].Animal Science Journal,2016,87(3):354-360.
[8]Meng C,Guo N,Wei Q,et al.Expression of repulsive guidance molecule b(RGMb)in the uterus and ovary during the estrous cycle in rats[J].Acta Histochem,2014,116(8):1231-1236.
[9]Wang Y,Meng C,Wei Q,et al.Expression and regulation of scavenger receptor class B type 1 in the rat ovary and uterus during the estrous cycle[J].Acta Histochem,2015,117(3):297-304.
[10]Kurusu S,Sonoda N,Nakahara M,et al.Evidence suggesting multiple promoting roles of luteal group IVA phospholipase A(2)in prostaglandin F(2alpha)-induced regression in pseudopregnant rats[J].Prostaglandins and Other Lipid Mediators,2010,93(1/2):37-43.
[11]Kurowicka B,Gajewska A,Franczak A.Effect of early thermal experience on pituitary-gonadal axis in female rats[J].Reproductive Biology,2006,6(1):63-77.
[12]Stocco C,Telleria C,Gibori G.The molecular control of corpus luteum formation,function,and regression[J].Endocrine Reviews,2007,28(1):117-149.
[13]Li M,Xue K,Ling J,et al.The orphan nuclear receptor NR4A1 regulates transcription of keys teroidogenic enzymes in ovarian theca cells[J].Molecular and Cellular Endocrinology,2010,319(1/2):39-46.
[14]Li Y,Bourbon P M,Grant M A,et al.Requirement of novel amino acid fragments of orphan nuclear receptor TR3/Nur77 for its functions in angiogenesis[J].Oncotarget,2015,6(27):24261-24276.
[15]Xue K,Liu J Y,Murphy B D,et al.Orphan nuclear receptor NR4A1 is a negative regulator of DHT-induced rat preantral follicular growth[J].Molecular Endocrinology,2012,26(12):2004-2015.
[16]Zhang X.Targeting Nur77 translocation[J].Oncologic,Endocrine and Metabolic,2007,11(1):69-79.
[17]翟永宁,刁飞扬,刘嘉茵.核受体NR4A1蛋白在大鼠卵泡发育中的定位及其表达规律[J].医学研究生学报,2007,20(1):32-35.Zhai Y N,Diao F Y,Liu J Y.The localization and expression pattern of nuclear receptor NR4A1 protein in rat ovary[J].Journal of Medical Postgraduates,2007,20(1):32-35(in Chinese with English abstract).
[18]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.Ma H M,Liu J Y.Expression of nuclear hormone receptor NR4A1 in the human ovarian cycle[J].Jiangsu Medical Journal,2007,33(5):439-440(in Chinese with English abstract).
[19]贾娟娟,滚双宝,林长光,等.胎次、配种及分娩月份对母猪繁殖性能的影响[J].甘肃农业大学学报,2015,50(1):1-4.Jia J J,Gun S B,Lin C G,et al.The influence of different parities,mating months and birth months on reproductive performance of sows[J].Journal of Gansu Agricultural University,2015,50(1):1-4(in Chinese with English abstract).
[20]陈丽,张彬,李丽立.热应激对母猪繁殖性能的影响及防治措施[J].饲料博览,2015(9):13-17.Chen L,Zhang B,Li L L.The effects of heat stress on reproductive performance of sow and control meeasures[J].Feed Review,2015(9):13-17(in Chinese).
[21]Sakatani M,Balboula A Z,Yamanaka K,et al.Effect of summer heat environment on body temperature,estrous cycles and blood antioxidant levels in Japanese black cow[J].Animal Science Journal,2012,83(5):394-402.
[2]de S Torres-Júnior J R,de FA Pires M,de SaW F,et al.Effect of maternal heat-stress on follicular growth and oocyte competence in Bos indicus cattle[J].Theriogenology,2008,69(2):155-166.
[3]Hansen P J.Exploitation of genetic and physiological determinants of embryonic resistance to elevated temperature to improve embryonic survival in dairy cattle during heat stress[J].Theriogenology,2007,68(Suppl 1):S242-S249.
[4]Mizutani T,Ishikane S,Kawabe S,et al.Transcriptional regulation of genes related to progesterone production[J].Endocrine Journal,2015,62(9):757-763.
[5]Sudeshna T,Anand K,Medhamurthy R.Analysis of 20alpha-hydroxysteroid dehydrogenase expression in the corpus luteum of the buffalo cow:effect of prostaglandin F2-alpha treatment on circulating 20alpha-hydroxyprogesterone levels[J].Reproductive Biology and Endocrinology,2013,11(1):111-118.
[6]Guo N,Meng C,Bai W,et al.Prostaglandin F2alpha induces expression of activating transcription factor 3(ATF3)and activates MAPK signaling in the rat corpus luteum[J].Acta Histochem,2015,117(2):211-218.
[7]Macias-Cruz U,Gastelum M A,Alvarez F D,et al.Effects of summer heat stress on physiological variables,ovulation and progesterone secretion in Pelibuey ewes under natural outdoor conditions in an arid region[J].Animal Science Journal,2016,87(3):354-360.
[8]Meng C,Guo N,Wei Q,et al.Expression of repulsive guidance molecule b(RGMb)in the uterus and ovary during the estrous cycle in rats[J].Acta Histochem,2014,116(8):1231-1236.
[9]Wang Y,Meng C,Wei Q,et al.Expression and regulation of scavenger receptor class B type 1 in the rat ovary and uterus during the estrous cycle[J].Acta Histochem,2015,117(3):297-304.
[10]Kurusu S,Sonoda N,Nakahara M,et al.Evidence suggesting multiple promoting roles of luteal group IVA phospholipase A(2)in prostaglandin F(2alpha)-induced regression in pseudopregnant rats[J].Prostaglandins and Other Lipid Mediators,2010,93(1/2):37-43.
[11]Kurowicka B,Gajewska A,Franczak A.Effect of early thermal experience on pituitary-gonadal axis in female rats[J].Reproductive Biology,2006,6(1):63-77.
[12]Stocco C,Telleria C,Gibori G.The molecular control of corpus luteum formation,function,and regression[J].Endocrine Reviews,2007,28(1):117-149.
[13]Li M,Xue K,Ling J,et al.The orphan nuclear receptor NR4A1 regulates transcription of keys teroidogenic enzymes in ovarian theca cells[J].Molecular and Cellular Endocrinology,2010,319(1/2):39-46.
[14]Li Y,Bourbon P M,Grant M A,et al.Requirement of novel amino acid fragments of orphan nuclear receptor TR3/Nur77 for its functions in angiogenesis[J].Oncotarget,2015,6(27):24261-24276.
[15]Xue K,Liu J Y,Murphy B D,et al.Orphan nuclear receptor NR4A1 is a negative regulator of DHT-induced rat preantral follicular growth[J].Molecular Endocrinology,2012,26(12):2004-2015.
[16]Zhang X.Targeting Nur77 translocation[J].Oncologic,Endocrine and Metabolic,2007,11(1):69-79.
[17]翟永宁,刁飞扬,刘嘉茵.核受体NR4A1蛋白在大鼠卵泡发育中的定位及其表达规律[J].医学研究生学报,2007,20(1):32-35.Zhai Y N,Diao F Y,Liu J Y.The localization and expression pattern of nuclear receptor NR4A1 protein in rat ovary[J].Journal of Medical Postgraduates,2007,20(1):32-35(in Chinese with English abstract).
[18]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.Ma H M,Liu J Y.Expression of nuclear hormone receptor NR4A1 in the human ovarian cycle[J].Jiangsu Medical Journal,2007,33(5):439-440(in Chinese with English abstract).
[19]贾娟娟,滚双宝,林长光,等.胎次、配种及分娩月份对母猪繁殖性能的影响[J].甘肃农业大学学报,2015,50(1):1-4.Jia J J,Gun S B,Lin C G,et al.The influence of different parities,mating months and birth months on reproductive performance of sows[J].Journal of Gansu Agricultural University,2015,50(1):1-4(in Chinese with English abstract).
[20]陈丽,张彬,李丽立.热应激对母猪繁殖性能的影响及防治措施[J].饲料博览,2015(9):13-17.Chen L,Zhang B,Li L L.The effects of heat stress on reproductive performance of sow and control meeasures[J].Feed Review,2015(9):13-17(in Chinese).
[21]Sakatani M,Balboula A Z,Yamanaka K,et al.Effect of summer heat environment on body temperature,estrous cycles and blood antioxidant levels in Japanese black cow[J].Animal Science Journal,2012,83(5):394-402.