FSH及Wnt信号通路对绵羊卵泡颗粒细胞的增殖及雌激素分泌的影响

doi:10.11843/j.issn.0366-6964.2016.01.009

摘要/Abstract

摘要：

旨在研究Wnt信号通路对绵羊卵泡颗粒细胞增殖及雌激素分泌的影响，探究其是否与促卵泡素（Follicular stimulating hormone，FSH）有关。在添加有FSH以及不同浓度Wnt信号通路抑制剂IWR-1的培养基中培养绵羊卵巢卵泡颗粒细胞168 h，利用Guava ViaCount^® Reagent测定细胞数目的变化，利用绵羊ELISA试剂盒检测雌激素浓度的变化，利用qRT-PCR检测FSH靶基因CYP19和CCND2的相对表达量差异。结果表明，无论有无FSH，抑制剂IWR-1使颗粒细胞数目显著减少（P<0.05）；当有FSH时，抑制剂IWR-1的添加导致雌激素浓度显著降低（P<0.05）。IWR-1的浓度为1.0 μmol•L^-1时，对颗粒细胞的抑制效果最明显。IWR-1还导致CYP19和CCND2 mRNA的表达量降低。综上表明，Wnt信号通路对绵羊卵泡颗粒细胞的生物学功能具有促进作用，并且这种促进作用可能与FSH有关。

Abstract:

This study aims to investigate the effect of Wnt signaling pathway on proliferation and estrogen secretion of sheep granulosa cells and to explore whether the effect was related to FSH(Follicular stimulating hormone，FSH).With or without FSH，sheep granulosa cells were cultured in medium with different concentrations of Wnt signaling pathway inhibitor IWR-1 for 168 h.Then，Guava ViaCount^®Reagent was used to detect the changes of cell number and sheep ELISA kit was used to detect the changes of estrogen concentrations.The relative expression of CYP19 and CCND2，which are the target genes of FSH，was analyzed by qRT-PCR.The results showed that IWR-1 could cause a significant(P<0.05) reduction in the number of granule cells whether FSH existed or not.With the presence of FSH，the addition of IWR-1 could significantly(P<0.05) decrease estrogen concentration.When the concentration of IWR-1 was 1.0 μmol•L^-1，its inhibitory effect on granulosa cells was of most obvious.Besides，IWR-1 decreased the abundance of CYP19 mRNA and CCND2 mRNA.In conclusion，Wnt signaling pathway can improve the biological function of sheep granulosa cells，and this function may be related to FSH.

中图分类号:

S826.2

赵妙妙，姜晓龙，陈建伟，黄洋，姚晓磊，曹霞，李鹏飞，吕丽华. FSH及Wnt信号通路对绵羊卵泡颗粒细胞的增殖及雌激素分泌的影响[J]. 畜牧兽医学报, 2016, 47(1): 64-70.

ZHAO Miao-miao，JIANG Xiao-long，CHEN Jian-wei，HUANG Yang，YAO Xiao-lei，CAO Xia，LI Peng-fei，Lü Li-hua. Effects of FSH and Wnt Signaling Pathway on Proliferation and Estrogen Secretion of Sheep Granulosa Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(1): 64-70.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2016.01.009

https://www.xmsyxb.com/CN/Y2016/V47/I1/64

参考文献

［1］NUSSE R，VARMUS H E.Many tumors induced by the mouse mammary tumor virus contain a provirns integrated in the same region of the host genome［J］.Cell，1982，31(1)：99-109.
［2］SEGDITSAS S，TOMLINSON I.Colorectal cancer and genetic alterations in the Wnt pathway［J］.Oncogene，2006，25(57)：7531-7537.
［3］WODARZ A，NUSSE R.Mechanisms of Wnt signaling in development［J］.Annu Rev Cell Dev Biol，1998,14：59-88.
［4］KIKUCHI A，YAMAMOTO H，KISHIDA S.Multiplicity of the interactions of Wnt proteins and their receptors［J］. Cell Signal，2007,19(4)：659-671.
［5］尹定子，宋海云.Wnt信号通路:调控机理和生物学意义［J］.中国细胞生物学学报，2011，33(2)：103-111.
YIN D Z，SONG H Y.Regulation of Wnt signaling:Mechanism and Biological significance［J］. Chinese Journal of Cell Biology，2011，33(2)：103-111.(in Chinese)
［6］刘烜，王海生，刘淑萍.Wnt信号转导通路与细胞凋亡［J］.内蒙古医学杂志，2011，43(1)：50-53.
LIU X，WANG H S，LIU S P.Wnt signal transduction pathway and apoptosis［J］.Inner Mongolia Medical Journal，2011，43(1)：50-53.(in Chinese)
［7］WILLERT K，BROWN J D，DANENBERG E，et al.Wnt proteins are lipid-modified and can act as stem cell growth factors［J］.Nature，2003，423(6938)：448-452.
［8］BOYER A，GOFF A K，BOERBOOM D.WNT signaling in ovarian follicle biology and tumorigenesis［J］. Trends Endocrinol Metab，2010，21(1)：25-32.
［9］CHEN B，DODGE M E，TANG W，et al.Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer［J］.Nat Chem Biol， 2009，5(2)：100-107.
［10］LU J，MA Z，HSIEH J C，et al.Structure-activity relationship studies of small-molecule inhibitors of Wnt response［J］.Bioorg Med Chem Lett，2009，19(14)：3825-3827.
［11］GUPTA P S，FOLGER J K,RAJPUT S K，et al.Regulation and regulatory role of WNT signaling in potentiating FSH action during bovine dominant follicle selection［J］.PLoS One，9(6)：e100201.
［12］VAINIO S，HEIKKILÄ M，KISPERT A，et al.Female development in mammals is regulated by Wnt-4 signalling［J］.Nature，1999，397（6718）：405-409.
［13］BOERBOOM D，PAQUET M，HSIEH M，et al.Misregulated Wnt/beta-catenin signaling leads to ovarian granulosa cell tumor development［J］.Cancer Res，2005，65（20）：9206-9215.
［14］高庆华，周虚，王春清，等.促卵泡激素和胰岛素对牛卵泡颗粒细胞长期培养的影响［J］.中国兽医学报，2006，26(4)：442-443，447.
GAO Q H，ZHOU X，WANG C Q，et al.Effect of FSH and insulin on bovine granulosa cells in long term serum-cultrue［J］. Chinese Journal of Veterinary Science，2006，26(4)：442-443，447.(in Chinese)
［15］李鹏飞，岳文斌，李富禄，等.可卡因-苯丙胺调控转录肽(CART)对猪卵巢卵泡颗粒细胞雌激素产生的影响［J］.畜牧兽医学报，2012，43(12)：1879-1886.
LI P F，YUE W B，LI F L，et al.Effects of CART on estradiol production of pig ovarian follicular granulosa cells in vitro culture［J］.Acta Veterinaria et Zootechnica Sinica，2012，43(12)：1879-1886.(in Chinese)
［16］姜晓龙.DAPT 阻断Notch信号通路对绵羊卵泡颗粒细胞功能的影响［D］.太谷：山西农业大学,2013.
JIANG X L.Effect of Notch signaling pathway blocked with DAPT on function of sheep granulosa cells in vitro［D］.Taigu：Shanxi Agricultural University,2013.(in Chinese)
［17］王锋.卵泡生长发育成熟及其内分泌调控［J］.动物医学进展，1994，19(1)：1-8.
WANG F.Mature follicle growth and endocrine regulation［J］.Progress in Veterinary Medicine，1994，19(1)：1-8.(in Chinese)
［18］SEN A，LÜ L，BELLO N，et al.Cocaine- and amphetamine-regulated transcript accelerates termination of follicle-stimulating hormone-induced extracellulary regulated kinase 1/2 and Akt activation by regulating the expression and degradation of specific mitogen-activated protein kinase phosphatases in bovine granulosa cells［J］.Mol Endocrinol，2008，22(12)：2655-2676.
［19］LÜ L H，JIMENEZ-KRASSEL F，SEN A，et al.Evidence supporting a role for cocaine- and amphetamineregulated transcript(CARTPT) in control of granulosa cell estradiol production associated with dominant follicle selection in cattle［J］.Biol Reprod，2009，81(3)：580-586.
［20］HUGHES F M J R，GOROSPE W C.Biochemical identification of apoptosis(programmed cell death) in granulosa cells：evidence for a potential mechanism underlying follicular atresia［J］.Endocrinology，1991，129(5)：2415-2422.
［21］LÜ L H，JIMENEZ-KRASSEL F，SEN A，et al.Evidence for a local inhibitory effect of the CART peptide on LH-induced thecal and rostenedione production［J］.Biol Reprod，2007，77(Suppl 1)：179.
［22］HUNZICKER-DUNN M，MAIZELS E T.FSH signaling pathways in immature granulosa cells that regulate target gene expression：branching out from protein kinase A［J］.Cell Signal，2006，18(9)：1351-1359.
［23］CAMPBELL B K,SCARAMUZZI R J,WEBB R.Induction and maintenance of oestradiol and immunoreactive inhibin production with FSH by ovine granulosa cells cultured in serum-free media［J］.J Reprod Fertil，1996，106(1)：7-16.
［24］李鹏飞，岳文斌，庞钰莹，等.FSH和胰岛素对绵羊卵巢卵泡颗粒细胞体外培养的影响［J］.畜牧兽医学报，2013，44(9)：1386-1391.
LI P F，YUE W B，PANG Y Y，et al.Effects of FSH and insulin on sheep ovarian follicular granulosa cells in vitro culture［J］.Acta Veterinaria et Zootechnica Sinica，2013，44(9)：1386-1391.(in Chinese)
［25］LAPOINTE E，BOERBOOM D.WNT signaling and the regulation of ovarian steroidogenesis［J］.Front Biosci （Schol Ed），2011，3：276-285.
［26］ZHAO Y，MENDELSON C R，SIMPSON E R.Characterization of the sequences of the human CYP19(aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes［J］.Mol Endocrinol，1995，9(3)：340-349.
［27］LUO W，WILTBANK M C.Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1 in bovine granulosa cells［J］.Biol Reprod，2006，75(2)：217-225.
［28］HAN Y，XIA G，TSANG B K.Regulation of cyclin D2 expression and degradation by follicle-stimulating hormone during rat granulosa cell proliferation in vitro［J］.Biol Reprod，2013，88(3)：57.
［29］SEN A，BETTEGOWDA A，JIMENEZ-KRASSEL F，et al.Cocaine-and amphetamine regulated transcript regulation of follicle-stimulating hormone signal transduction in bovine granulosa cells［J］. Endocrinology，2007，148(9)：4400-4410.

[1]	王海波, 占今舜, 谷志勇, 陈新锋, 潘月, 贾浩滨, 钟小军, 李开嵘, 赵生国, 霍俊宏. 湖羊与三元杂交绵羊夏杜湖、夏澳湖肉质特性比较研究[J]. 畜牧兽医学报, 2024, 55(1): 110-119.
[2]	韩浩园, 李世凯, 杨瑞巧, 李曼曼, 李君, 哈斯, 赵金艳, 魏红芳, 权凯. 基于转录组测序挖掘槐山羊高繁关键候选基因[J]. 畜牧兽医学报, 2023, 54(12): 5077-5090.
[3]	栗登攀, 马克岩, 韩金涛, 白雅琴, 李讨讨, 马友记. 永登七山羊全基因组选择信号检测分析[J]. 畜牧兽医学报, 2023, 54(11): 4577-4588.
[4]	赵雪洋, 李丹妮, 王钰晨, 郭磊, 王丽, 黄杰, 焦仪强, 安小鹏, 张希云, 张磊, 宋宇轩. 东湖杂交羊产羔性状的GWAS分析及候选基因GRID2的验证[J]. 畜牧兽医学报, 2023, 54(11): 4625-4635.
[5]	张寒月, 赵丹, 梁煜, 赵弼时, 范蒙丹, 乔利英, 刘建华, 杨凯捷, 潘洋洋, 刘文忠. miR-150靶向AOC3调控绵羊前体脂肪细胞分化的研究[J]. 畜牧兽医学报, 2023, 54(8): 3262-3274.
[6]	刘林丽, 彭雪兰, 李博, 程乐凡, 次仁拉姆, 张恩平. 过表达UCP3基因对萨湖羊前体脂肪细胞分化的影响[J]. 畜牧兽医学报, 2023, 54(8): 3275-3285.
[7]	王万年, 陈思佳, 郜金荣, 温中豪, 袁梦娇, 张洪志, 庞志旭, 乔利英, 刘文忠. 基于多层感知机的绵羊限性性状基因组选择模拟研究[J]. 畜牧兽医学报, 2023, 54(7): 2824-2835.
[8]	董亚洁, 郝晓静, 吴晋强, 王荣, 张鹏翔, 王海东, 赫晓燕. 基于绵羊角质形成细胞探究SHH通过Krox20调节IGFBP5的表达影响羊毛弯曲[J]. 畜牧兽医学报, 2023, 54(6): 2365-2375.
[9]	毕亚珍, 尚明玉, 胡文萍, 张莉. 绵羊生长性状间的相关和回归分析及TRHDE基因多态性与生长性状的关联分析[J]. 畜牧兽医学报, 2023, 54(4): 1415-1428.
[10]	李隐侠, 牙生江·那斯尔, 赛里克·都曼, 钱勇, 曹少先, 王伟列, 孟春花, 张俊, 张建丽. SNP芯片评估柯尔克孜羊群体遗传多样性和遗传结构[J]. 畜牧兽医学报, 2023, 54(2): 572-583.
[11]	李玲, 李业芳, 梁奔梦, 孙玉江, 马月辉, 马青, 蒋琳, 刘书琴. 基于SNP标记的滩羊亲子鉴定研究[J]. 畜牧兽医学报, 2022, 53(9): 2912-2919.
[12]	周世卫, 吴庭杰, 王倩, 蔡蓓, 韩赛铮, 吴彦虎, 王小龙, 陈玉林. 绵羊FecB突变检测方法的建立及高繁滩羊核心群的构建[J]. 畜牧兽医学报, 2022, 53(9): 2920-2929.
[13]	丁自强, 葛闻博, 段宏伟, 吕建树, 曾建林, 王文娟, 牛甜, 张勇, 赵兴绪, 胡俊杰. 双氢睾酮通过调控孕酮、雌二醇和细胞凋亡参与绵羊子宫功能[J]. 畜牧兽医学报, 2022, 53(9): 2993-3005.
[14]	李亭亭, 刘秋月, 李向臣, 王海涛. 绵羊经济性状相关基因研究进展及其应用[J]. 畜牧兽医学报, 2022, 53(8): 2417-2434.
[15]	徐颂为, 向华, 张焕容, 杨璐, 王宪军, 撒朗文朱, 朱江江. 干扰SERBP1表达对山羊鼻甲骨原代细胞增殖、周期及凋亡的作用[J]. 畜牧兽医学报, 2022, 53(8): 2708-2720.