BMP4诱导的Smad9在小鼠卵泡发育中的作用及其机制的初步研究

doi:10.16431/j.cnki.1671-7236.2018.10.028

摘要/Abstract

摘要：

本研究旨在探讨Smad9在小鼠卵泡发育中的作用，为卵泡生长发育及其调控机制的研究提供新思路。将6周龄的雌性昆明小鼠随机分成3组，分别腹腔注射生理盐水、骨形态发生蛋白-4（BMP4）和Smad9抑制剂（LDN-193189），依次作为对照组、BMP4组和LDN组。48 h后收集卵巢制作石蜡切片，HE染色后观察卵泡发育情况并对各级卵泡进行计数；采用Western blotting和实时荧光定量PCR技术检测Smad9蛋白和mRNA水平。同时通过ELISA试验测定各组小鼠血清中雌二醇（E₂）、孕酮（P₄）、黄体生成素（LH）、卵泡刺激素（FSH）和芳香化酶等的浓度，并检测了CYP19a1、LHR、PRLR和FSHR基因的表达情况，进一步探究Smad9可能的作用机制。结果显示，与对照组相比，BMP4组Smad9蛋白和mRNA水平较高，而LDN组较低；BMP4组有腔卵泡显著增加（P<0.05），而LDN组显著减少（P<0.05）。此外，ELISA试验结果显示，BMP4组血清E₂、FSH和芳香化酶含量增加，而P₄和LH含量降低；LDN组中E₂和芳香化酶的含量降低。实时荧光定量PCR结果显示，BMP4组FSHR和CYP19a1基因的mRNA水平升高，而LHR和PRLR基因的mRNA水平降低；LDN组PRLR基因的mRNA水平升高，而CYP19a1基因的mRNA水平降低。由以上试验结果可以得出，BMP4诱导的Smad9能促进小鼠有腔卵泡的发育，而其可能主要通过影响E₂、PRL和芳香化酶等的产生而起作用。

关键词: BMP/Smads信号通路; Smad9; 卵泡发育; 小鼠

Abstract:

This study was conducted to explore the role of Smad9 on follicular development in mice and provide new ideas for the study of follicular growth and its regulation mechanism.Six-week-old female Kunming mice were randomly divided into 3 groups and injected intraperitoneally with physiological saline,bone morphogenetic protein-4 (BMP4) and Smad9 inhibitor (LDN-193189),respectively,which were used as the control group,BMP4 group and LDN group.After 48 hours,the ovaries were collected to make paraffin sections.Follicles were observed after HE staining and the follicles at different stages of development were counted.Western blotting and Real-time PCR were used to detect Smad9 protein and mRNA levels.At the same time,the concentrations of estradiol (E₂),progesterone (P₄),luteinizing hormone (LH),follicle stimulating hormone (FSH) and aromatase in the serum of each group of mice were measured by ELISA,and the expression of genes such as CYP19a1,LHR,PRLR and FSHR were detected to further explore possible mechanisms of Smad9.The results showed that compared with control group,the Smad9 protein and mRNA levels were higher in BMP4 group and lower in LDN group;The number of antral follicles in BMP4 group was significantly increased (P < 0.05),while LDN group was significantly reduced (P < 0.05).In addition,ELISA results showed that the levels of serum E₂,FSH and aromatase increased,while the levels of P₄ and LH decreased in BMP4 group;The levels of E₂ and aromatase decreased in LDN group.The results of Real-time PCR showed that the mRNA levels of FSHR and CYP19a1 genes increased,while the mRNA levels of LHR and PRLR genes decreased in BMP4 group;The mRNA level of PRLR gene increased,while the mRNA level of CYP19a1 gene decreased in LDN group.From the above experimental results,it could be concluded that BMP4-induced Smad9 promoted antral follicles development,and it might work via affecting the production of E₂,PRL and aromatase.

Key words: BMP/Smads signaling pathway; Smad9; follicular development; mice

中图分类号:

S814

王和建, 张莉, 余道伦, 陈杰, 邢朝凤, 李洁, 李君, 蔡亚非. BMP4诱导的Smad9在小鼠卵泡发育中的作用及其机制的初步研究[J]. 《中国畜牧兽医》, 2018, 45(10): 2902-2909.

WANG Hejian, ZHANG Li, YU Daolun, CHEN Jie, XING Chaofeng, LI Jie, LI Jun, CAI Yafei. Preliminary Study on the Role of BMP4-induced Smad9 in Mouse Follicular Development and Its Mechanism[J]. , 2018, 45(10): 2902-2909.

参考文献

[1] WANG S B,XING B S,YI L,et al.Expression of frizzled 2 in the mouse ovary during oestrous cycle[J].Journal of Animal Physiology and Animal Nutrition,2010,94(4):437-445.
[2] LE GOOD J A,JOUBIN K,GIRALDEZ A J,et al.Nodal stability determines signaling range[J].Current Biology,2005,15(1):31-36.
[3] THISSE B,THISSE C.Functions and regulations of fibroblast growth factor signaling during embryonic development[J].Developmental Biology,2005,287(2):390-402.
[4] 王伟,王少兵,徐银学.BMP/Smad信号通路与哺乳动物卵泡发生[J].遗传,2009,31(3):245-254.WANG W,WANG S B,XU Y X.Signal transduction of BMP/Smad and its relationship with mammalian folliculogenesis[J].Hereditas (Beijing),2009,31(3):245-254.(in Chinese)
[5] HOODLESS P A,WRANA J L.Mechanism and function of signaling by the TGF beta superfamily[J].Current Topics in Microbiology and Immunology,1998,228:235-272.
[6] OHTA Y,NAKAGAWA K,IMAI Y,et al.Cyclic AMP enhances Smad-mediated BMP signaling through PKA-CREB pathway[J].Journal of Bone and Mineral Metabolism,2008,26(5):478-484.
[7] LEMBONG J,YAKOBY N,SHVARTSMAN S Y.Spatial regulation of BMP signaling by patterned receptor expression[J].Tissue Engineering Part A,2008,14(9):1469-1477.
[8] MASSAGUE J,SEOANE J,WOTTON D.Smad transcription factors[J].Genes & Development,2005,19(23):2783-2810.
[9] TSUKAMOTO S,MIZUTA T,FUJIMOTO M,et al.Smad9 is a new type of transcriptional regulator in bone morphogenetic protein signaling[J].Scientific Reports,2014,4:7596.
[10] KATAKAWA Y,FUNABA M,MURAKAMI M.Smad8/9 is regulated through the BMP pathway[J].Journal of Cellular Biochemistry,2016,117(8):1788-1796.
[11] XU J,LI J,WANG H,et al.A novel SMAD family protein,SMAD9 is involved in follicular initiation and changes egg yield of geese via synonymous mutations in exon1 and intron2[J].Molecular Biology Reports,2015,42(1):289-302.
[12] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2(-Delta Delta CT) method[J].Methods,2001,25(4):402-408.
[13] 杨佳栋,刘月琴,张英杰.山羊BMPR-ⅠB基因编码蛋白结构及功能的生物信息学分析[J].中国畜牧兽医,2017,44(5):1259-1266.YANG J D,LIU Y Q,ZHANG Y J.Bioinformatics analysis of the structure and function of BMPR-ⅠB gene coding protein in goat[J].China Animal Husbandry & Veterinary Medicine,2017,44(5):1259-1266.(in Chinese)
[14] 谢健,喻昌燕,许瑶,等.可乐猪乳头数性状全基因组关联分析[J].中国畜牧兽医,2016,43(11):2866-2872.XIE J,YU C Y,XU Y,et al.Genome-wide association study of teats number traits in Kele pigs[J].China Animal Husbandry & Veterinary Medicine,2016,43(11):2866-2872.(in Chinese)
[15] 程莉芬,姬爱国,王清祥.骨形态发生蛋白15基因与绵羊繁殖性能关系的研究进展[J].中国畜牧兽医,2017,44(8):2392-2397.CHENG L F,JI A G,WANG Q X.Research progress on the relationship between BMP15 gene and reproduction performance of sheep[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2392-2397.(in Chinese)
[16] ISRAEL D I,NOVE J,KERNS K M,et al.Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells[J].Growth Factors,1992,7(2):139-150.
[17] CHILDS A J,KINNELL H L,COLLINS C S,et al.BMP signaling in the human fetal ovary is developmentally regulated and promotes primordial germ cell apoptosis[J].Stem Cells,2010,28(8):1368-1378.
[18] OTSUKA F,MCTAVISH K J,SHIMASAKI S.Integral role of GDF-9 and BMP-15 in ovarian function[J].Molecular Reproduction and Development,2011,78(1):9-21.
[19] DING X,ZHANG X,MU Y,et al.Effects of BMP4/SMAD signaling pathway on mouse primordial follicle growth and survival via up-regulation of Sohlh2 and c-kit[J].Molecular Reproduction and Development,2013,80(1):70-78.
[20] TANWAR P S,O'SHEA T,MCFARLANE J R.In vivo evidence of role of bone morphogenetic protein-4 in the mouse ovary[J].Animal Reproduction Science,2008,106(3-4):232-240.
[21] YU P B,DENG D Y,LAI C S,et al.BMP type Ⅰ receptor inhibition reduces heterotopic ossification[J].Nature Medicine,2008,14(12):1363-1369.
[22] NAKAYAMA T,SNYDER M A,GREWAL S S,et al.Xenopus Smad8 acts downstream of BMP-4 to modulate its activity during vertebrate embryonic patterning[J].Development,1998,125(5):857-867.
[23] CUNY G D,YU P B,LAHA J K,et al.Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors[J].Bioorganic & Medicinal Chemistry Letters,2008,18(15):4388-4392.
[24] NILSSON E E,SKINNER M K.Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development[J].Biology of Reproduction,2003,69(4):1265-1272.
[25] DA CUNHA E V,MELO L R F,SOUSA G B,et al.Effect of bone morphogenetic proteins 2 and 4 on survival and development of bovine secondary follicles cultured in vitro[J].Theriogenology,2018,110:44-51.
[26] SHIMASAKI S,ZACHOW R J,LI D,et al.A functional bone morphogenetic protein system in the ovary[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(13):7282-72877.
[27] MULSANT P,LECERF F,FABRE S,et al.Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Merino ewes[J].Proceedings of the National Academy of Sciences of the United States of America,2001,98(9):5104-5109.
[28] MONGET P,FABRE S,MULSANT P,et al.Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals[J].Domestic Animal Endocrinology,2002,23(1-2):139-154.
[29] RAJESH G,MISHRA S R,PAUL A,et al.Transcriptional and translational abundance of bone morphogenetic protein (BMP) 2,4,6,7 and their receptors BMPR1A,1B and BMPR2 in buffalo ovarian follicle and the role of BMP4 and BMP7 on estrogen production and survival of cultured granulosa cells[J].Research in Veterinary Science,2018,118:371-388.
[30] MILLER W L.Molecular biology of steroid hormone synthesis[J].Endocrine Reviews,1988,9(3):295-318.
[31] SHIMASAKI S,MOORE R K,OTSUKA F,et al.The bone morphogenetic protein system in mammalian reproduction[J].Endocrine Reviews,2004,25(1):72-101.
[32] 喻长法,张仙森,李香娟,等.高泌乳素血症与不孕的相关性研究[J].中国优生与遗传杂志,2009,17(4):120-121.YU C F,ZHANG X S,LI X J,et al.The study of relationship between hyperprolactinemia and infertility.Chinese Journal of Birth Health & Heredity,2009,17(4):120-121.(in Chinese)
[33] 陆冬芳.血清泌乳素水平与女性不孕的关系探讨[J].中国现代医生,2010,48(30):155-156.LU D F.The relationship between serum prolactin level and female infertility[J].China Modern Doctor,2010,48(30):155-156.(in Chinese)
[34] HESTER M,THOMPSON J C,MILLS J,et al.Smad1 and Smad8 function similarly in mammalian central nervous system development[J].Molecular and Cellular Biology,2005,25(11):4683-4692.
[35] HUANG Z,WANG D,IHIDA-STANSBURY K,et al.Defective pulmonary vascular remodeling in Smad8 mutant mice[J].Human Molecular Genetics,2009,18(15):2791-2801.
[36] SHINTANI M,YAGI H,NAKAYAMA T,et al.A new nonsense mutation of SMAD8 associated with pulmonary arterial hypertension[J].Journal of Medical Genetics,2009,46(5):331-337.
[37] WEI C Y,WANG H P,ZHU Z Y,et al.Transcriptional factors smad1 and smad9 act redundantly to mediate zebrafish ventral specification downstream of smad5[J].The Journal of Biological Chemistry,2014,289(10):6604-6618.
[38] KAWAI S,FAUCHEU C,GALLEA S,et al.Mouse Smad8 phosphorylation downstream of BMP receptors ALK-2,ALK-3,and ALK-6 induces its association with Smad4 and transcriptional activity[J].Biochemical and Biophysical Research Communications,2000,271(3):682-687.