SMAD1基因对黔北麻羊卵巢颗粒细胞的影响及组织表达分析

doi:10.11843/j.issn.0366-6964.2020.07.013

摘要/Abstract

摘要： 旨在验证SMAD1基因在转录组测序中单羔组对多羔组上调的结果，并探究SMAD1基因对黔北麻羊产羔性状的影响。本试验选取健康、体重45 kg、4岁左右的单、多羔黔北麻羊母羊为研究对象，采用qRT-PCR法检测SMAD1基因在单、多羔组黔北麻羊母羊子宫、输卵管、垂体、下丘脑及卵巢组织的表达水平，PCR扩增黔北麻羊SMAD1基因CDS区，构建pEGFP-N3-SAMD1重组质粒，利用脂质体转染法转染pEGFP-N3-SAMD1重组质粒进入卵巢颗粒细胞，分为超表达SMAD1试验组和pEGFP-N3空载体对照组，通过检测培养基中雌二醇和孕酮的浓度，利用CCK-8法和Annexin V-FITC法检测超表达SMAD1基因对卵巢颗粒细胞增殖、凋亡的影响，qRT-PCR检测超表达SMAD1基因对SAMD1、GnRHR、FSHR、BMP4、TIMP3基因mRNA表达的影响，来探究SMAD1基因对卵巢颗粒细胞的影响。结果表明，SMAD1基因在黔北麻羊各组织中均有表达。组间比较可知，卵巢和下丘脑组织单羔组极显著高表达于多羔组（P<0.01），输卵管组织中单羔组显著高表达于多羔组（P<0.05），其他组织未达到差异显著性（P>0.05）。本研究成功克隆了SMAD1基因CDS序列，未发现突变位点，并将pEGFP-N3-SAMD1重组质粒转染进入卵巢颗粒细胞，转染试验组培养基中E2含量在12、48 h显著高于对照组（P<0.05），P4含量在12 h显著高于对照组（P<0.05）；超表达SMAD1基因对细胞增殖在24 h达到显著促进作用（P<0.05），48、72 h达到极显著促进作用（P<0.01），并且发现超表达SMAD1基因对卵巢颗粒细胞的凋亡具有抑制作用，与SMAD1基因对卵巢颗粒细胞增殖具有促进的结果是相符的。SMAD1基因超表达后繁殖相关基因GnRHR、FSHR、BMP4、TIMP3 mRNA的表达极显著降低（P<0.01）。综上表明，SMAD1基因超表达可提高E2、P4的生成，有助于黔北麻羊发情及妊娠的维持，促进细胞增殖，抑制细胞凋亡，也极显著抑制繁殖相关基因mRNA的表达，而这些繁殖相关基因都能够调控卵泡数、卵泡的发育与形成以及排卵。因此，SMAD1基因能够抑制这些基因的表达，致使黔北麻羊单羔组受胎率低，进而降低产羔数，这为产羔性状相关基因的研究提供了理论基础，初步认为SMAD1基因可作为探究影响山羊产羔性状的候选基因。

关键词: 黔北麻羊, SMAD1基因, 卵巢颗粒细胞, 差异表达, 产羔性状

Abstract: The purpose of this study was to verify the results of SMAD1 gene up-regulation comparing single lamb group to multiple lambs group in transcriptome sequencing, and to explore the effect of SMAD1 gene on lambing traits of Qianbei Ma goats. In this experiment, healthy, 4-year-old single, multi-lamb Qianbei Ma goats ewes with 45 kg were selected as the research objects. qRT-PCR method was used to detect the expression of SMAD1 gene in uterus, fallopian tubes, pituitary, hypothalamus and ovarian tissues of Qianbei Ma goats ewes in the single and multi-lamb groups. CDS region of SMAD1 gene of Qianbei Ma goats was amplified, recombinant plasmid pEGFP-N3-SAMD1 was constructed and was transfected into ovarian granulosa cells by liposome transfection method. The SMAD1 overexpression test group and pEGFP-N3 empty vector control group were set up. By detecting the concentration of estradiol and progesterone in the culture medium, the effect of overexpressing SMAD1 gene on the proliferation and apoptosis of ovarian granulosa cells was detected by CCK-8 and Annexin V-FITC methods. qRT-PCR was used to detect the effect of overexpressing SMAD1 gene on the mRNA expressions of SAMD1, GnRHR, FSHR, BMP4 and TIMP3 genes to explore the influence of SMAD1 gene on ovarian granulosa cells. The results showed that SMAD1 gene was expressed in various tissues of Qianbei Ma goats. Comparison between the groups showed that the expression of SMAD1 gene in ovary and hypothalamus tissues were extremely significantly higher in the single lamb group than those in the multiple lambs group (P<0.01), in the fallopian tube tissues, it was significantly higher in the single lamb group than that in the multiple lambs group (P<0.05), and there were no significant difference in other tissues between the groups(P>0.05). The CDS sequence of SMAD1 gene was successfully cloned, no mutation site was found. The recombinant plasmid pEGFP-N3-SAMD1 was transfected into ovarian granulosa cells. The E2 content in the culture medium in transfection test group was significantly higher than that in the control group at 12 and 48 h (P<0.05), the content of P4 in transfection test group was significantly higher than that in the control group at 12 h (P<0.05); Overexpressing SMAD1 gene significantly promoted cell proli-feration at 24 h (P<0.05), and extremely significantly promoted cell proliferation at 48 and 72 h (P<0.01). It was found that overexpression of SMAD1 gene inhibited the apoptosis of ovarian granulosa cells, which was consistent with the result of SMAD1 gene promoting the proliferation of ovarian granulosa cells. However, the mRNA expressions of GnRHR, FSHR, BMP4, and TIMP3 were extremely significantly decreased after overexpressing SMAD1 gene(P<0.01). In summary, the overexpression of SMAD1 gene can increase the production of E2 and P4, help to maintain the estrus and pregnancy of Qianbei Ma goats, promote cell proliferation, inhibit cell apoptosis, and also significantly inhibit the expression of reproduction-related genes mRNA, and these reproduction-related genes can regulate the number, development and formation of follicles and ovulation. Therefore, the SMAD1 gene can suppress the expressions of reproduction-related genes, which results in a low conception rate in the single lamb group of Qianbei Ma goats, and further reduces the number of lambs. The result of this study provide a theoretical basis for studying the lambing traits related genes, and SMAD1 gene can be used as a candidate gene to explore goat lambing traits.

Key words: Qianbei Ma goats, SMAD1 gene, ovarian granulosa cells, differential expression, lambing traits

中图分类号:

S827.3

敖叶, 陈祥, 周志楠, 张艳, 洪磊, 韦仕南, 吴雨, 唐文. SMAD1基因对黔北麻羊卵巢颗粒细胞的影响及组织表达分析[J]. 畜牧兽医学报, 2020, 51(7): 1607-1618.

AO Ye, CHEN Xiang, ZHOU Zhinan, ZHANG Yan, HONG Lei, WEI Shinan, WU Yu, TANG Wen. Effect of SMAD1 Gene on Ovarian Granulosa Cells and Its Tissue Expression Analysis in Qianbei Ma Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1607-1618.

参考文献 40

[1]	乔虎军,王国祥,郝鑫.转化生长因子β/Smad信号通路和骨关节炎研究进展[J].中国运动医学杂志,2019,38(2):143-151.QIAO H J,WANG G X,HAO X.Research progress of transforming growth factor β/Smad signaling pathway and osteoarthritis[J].Chinese Journal of Sports Medicine,2019,38(2):143-151.(in Chinese)
[2]	SAVAGE C,DAS P,FINELLI A L,et al.Caenorhabditis elegans genes sma-2,sma-3,and sma-4 define a conserved family of transforming growth factor beta pathway components[J].Proc Natl Acad Sci U S A,1996,93(2):790-794.
[3]	宁越,米雪,陈星伊,等.SMAD1基因的沉默和过表达及对秦川牛原代成肌细胞生肌的影响[J].中国农业科学, 2019,52(10):1818-1829.NING Y,MI X,CHEN X Y,et al.Silencing and overexpressing SMAD family member 1(SMAD1) gene and its effect on myogenesis in primary myoblast ofQinchuan Cattle (Bos taurus)[J].Scientia Agricultura Sinica,2019,52(10):1818-1829.(in Chinese)
[4]	SHI Y G,MASSAGUÉ J.Mechanisms of TGF-β signaling from cell membrane to the nucleus[J].Cell, 2003, 113(6):685-700.
[5]	DIJKE P T,HILL C S.New insights into TGF-β-Smad signalling[J].Trends Biochem Sci,2004,29(5):265-273.
[6]	CHEN G Q,DENG C X,LI Y P.TGF-β and BMP signaling in osteoblast differentiation and bone formation[J].Int J Biol Sci,2012,8(2):272-288.
[7]	陈兵,易斌,鲁开智.Smad蛋白家族调控细胞分化的研究进展[J].医学研究生学报,2013,26(5):544-547.CHEN B,YI B,LU K Z.Advances in researches on Smad proteins in cell differentiation[J].Journal of Medical Postgraduates,2013,26(5):544-547.(in Chinese)
[8]	MASSAGUÉ J,WOTTON D.Transcriptional control by the TGF-β/Smad signaling system[J].EMBO J,2000,19(8):1745-1754.
[9]	苟海昕,詹红生,赵咏芳,等.补肾中药对自然衰老雄性大鼠骨髓微环境中Smad1基因表达的影响[J].中国中医骨伤科杂志,2007,15(2):26-28.GOU H X,ZHAN H S,ZHAO Y F,et al.The Effect of invigorating-kidney herbs on Smad1 gene expre-ssion in bone marrow microenvironment of ageing male rat[J].Chinese Journal of Traditional Medical Traumatology & Orthopeics,2007,15(2):26-28.(in Chinese)
[10]	WANG C Y,CORE A B,CANALI S,et al.Smad1/5 is required for erythropoietin-mediated suppression of hepcidin in mice[J].Blood,2017,130(1):73-83.
[11]	LIU P,LI X S,SONG F H,et al.Testosterone promotes tube formation of endothelial cells isolated from veins via activation of Smad1 protein[J].Mol Cell Endocrinol,2017,446:21-31.
[12]	BONDESTAM J,KAIVO-OJA N,KALLIO J,et al.Engagement of activin and bone morphogenetic protein signaling pathway Smad proteins in the induction of inhibin B production in ovarian granulosa cells[J].Mol Cell Endocrinol,2002,195(1-2):79-88.
[13]	XU S S,GAO L,XIE X L,et al.Genome-wideassociation analyses highlight the potential for different genetic mechanisms for litter size among sheep breeds[J].Front Genet,2018,9:118.
[14]	BAHIRE S V,RAJPUT P K,KUMAR V,et al.Quantitative expression of mRNA encoding BMP/SMAD signalling genes in the ovaries of Booroola carrier and non-carrier GMM sheep[J].Reprod Domest Anim,2019,54(10):1375-1383.
[15]	LV X Y,GAO W,JIN C Y,et al.Preliminary study on microR-148a and microR-10a in dermal papilla cells of Hu sheep[J].BMC Genet,2019,20(1):70.
[16]	PANGAS S A,LI X H,UMANS L,et al.Conditional deletion of Smad1 and Smad5 in somatic cells of male and female gonads leads to metastatic tumor development in mice[J].Mol Cell Biol,2008,28(1):248-257.
[17]	孙洪新,张英杰,刘月琴,等.绵羊CYP19基因卵巢启动子的克隆及其表达活性研究[J].畜牧兽医学报,2015,46(9):1540-1548.SUN H X,ZHANG Y J,LIU Y Q,et al.Cloning and expression assay of sheep CYPl9 gene ovarian promoter[J].Acta Veterinaria et Zootechnica Sinica,2015,46(9):1540-1548.(in Chinese)
[18]	田志龙,汤继顺,孙庆,等.绵羊SMAD1基因组织表达及其多态性与产羔数关联分析[J].中国农业科学,2019,52(4):755-766.TIAN Z L,TANG J S,SUN Q,et al.Tissue expre-ssion and polymorphism of sheep SMAD1 gene and their association with litter size[J].Scientia Agricultura Sinica,2019,52(4):755-766.(in Chinese)
[19]	李兰兰,栾兆进,刘开东,等.Smad1基因在敖汉细毛羊发情不同时期卵巢中的表达规律研究[J].黑龙江畜牧兽医,2017(11):93-96,294.LI L L,LUAN Z J,LIU K D,et al.Study of expre-ssion pattern of Smad1 gene of ovary of Aohan fine-wool sheep in different oestrus periods[J].Heilongjiang Animal Science and Veterinary Medicine, 2017(11):93-96,294.(in Chinese)
[20]	WANG W,WANG L,LI X X,et al.Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells[J].J Zhejiang Univ Sci B,2010,11(9):719-727.
[21]	MOTTERSHEAD D G,PULKKI M M,MUGGALLA P,et al.Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[J].Mol Cell Endocrinol,2008,283(1-2):58-67.
[22]	CUI X R,JING X,WU X Q,et al.Abnormal expre-ssion levels of BMP15/Smad1 are associated with granulosa cell apoptosis in patients with polycystic ovary syndrome[J].Mol Med Rep,2017,16(6):8231-8236.
[23]	MAKITA M,MIYANO T.Steroid hormones promote bovine oocyte growth and connection with granulosa cells[J].Theriogenology,2014,82(4):605-612.
[24]	RIVAS R E C,NIETO M P C,KAMIYOSHI M.Effects of steroid hormone in avian follicles[J].Asian Australas J Anim Sci,2016,29(4):487-499.
[25]	REGAN S L,MCFARLANE J R,O'SHEA T,et al.Flow cytometric analysis of FSHR,BMRR1B,LHR and apoptosis in granulosa cells and ovulation rate in merino sheep[J].Reproduction,2015,150(2):151-163.
[26]	LOOPER M L,VIZCARRA J A,WETTEMANN R P,et al.Influence of estradiol,progesterone,and nutrition on concentrations of gonadotropins and GnRH receptors,and abundance of mRNA for GnRH receptors and gonadotropin subunits in pituitary glands of beef cows[J].J Anim Sci,2003,81(1):269-278.
[27]	彭甲银.奶山羊卵巢颗粒细胞中TIMP3基因的表达调控及功能研究[D].杨凌:西北农林科技大学,2016.PENG J Y.Expressionregulation and function of TIMP3 in dairy goat ovarian granulosa cells[D]. Yangling:Northwest A&F university,2016.(in Chinese)
[28]	PENG J Y,GAO K X,GAO T Y,et al.Expression and regulationof tissue inhibitors of metalloproteinases (TIMP1 and TIMP3) in goat oviduct[J].Theriogenology,2015,84(9):1636-1643.
[29]	STEVENS T A,MEECH R.BARX2 and estrogenreceptor-α (ESR1) coordinately regulate the production ofalternatively spliced ESR1 isoforms and control breast cancer cell growth and invasion[J]. Oncogene,2006,25(39):5426-5435.
[30]	SAKAGUCHI K,HUANG W P,YANG Y H,et al.Relationship between in vitro growth of bovine oocytes and steroidogenesis of granulosa cells cultured in medium supplemented with bone morphogenetic protein-4 and follicle stimulating hormone[J].Theriogenology,2017,97:113-123.
[31]	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.
[32]	KNIGHT P G,GLISTER C.TGF-β superfamily members and ovarian follicle development[J]. Reproduction,2006,132(2):191-206.
[33]	NILSSON E E,SKINNER M K.Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development[J].Biol Reprod,2003,69(4):1265-1272.
[34]	HATIF S A,AL-MUTAR H A K,HUSSAIN K A.Clinical study with investigation of polymorphism FSHR gene in twining goats in Iraq[J].Adv Anim Vet Sci,2017,5(5):192-195.
[35]	DE CASTRO F,RUIZ R,MONTORO L,et al.Role of follicle-stimulating hormone receptor Ser680Asn polymorphism in the efficacy of follicle-stimulating hormone[J].Fertil Steril,2003,80(3):571-576.
[36]	郑梦雪.促卵泡刺激素通过调控颗粒细胞分泌干细胞因子促进卵泡发育成熟的研究[D].银川:宁夏医科大学,2017.ZHENG M X.The research of follicle stimulating hormone regulates the expression of stem cell factor in granulosa cells to promote the follicular development[D].Yinchuan:Ningxia Medical University,2017.(in Chinese)
[37]	SALEHNIA M,PAJOKH M,GHORBANMEHR N.Short term organ culture of mouse ovary in the medium supplemented with bone morphogenetic protein 15 and follicle stimulating hormone:a morphological, hormonal and molecular study[J].J Reprod Infertil,2016,17(4):199-207.
[38]	唐杰.山羊促性腺激素及其受体mRNA丰度的研究[D].保定:河北农业大学,2007.TANG J.The study on gonadotrophin and the mRNA's abundance of its receptor in goat[D].Baoding:Agricultural University of Hebei,2007.(in Chinese)
[39]	CUI H X,ZHAO S M,CHENG M L,et al.Cloning and expression levels of genes relating to the ovulation rate of the Yunling black goat[J].Biol Reprod,2009,80(2):219-226.
[40]	NORWITZ E R,JEONG K H,CHIN W W.Molecular mechanisms of gonadotropin-releasing hormone receptor gene regulation[J].J Soc Gynecol Investig,1999,6(4):169-178.