TGFβ2和PPP3CA基因在黔北麻羊性腺轴组织表达及对卵巢颗粒细胞的调控研究

doi:10.11843/j.issn.0366-6964.2020.11.009

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (11): 2699-2709.doi: 10.11843/j.issn.0366-6964.2020.11.009

TGFβ2和PPP3CA基因在黔北麻羊性腺轴组织表达及对卵巢颗粒细胞的调控研究

敖叶^1,2, 陈祥^1,2*, 周志楠^1,2, 骆金红³, 韦仕南^1,2, 张艳^1,2, 惠茂茂^1,2, 吴雨^1,2, 杨沛方^1,2

1. 贵州大学高原山地动物遗传育种与繁殖教育部重点实验室, 贵州省动物遗传育种与繁殖重点实验室, 贵阳 550025;
2. 贵州大学动物科学学院, 贵阳 550025;
3. 贵州省草业研究所, 贵阳 550006

收稿日期:2020-05-06 出版日期:2020-11-25 发布日期:2020-11-20
通讯作者: 陈祥,主要从事动物繁殖生物技术研究,E-mail:xchen2@gzu.edu.cn
作者简介:敖叶(1992-),男,土家族,贵州铜仁人,硕士生,主要从事动物繁殖与生物技术研究,E-mail:516097146@qq.com
基金资助:
国家自然科学基金（31760652）；国家重点研发计划项目（2017YFD0501900）；贵州省优秀青年科技人才项目（[2017]5624）

Expression of TGFβ2 and PPP3CA Genes in Gonadal Axis of Qianbei Ma Goat and Their Regulation on Ovarian Granulosa Cells

AO Ye^1,2, CHEN Xiang^1,2*, ZHOU Zhinan^1,2, LUO Jinhong³, WEI Shinan^1,2, ZHANG Yan^1,2, HUI Maomao^1,2, WU Yu^1,2, YANG Peifang^1,2

1. Guizhou Key Laboratory of Animal Genetics, Breeding and Reproduction, Key Laboratory of Plateau Mountain Animal Genetics, Breeding and Reproduction of Ministry of Education, Guizhou University, Guiyang 550025, China;
2. College of Animal Science, Guizhou University, Guiyang 550025, China;
3. Guizhou Province Grass Industry Research Institute, Guiyang 550006, China

Received:2020-05-06 Online:2020-11-25 Published:2020-11-20

摘要/Abstract

摘要： 旨在对转录组测序中单羔组对多羔组TGFβ2和PPP3CA基因的上、下调结果进行验证，并探究黔北麻羊TGFβ2和PPP3CA基因对卵巢颗粒细胞的调控机制。本研究以单、多羔黔北麻羊母羊为试验对象。通过RT-qPCR技术检测TGFβ2与PPP3CA基因在单、多羔黔北麻羊性腺轴中的表达水平；构建目的基因真核表达载体。培养黔北麻羊卵巢颗粒细胞，并利用脂质体转染法将pEGFP-N3-TGFβ2、pEGFP-N3-PPP3CA重组质粒导入卵巢颗粒细胞，检测培养基中雌二醇（E2）、孕酮（P4）的浓度。利用CCK-8和Annexin V-FITC法检测TGFβ2、PPP3CA基因对卵巢颗粒细胞增殖、凋亡的影响。随后，利用RT-qPCR从细胞水平检测超表达TGFβ2、PPP3CA基因对TGFβ2、PPP3CA、GnRHR、FSHR、BMP4、TIMP3基因表达的影响。RT-qPCR结果表明，TGFβ2与PPP3CA基因在被检测的组织中均有表达，且在单羔组中的表达量普遍高于多羔组；免疫荧光法证实，所培养的细胞为黔北麻羊卵巢颗粒细胞；TGFβ2与PPP3CA基因真核表达质粒导入细胞后，在极显著提高TGFβ2与PPP3CA基因表达的同时，能够极显著地抑制卵巢颗粒细胞中FSHR、GnRHR、BMP4、TIMP3基因的表达（P<0.01）。细胞增殖与凋亡结果显示，TGFβ2与PPP3CA基因能够促进颗粒细胞凋亡，抑制其增殖。酶联免疫吸附试验结果表明，TGFβ2与PPP3CA基因能够显著促进E2的分泌，但对P4的分泌无显著影响。本研究成功构建了TGFβ2、PPP3CA基因真核表达载体，荧光定量结果表明，超表达TGFβ2、PPP3CA基因抑制了繁殖相关基因的表达，提示TGFβ2、PPP3CA基因对山羊繁殖相关基因表达有一定的影响。为进一步研究TGFβ2、PPP3CA基因对黔北麻羊繁殖力的影响提供基础数据。

关键词: 黔北麻羊, TGFβ2基因, PPP3CA基因, 卵巢颗粒细胞, 差异表达

Abstract: The aim of this study was to verify the up-regulation and down-regulation of TGFβ2 and PPP3CA genes in transcriptome sequencing in monotocous group compared to polytocous group, and to explore the regulatory mechanism of TGFβ2 and PPP3CA genes on ovarian granulosa cells of Qianbei Ma goat. The monotocous and polytocous Qianbei Ma goat ewes were used as the research object. The expression levels of TGFβ2 and PPP3CA genes in the gonadal axis of monotocous and polytocous Qianbei Ma goat were detected by RT-qPCR technology; The eukaryotic expression vector of target genes were constructed. Qianbei Ma goat ovarian granular cells were cultured, the pEGFP-N3-TGFβ2 and pEGFP-N3-PPP3CA recombinant plasmids were transfected into ovarian granular cells by liposome transfection method, and the concentrations of estradiol(E2) and progesterone(P4) in the medium were detected. The effects of TGFβ2 and PPP3CA genes on the proliferation and apoptosis of ovarian granulosa cells were detected using CCK-8 and Annexin V-FITC methods. Then RT-qPCR was used to detect the effect of overexpressing TGFβ2, PPP3CA genes on TGFβ2, PPP3CA, GnRHR, FSHR, BMP4, TIMP3 genes expression at the cell level. RT-qPCR results showed that TGFβ2 and PPP3CA genes were expressed in the tested tissues, and the expression levels in the monotocous group were generally higher than those in the polytocous group; Immunofluorescence result confirmed that the cultured cells were Qianbei Ma goat ovarian granulosa cells; After the eukaryotic expression plasmids of TGFβ2 and PPP3CA genes were transfected into ovarian granulosa cells, the expressions of TGFβ2 and PPP3CA genes significantly increased while the expressions of FSHR, GnRHR, BMP4, TIMP3 genes were significantly inhibited in ovarian granulosa cells (P<0.01). The results of cell proliferation and apoptosis showed that TGFβ2 and PPP3CA genes could promote the apoptosis of granulosa cells and inhibit their proliferation. The result of enzyme-linked immunosorbent assay showed that TGFβ2 and PPP3CA genes could significantly promote the secretion of E2, but had no significant effect on the secretion of P4. The eukaryotic expression vector of TGFβ2 and PPP3CA genes were successfully constructed in this study. Fluorescence quantitative results showed that overexpression of TGFβ2 and PPP3CA genes inhibited the expression of reproduction-related genes, suggesting that TGFβ2 and PPP3CA genes had a certain effect on goat reproduction-related genes expression. These results will provide basic data for further research on the influence of TGFβ2 and PPP3CA genes on the fecundity of Qianbei Ma goat.

Key words: Qianbei Ma goats, TGFβ2 gene, PPP3CA gene, ovarian granulosa cells, differential expression

中图分类号:

敖叶, 陈祥, 周志楠, 骆金红, 韦仕南, 张艳, 惠茂茂, 吴雨, 杨沛方. TGFβ2和PPP3CA基因在黔北麻羊性腺轴组织表达及对卵巢颗粒细胞的调控研究[J]. 畜牧兽医学报, 2020, 51(11): 2699-2709.

AO Ye, CHEN Xiang, ZHOU Zhinan, LUO Jinhong, WEI Shinan, ZHANG Yan, HUI Maomao, WU Yu, YANG Peifang. Expression of TGFβ2 and PPP3CA Genes in Gonadal Axis of Qianbei Ma Goat and Their Regulation on Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11): 2699-2709.

参考文献 39

[1]	李琴,王启贵.TGF-β家族成员调控家禽卵泡发育的研究进展及展望[J].中国家禽,2019,41(19):1-5.LI Q,WANG Q G.The role of TGF-β family members in avian follicle development[J].China Poultry,2019,41(19):1-5.(in Chinese)
[2]	MORIKAWA M,DERYNCK R,MIYAZONO K.TGF-β and the TGF-β family:context-dependent roles in cell and tissue physiology[J].Cold Spring Harb Perspect Biol,2016,8(5):a021873.
[3]	TABECKA-LONCZYNSKA A,MYTYCH J,SOLEK P,et al.Local regulators of seasonal reproduction processes in uterus masculinus of an adult male European bison (Bison bonasus,Linnaeus 1758)[J].J Physiol Pharmacol,2018,69(5):747-753.
[4]	张勇,秦娜,于斌.TGF-β/Smads信号转导通路的研究进展[J].广西医科大学学报,2009,26(1):155-157.ZHANG Y,QIN N,YU B.Research progress of TGF-β/Smads signal transduction pathway[J].Journal of Guangxi Medical University, 2009,26(1):155-157.(in Chinese)
[5]	BOTTOMS S E,HOWELL J E,REINHARDT A K,et al.TGF-β isoform specific regulation of airway inflammation and Remodelling in a murine model of asthma[J].PLoS One,2010,5(3):e9674.
[6]	DE CAESTECKER M.The transforming growth factor-β superfamily of receptors[J].Cytok Growth Factor Rev,2004,15(1):1-11.
[7]	KIM S K,WHITLEY M J,KRZYSIAK T C,et al.Structural adaptation in its orphan domain engenders Betaglycan with an alternate mode of growth factor binding relative to Endoglin[J].Structure,2019,27(9):1427-1442.e4.
[8]	MASSAGUÉ J,CHEN Y G.Controlling TGF-β signaling[J].Genes Dev,2000,14(6):627-644.
[9]	O'LEARY S,ARMSTRONG D T,ROBERTSON S A.Transforming growth factor-β (TGFβ) in porcine seminal plasma[J].Reprod Fertil Dev,2011,23(6):748-758.
[10]	COLLINS J J P,KUNZMANN S,KUYPERS E,et al.Antenatal glucocorticoids counteract LPS changes in TGF-β pathway and caveolin-1 in ovine fetal lung[J].Am J Physiol Lung Cell Mol Physiol,2013,304(6):L438-L444.
[11]	ELHAMOULY M,NII T,ISOBE N,et al.Expression of pro-and anti-inflammatory cytokines and chemokines during the ovulatory cycle and effects of aging on their expression in the uterine mucosa of laying hens[J].Cytokine,2018,111:303-308.
[12]	RUSNAK F,MERTZ P.Calcineurin:form and function[J].Physiol Rev,2000,80(4):1483-1521.
[13]	刘卫军,沈瑛,丁健.蛋白磷酸酶2A的结构、功能和活性调节[J].生物化学与生物物理学报,2003,35(2):105-112.LIU W J,SHEN Y,DING J.Protein phosphatase 2A:its structure,function and activity regulation[J].Acta Biochimica et Biophysica Sinica,2003,35(2):105-112.(in Chinese)
[14]	WAN L,MA J S,XU G Y,et al.Molecular cloning,structural analysis and tissue expression of Protein Phosphatase 3 Catalytic Subunit Alpha Isoform (PPP3CA) gene in Tianfu goat muscle[J].Int J Mol Sci,2014,15(2):2346-2358.
[15]	BÄR L,GROßMANN C,GEKLE M,et al.Calcineurin inhibitors regulate fibroblast growth factor 23(FGF23) synthesis[J].Naunyn Schmiedebergs Arch Pharmacol,2017,390(9):1117-1123.
[16]	单艳菊,宋迟,束婧婷,等.钙调磷酸酶催化亚基A基因(PPP3CA)在不同品种鸭发育早期肌肉中的表达及其与肌纤维特性的相关性[J].农业生物技术学报,2015,23(2):236-243.SHAN Y J,SONG C,SHU Q T,et al.Expression profile of protein phosphatase 3 catalytic A gene(PPP3CA) mRNA in muscles of two duck breeds (Anas platyrhynchos domestica) and its relationship with myofiber traits during early development[J].Journal of Agricultural Biotechnology,2015,23(2):236-243.(in Chinese)
[17]	DIAS M M,SOUZA F R P,TAKADA L,et al.Study of lipid metabolism-related genes as candidate genes of sexual precocity in Nellore cattle[J].Genet Mol Res,2015,14(1):234-243.
[18]	E G X,ZHAO Y J,HUANG Y F.Selection signatures of litter size in Dazu black goats based on a whole genome sequencing mixed pools strategy[J].Mol Biol Rep,2019,46(5):5517-5523.
[19]	REDGROVE K A,BERNSTEIN I R,PYE V J,et al.Dynamin 2 is essential for mammalian spermatogenesis[J].Sci Rep,2016,6(1):35084.
[20]	王凯.神经钙调蛋白磷酸酶PPP3CA特异性siRNA对VEGF和FGF2诱导的羊胎盘动脉血管内皮细胞殖的影响[D].大连:大连医科大学,2008.WANG K.Effect of PPP3CA siRNA on VEGF-and FGF 2-stimulated cell proliferationin ovine fetoplacental artery endothelial cells[D].Dalian:Dalian Medical University,2008.(in Chinese)
[21]	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 CYP19 gene ovarian promoter[J].Acta Veterinaria et Zootechnica Sinica,2015,46(9):1540-1548.(in Chinese)
[22]	YIN J W,CHANG H M,YI Y Y,et al.TGF-β1 Increases GDNF production by upregulating the expression of GDNF and furin in human granulosa-lutein cells[J].Cells,2020,9(1):185.
[23]	孙玉英,陈淑萍,谈勇.滋阴补阳序贯法联合西药对卵巢储备功能下降排卵障碍性不孕大鼠TGF-β1/Smads信号通路的影响[J].中南大学学报:医学版,2018,43(10):1068-1074.SUN Y Y,CHEN S P,TAN Y.Influence of nourishing yin and tonifying yang sequential therapy combined with Western medicine on TGF-β1/Smads signaling pathway in anovulatory infertility rats with diminished ovarian reserve[J].Journal of Central South University:Medical Science,2018,43(10):1068-1074.(in Chinese)
[24]	GRANADOS-APARICI S,HARDY K,FRANKS S,et al.SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles[J].Sci Rep,2019,9(1):6513.
[25]	PANGAS S A.Growth factors in ovarian development[J].Semin Reprod Med,2007,25(4):225-234.
[26]	FERNANDES-SILVA H,VAZ-CUNHA P,BARBOSA V B,et al.Retinoic acid regulates avian lung branching through a molecular network[J].Cell Mol Life Sci,2017,74(24):4599-4619.
[27]	DADARWAL D,GONZÁLEZ-CANO P,DICKINSON R,et al.Characterization of cytokine gene expression in uterine cytobrush samples of non-endometritic versus endometritic postpartum dairy cows[J].Theriogenology,2019,126:128-139.
[28]	SONG Y,WANG K,CHEN D B,et al.Suppression of protein phosphatase 2 differentially modulates VEGF-and FGF2-induced signaling in ovine Fetoplacental artery endothelial cells[J].Placenta,2009,30(10):907-913.
[29]	REDGROVE K A,BERNSTEIN I R,PYE V J,et al.Dynamin 2 is essential for mammalian spermatogenesis[J].Sci Rep,2016,6(1):35084.
[30]	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.
[31]	GONG Z D,CHE T J,LAI L J,et al.FSH receptor binding inhibitor restrains follicular development and possibly attenuates carcinogenesis of ovarian cancer through down-regulating expression levels of FSHR and ERβ in normal ovarian tissues[J]. Gene, 2018,668:174-181.
[32]	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.
[33]	STAMATIADES G A,KAISER U B.Gonadotropin regulation by pulsatile GnRH:signaling and gene expression[J].Mol Cell Endocrinol, 2017,463:131-141.
[34]	RICHARDS J A S,RUSSELL D L,OCHSNER S,et al.Novel signaling pathways that control ovarian follicular development, ovulation, and luteinization[J].Recent Prog Horm Res,2002,57:195-220.
[35]	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.
[36]	KNIGHT P G,GLISTER C.TGF-β superfamily members and ovarian follicle development[J]. Reproduction, 2006, 132(2):191-206.
[37]	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.
[38]	PENG J Y,GAO K X,GAO T Y,et al.Expression and regulation of tissue inhibitors of metalloproteinases (TIMP1 and TIMP3) in goat oviduct[J].Theriogenology,2015,84(9):1636-1643.
[39]	敖叶,陈祥,周志楠,等.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 Y,CHEN X,ZHOU Z N,et al.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.(in Chinese)

TGFβ2和PPP3CA基因在黔北麻羊性腺轴组织表达及对卵巢颗粒细胞的调控研究

Expression of TGFβ2 and PPP3CA Genes in Gonadal Axis of Qianbei Ma Goat and Their Regulation on Ovarian Granulosa Cells

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 39

相关文章 15

编辑推荐

Metrics

本文评价

[1]	刘阳光, 章会斌, 文浩宇, 谢帆, 赵世明, 丁月云, 郑先瑞, 殷宗俊, 张晓东. 猪卵泡液外泌体处理卵巢颗粒细胞的SNP/Indel筛选分析[J]. 畜牧兽医学报, 2024, 55(2): 576-586.
[2]	高娅薇, 彭弟, 孙朝阳, 晏子越, 崔凯, 马泽芳. 基于转录组数据挖掘外源褪黑激素影响水貂卵巢发育的分子机制[J]. 畜牧兽医学报, 2024, 55(2): 607-618.
[3]	段香茹, 康佳, 杨若晨, 单新雨, 李太春, 赵雯, 张英杰, 刘月琴. L-半胱氨酸对绵羊卵巢颗粒细胞增殖、凋亡和类固醇激素分泌的影响[J]. 畜牧兽医学报, 2024, 55(1): 179-191.
[4]	姚颖, 周应聪, 杜培岩, 李一娟, 钱文洁, 李柳杨, 余志鹏, 崔燕, 余四九, 樊江峰. 基于TMT技术的牦牛妊娠期血清蛋白质组学分析[J]. 畜牧兽医学报, 2024, 55(1): 192-206.
[5]	王子渲, 王巧, 张锦, Astrid Lissette Barreto Sánchez, 郑麦青, 李庆贺, 崔焕先, 安炳星, 赵桂苹, 文杰, 李和刚. 基于脾脏转录组筛选北京油鸡和广明白鸡抗热应激相关功能基因[J]. 畜牧兽医学报, 2023, 54(5): 1905-1914.
[6]	欧正淼, 周家文, 刘莉莉, 吴芸, 陈粉粉. 基于RNA-Seq的无量山乌骨鸡肝脏组织脂代谢相关基因筛选及其表达分析[J]. 畜牧兽医学报, 2023, 54(3): 976-988.
[7]	胡亚美, 宋湘容, 黄亮, 张璐通, 高磊, 庞卫军, 杨公社, 褚瑰燕. FGF21增强线粒体功能抑制猪卵巢颗粒细胞凋亡[J]. 畜牧兽医学报, 2023, 54(3): 1034-1045.
[8]	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.
[9]	蔡佳炜, 张琛, 靳荣帅, 鲍志远, 张希宇, 王璠, 翟频, 赵博昊, 陈阳, 汤先伟, 吴信生. 热应激下公兔睾丸组织形态和精液转录组分析[J]. 畜牧兽医学报, 2023, 54(11): 4653-4663.
[10]	蒋盛强, 胡靖, 陈红英. H1N1亚型流感病毒感染A549细胞的环状RNA表达分析[J]. 畜牧兽医学报, 2023, 54(11): 4724-4734.
[11]	张寅梁, 岳巧娴, 黄晨轩, 陈辉, 王德贺, 周荣艳. 产蛋初期和后期蛋鸡胫骨转录谱的构建及骨代谢相关基因分析[J]. 畜牧兽医学报, 2023, 54(10): 4164-4173.
[12]	吴春琳, 钟乐苗, 赵妍, 李文迹, 黄晓紫, 吴异健. 鸡毒支原体MG-HY株感染鸡气管的转录组学分析[J]. 畜牧兽医学报, 2022, 53(8): 2652-2662.
[13]	毛彦妮, 常佳伟, 李娜, 王鑫, 康馨匀, 马强, 马靓, 王桂琴. 金黄色葡萄球菌在生物被膜态与浮游态的转录组差异表达分析[J]. 畜牧兽医学报, 2022, 53(8): 2697-2707.
[14]	张俊珍, 李彩娥, 刘博, 李建慧, 张蒙, 李亚莉, 常强强. 不同生理阶段边鸡卵巢转录谱的构建及卵泡发育相关基因的分析[J]. 畜牧兽医学报, 2022, 53(2): 423-435.
[15]	王磊, 何莉娜, 唐雪, 李碧筠, 黄思艺, 王钰锟, 徐德军, 赵中权. miR-495-3p对山羊卵巢颗粒细胞功能的影响[J]. 畜牧兽医学报, 2022, 53(2): 436-446.