WNT2在绵羊卵泡颗粒细胞的表达及功能研究

doi:10.11843/j.issn.0366-6964.2020.01.009

Abstract

Abstract: This study aimed to investigate the expression and function of WNT2 in ovine follicular granulosa cells (GCs). In this study, both ovaries of 20 healthy ewes aged 4-6 months were collected and the expression of WNT2 protein in follicles was localized by immunohistochemistry; qRT-PCR and Western blot were used to detect the differences of its expression in follicular granulosa cells at different developmental stages; The Wnt2 expression was silenced in GCs, and the relative expression of Wnt2 gene and the key gene CTNNB1 involved in the classical WNT signaling pathway were analyzed using qRT-PCR technology, and the apoptosis of GCs was detected. The results showed that:1) WNT2 protein was expressed in theca interna cells, granulosa cells and cumulus cells of ovine. 2) The results of qRT-PCR and Western blot were basically consistent, the expression of Wnt2 mRNA and protein in granulosa cells with different sizes were significantly different (P<0.05), and the expression in large follicle granulosa cell was significantly higher than that in medium follicles (P<0.05), and the expression in medium follicle granulosa cell was significantly higher than that in small follicles (P<0.05). 3) After silenced Wnt2 gene, the expressions of Wnt2 and CTNNB1 in Wnt2 siRNA group were significantly lower than those in nonsense sequence siRNA group (NC group) and blank control group (P<0.05), while the percentage of apoptosis in Wnt2 siRNA group was significantly higher than those in the other two groups(P<0.05). In conclusion, WNT2 can improve the biological function of ovaries granulosa cells in sheep by the WNT2/CTNNB1 signal pathway.

Key words: sheep, follicle, granulosa cells, WNT2, Wnt/β-catenin signaling pathway

CLC Number:

S826.3

XUE Lina, BI Xilin, WANG Kai, DANG Wenqing, HAN Qi, LUO Huidi, CAO Ningxian, Lü Lihua. Expression and Function Analysis of WNT2 in Ovine Follicular Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(1): 74-82.

References 31

[1]	DIAZ F J,WIGGLESWORTH K,EPPIG J J.Oocytes are required for the preantral granulosa cell to cumulus cell transition in mice[J].Dev Biol,2007,305(1):300-311.
[2]	曹俊国,陈敏,李文,等.哺乳动物卵泡发育调控分子机制研究进展[J].特产研究,2018,40(4):114-118.CAO J G,CHEN M,LI W,et al.Research progresses on the molecular mechanismsunderlying mammalian follicle development[J].Special Wild Economic Animal and Plant Research,2018,40(4):114-118.(in Chinese)
[3]	金艳梅.颗粒细胞对卵泡发育的影响[J].中国畜牧兽医,2010,37(8):69-72.JIN Y M.Effect of granulosa cells on follicle development[J].China Animal Husbandry & Veterinary Medicine,2010,37(8):69-72.(in Chinese)
[4]	薛丽娜,景炅婕,党文庆,等.绵羊Wnt2蛋白生物信息学分析[J].基因组学与应用生物学,2019,38(4):1497-1502.XUE L N,JING J J,DANG W Q,et al.Bioinformatics analysis of sheep Wnt2 protein[J].Genomics and Applied Biology,2019,38(4):1497-1502.(in Chinese)
[5]	WODARZ A,NUSSE R.Mechanisms of Wnt signaling in development[J].Annu Rev Cell Dev Biol,1998,14:59-88.
[6]	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.
[7]	LI L,JI S Y,YANG J L,et al.Wnt/β-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components[J].Mol Cell Endocrinol,2014,382(2):915-925.
[8]	苏尚,吴畏.Wnt/β-catenin信号通路对靶基因转录的调控[J].中国科学,2014,44(10):1029-1042.SU S,WU W.Regulation of target gene transcription by Wnt/β-catenin signaling[J].Scientia Sinica (Vitae),2014,44(10):1029-1042.(in Chinese)
[9]	于彩云,刘重慧,魏涛.靶向Wnt/β-catenin信号通路抑制剂研究进展[J].河南师范大学学报:自然科学版,2016,44(2):126-130.YU C X,LIU C H,WEI T.Research progress of targeted Wnt/β-catenin signaling pathway inhibitors[J].Journal of Henan Normal University:Natural Science Edition,2016,44(2):126-130.(in Chinese)
[10]	周团萍,崔毓桂,千日成.卵泡发育过程中的Wnt信号通路[J].国际生殖健康/计划生育杂志,2016,35(2):128-131.ZHOU T P,CUI Y G,QIAN R C.Wnt signal pathway during folliculogenesis[J].Journal of International Reproductive Health/Family Planning,2016,35(2):128-131.(in Chinese)
[11]	SEGDITSAS S,TOMLINSON I.Colorectal cancer and genetic alterations in the Wnt pathway[J].Oncogene,2006,25(57):7531-7537.
[12]	HU T H,YAO Y,YU S,et al.SDF-1/CXCR4 promotes epithelial-mesenchymal transition and progression of colorectal cancer by activation of the Wnt/β-catenin signaling pathway[J].Cancer Lett,2014,354(2):417-426.
[13]	WANG Y G,ZHENG T Y.Screening of hub genes and pathways in colorectal cancer with microarray technology[J].Pathol Oncol Res, 2014,20(3):611-618.
[14]	WANG H X,LI T Y,KIDDER G M,et al.WNT2 regulates DNA synthesis in mouse granulosa cells through beta-catenin[J].Biol Reprod,2010,82(5):867-875.
[15]	赵妙妙,姜晓龙,陈建伟,等.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 M M,JIANG X L,CHEN J W,et al.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.(in Chinese)
[16]	JING J J,JIANG X L,CHEN J W,et al.Notch signaling pathway promotes the development of ovine ovarian follicular granulosa cells[J].Anim Reprod Sci,2017,181:69-78.
[17]	毕锡麟,王锴,李鹏飞,等.牛卵泡颗粒细胞PRSS35表达模式及功能研究[J].畜牧兽医学报,2018,49(11):2394-2401.BI X L,WANG K,LI P F,et al.Expression profile and function analysis of PRSS35 in bovine follicle granulosa cells[J].Acta Veterinaria et Zootechnica Sinica,2018,49(11):2394-2401.(in Chinese)
[18]	ISHIKAWA T,TAMAI Y,ZORN A M,et al.Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis[J].Development,2002,128(1):25-33.
[19]	TOMIZUKA K,HORIKOSHI K,KITADA R,et al.R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling[J].Hum Mol Genet,2008,17(9):1278-1291.
[20]	DIERICK H,BEJSOVEC A.Cellular mechanisms of wingless/Wnt signal transduction[J].Curr Top Dev Biol,1999,43:153-190.
[21]	WANG H X,TEKPETEY F R,KIDDER G M.Identification of WNT/β-CATENIN signaling pathway components in human cumulus cells[J].Mol Hum Reprod,2009,15(1):11-17.
[22]	HOLSTEIN T W.The evolution of the Wnt pathway[J].Cold Spring Harb Perspect Biol,2012,4(7):a007922.
[23]	RICHEN A,LOCHHEAD P,KONTOGIANNEA M,et al.Wnt signaling in the ovary:identification and compartmentalized expression of wnt-2,wnt-2b,and frizzled-4 mRNAs[J].Endocrinology,2002,143(7):2741-2749.
[24]	ABEDINI A,ZAMBERLAM G,BOERBOOM D,et al.Non-canonical WNT5A is a potential regulator of granulosa cell function in cattle[J].Mol Cell Endocrinol, 2015,403:39-45.
[25]	QIAO G Y,DONG B W,ZHU C J,et al.Deregulation of WNT2/FZD3/β-catenin pathway compromises the estrogen synthesis in cumulus cells from patients with polycystic ovary syndrome[J].Biochem Biophys Res Commun,2017,439(1):847-854.
[26]	CASTAÑON B I,STAPP A D,GIFFORD C A,et al.Follicle-stimulating hormone regulation of estradiol production:possible involvement of WNT2 and β-catenin in bovine granulosa cells[J].J Ainim Sci,2012,90(11):3789-3797.
[27]	KAIDI A,WILLIAMS A C,PARASKEVA C.Interaction between β-catenin and HIF-1 promotes cellular adaptation to hypoxia[J].Nat Cell Biol,2007,9(2):210-217.
[28]	MANOLAGAS S C,ALMEIDA M.Gone with the Wnts:ü-catenin,T-cell factor,forkhead box O,and oxidative stress in age-dependent diseases of bone,lipid,and glucose metabolism[J].Mol Endocrinol,2007,21(11):2605-2614.
[29]	YOU L,HE B,XU Z D,et al.Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells[J].Oncogene,2004,23(36):6170-6174.
[30]	SHI Y H,HE B,KUCHENBECKER K M,et al.Inhibition of Wnt-2 and galectin-3 synergistically destabilizes β-catenin and induces apoptosis in human colorectal cancer cells[J].Int J Cancer,2007,121(6):1175-1181.
[31]	WANG H X,GILLIO-MEINA C,CHEN S L,et al.The canonical WNT2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells[J].Biol Reprod,2013,89(2):1-7.

Expression and Function Analysis of WNT2 in Ovine Follicular Granulosa Cells

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 31

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	CAO Jianhua, YANG Baigao, ZHANG Peipei, FENG Xiaoyi, ZHANG Hang, YU Zhou, NIU Yifan, HAO Haisheng, DU Weihua, ZHU Huabin, YANG Ling, ZHAO Xueming. Mechanisms of Negative Energy Balance Affects Follicular Development in Dairy Cattle [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 22-30.
[2]	WANG Haibo, ZHAN Jinshun, GU Zhiyong, CHEN Xinfeng, PAN Yue, JIA Haobin, ZHONG Xiaojun, LI Kairong, ZHAO Shengguo, HUO Junhong. Comparative Study on Meat Quality Characteristics of Three-Way Hybrid Sheep Charolais×Duper×Hu and Charolais×Australian White×Hu and Hu Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 110-119.
[3]	SHI Shengjie, WANG Liguang, GAO Lei, CAI Chuanjiang, HE Weixian, CHU Guiyan. Effect of miR-24-3p on Estradiol Synthesis in Porcine Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 169-178.
[4]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[5]	RU Meng, ZENG Wenhui, PENG Jianling, ZENG Qingjie, YIN Chao, CUI Yong, WEI Qing, LIANG Haiping, XIE Xianhua, HUANG Jianzhen. Research Progress on Follicles Development of Hens and Its Epigenetic Regulatory Mechanism [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3613-3622.
[6]	ZHANG Hanyue, ZHAO Dan, LIANG Yu, ZHAO Bishi, FAN Mengdan, QIAO Liying, LIU Jianhua, YANG Kaijie, PAN Yangyang, LIU Wenzhong. miR-150 Regulates Ovine Preadipocyte Differentiation by Targeting AOC3 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3262-3274.
[7]	LIU Linli, PENG Xuelan, LI Bo, CHENG Lefan, CIREN Lamu, ZHANG Enping. Effect of Overexpression of UCP3 Gene on the Differentiation of Sahu Sheep Preadipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3275-3285.
[8]	HE Mingyang, MA Yujing, WANG Yong, YANG Ruochen, LIU Yueqin, ZHANG Yingjie, DUAN Chunhui. Effects of Melatonin on Proliferation, Apoptosis of Ovarian Granulosa Cells, and Its Secretion of Steroid Hormones of Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3313-3324.
[9]	SONG Meijun, HAO Kexing, HAI Siyu, CHEN Yan, WANG Jing, HU Guangdong. Effects of SRIF-14 on Proliferation and Apoptosis of Endometrial Epithelial Cells in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3325-3334.
[10]	LI Yuexin, LIU Aiju, MA Xiaofei, ZHENG Zhong, HU Boxin, ZHI Yunxia, TIAN Shujun. Effect of TGFβR1 on the Function of Sheep Granulosa Cells Mediated by TGF-β/Smad Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3335-3347.
[11]	MA Youji, CHEN Pengfei, MA Qing, WU Yi. Effects of Different Exercise Amounts on Rumen Microflora Diversity of Tan Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3393-3405.
[12]	LIU Xinhuan, YUN Jialei, MAO Li, LI Jizong, HAO Fei, HE Miaofeng, YANG Leilei, ZHANG Wenwen, CHENG Zilong, SUN Min, LIU Maojun, WANG Shaohui, BAI Juan, LI Wenliang. Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3445-3454.
[13]	ZHANG Ying, JIN Hua, HAN Yang, SUI Dan, XIAO Xin, HAO Xiujing, LI Min. Analysis of Microbial Diversity in Nasal Cavity of Sheep with Respiratory Disease and Their Environment [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3455-3465.
[14]	CHEN Chun, KANG Zhaofeng, WEI Yue, LI Guanhong, WU Yanping, XIE Jinfang. Correlation of Wnt3a Gene Polymorphism with Skin Follicle Traits in Chongren Partridge Chicken [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2810-2823.
[15]	XIONG Chengkun, ZHANG Daoliang, YANG Yue, DING Hongyan, ZHAO Jie, LI Yu, WANG Xichun, FENG Shibin, ZHAO Chang, TANG Jishun, WU Jinjie. Effect of Rutin on Rumen Fermentation, Rumen Flora Structure and Antioxidant Properties in Perinatal Hu Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2898-2909.