可变剪接体WNT4-β对山羊卵泡颗粒细胞增殖和激素分泌的影响  

Effect of Alternative Splicing WNT 4-βon Follicular Granulosa Cell Proliferation and Hormone Secretion in Goats

作　　者：王鹏 韩海银[1] 李文韬 刘子嶷 储明星 刘玉芳[1,2] WANG Peng;HAN Haiyin;LI Wentao;LIU Ziyi;CHU Mingxing;LIU Yufang(College of Life Science and Food Engineering,Hebei University of Engineering,Handan 056021,China;Key Laboratory of Animal Genetics,Breeding and Reproduction of Ministry of Agriculture and Rural Affairs,Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]河北工程大学生命科学与食品工程学院,邯郸056021 [2]中国农业科学院北京畜牧兽医研究所农业农村部动物遗传育种与繁殖重点实验室,北京100193

出　　处：《畜牧兽医学报》2022年第10期3480-3489,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(32102509);财政部和农业农村部:国家现代农业产业技术体系(CARS-38);中国农业科学院科技创新工程(CAAS-ZDRW202106;ASTIP-IAS13)。

摘　　要：旨在研究WNT4的一个可变剪接体(WNT4-β)对山羊卵泡颗粒细胞增殖的影响。本研究选取4~6月龄健康母羊20只,采集双侧卵巢,体外分离卵泡颗粒细胞进行培养。通过免疫荧光染色技术确定WNT4-β的表达位置;在山羊颗粒细胞中过表达或干扰WNT4-β后,利用RT-qPCR、Westernblot检测WNT4-β和WNT信号通路中关键标记因子ROA1、RHOA及颗粒细胞增殖标记基因cyclin-D2、CDK4的表达变化;CCK-8技术检测颗粒细胞增殖情况;并通过ELISA分析颗粒细胞中生殖激素水平的变化。免疫荧光染色结果显示,WNT4-β只在山羊卵泡颗粒细胞中表达,在卵母细胞不表达;过表达WNT4-β后,WNT4-β和颗粒细胞增殖因子cyclin-D2、CDK4的mRNA相对表达量极显著增加(P<0.01),蛋白表达水平显著增加(P<0.05);WNT信号通路标记因子ROA1、RHOA mRNA表达水平显著增加(P<0.05),β-catenin蛋白表达水平显著增加(P<0.05);干扰WNT4-β后,WNT4-β、cyclin-D2、CDK4、ROA1和RHOA的mRNA表达显著降低(P<0.05),WNT4-β、cyclin-D2、CDK4及β-catenin蛋白表达显著降低(P<0.05)。CCK-8结果显示,过表达WNT4-β促进颗粒细胞增殖(P<0.05);ELISA结果显示,过表达WNT4-β后,颗粒细胞中雌二醇(estradiol,E_(2))水平显著增加(P<0.05),孕酮(progesterone,P_(4))水平升高但不显著(P>0.05);干扰WNT4-β后则结果相反,颗粒细胞增殖受到抑制(P<0.05),E_(2)和P_(4)的水平显著降低(P<0.05)。综上所述,WNT4可变剪接体WNT4-β通过调控WNT信号通路促进山羊卵泡颗粒细胞增殖及类固醇激素分泌,本研究为解析WNT4调控山羊颗粒细胞增殖的潜在分子机制提供理论基础。The aim of this study was to investigate the effect of an alternative splicing(WNT 4-β)of WNT 4 on the follicular granulosa cells proliferation of goat.In this study,20 healthy ewes from 4 to 6 months of age were selected,goat ovarian tissues were collected,follicular granulosa cells were isolated in vitro and cultured.The expression location of WNT4-βwas determined by immunofluorescence staining.Overexpression or interference of WNT 4-βin goat granulosa cells,RT-qPCR and Western blot were used to detect the expression of key marker factors(ROA 1 and RHOA)in the WNT signaling pathway,and granulosa cell proliferation marker genes cyclin-D2 and CDK 4.CCK-8 was used to detect the granulosa cells proliferation.ELISA was used to analyze the changes of reproductive hormone levels in granulosa cells.Immunofluorescence staining result showed that WNT 4 was expressed only in follicular granulosa cells of goat,but not in oocyte cells.WNT 4-βand granulosa cell proliferation factors cyclin-D2 and CDK 4 mRNA significantly increased(P<0.01)after overexpression of WNT 4-β,and significant increase in protein expression levels(P<0.05).The expression levels of WNT signaling pathway marker factors ROA 1 and RHOA mRNA significantly increased(P<0.05),andβ-catenin protein levels significantly increased(P<0.05).The mRNA expression of WNT 4-β,cyclin-D2,CDK 4,ROA 1 and RHOA significantly reduced after interfering with WNT 4-β(P<0.05).WNT4-β,cyclin-D2,CDK4 andβ-catenin proteins expression significantly reduced(P<0.05).The CCK-8 result showed that overexpression of WNT 4-βpromoted proliferation of granulosa cells(P<0.05).ELISA results showed that estradiol(E_(2))levels significantly increased(P<0.05)and progesterone(P_(4))levels increased but not significantly(P>0.05)in granulosa cells after overexpression of WNT 4-β.The opposite result was got after interfering with WNT 4-β,the granulosa cell proliferation was inhibited(P<0.05)and the levels of E_(2) and P_(4) significantly reduced(P<0.05).In conclusion,the WNT 4 alternative

关 键 词：山羊 卵泡发育 颗粒细胞增殖 可变剪接体 WNT4-β 

分 类 号：S827.3[农业科学—畜牧学]