摘要
采集牦牛卵巢组织,利用免疫组织化学法分析KDM4B在不同发育阶段牦牛卵泡中的表达与定位;根据牦牛卵泡直径大小,将卵泡分为大卵泡(≥7 mm)、中卵泡(3.0~6.9 mm)和小卵泡(≤2.9 mm)组,采用注射器抽取的方法分别收集各组别卵泡中卵丘卵母细胞复合体(cumulus-oocyte complex, COCs)及壁层颗粒细胞,对COCs进行体外成熟培养并统计各组别卵母细胞成熟率;分别提取各组别卵泡卵母细胞、卵丘颗粒细胞及壁层颗粒细胞总RNA,采用RT-qPCR方法检测卵泡细胞中KDM4B mRNA的相对表达量。结果显示,KDM4B在各发育阶段的牦牛卵泡中均有表达,主要定位于卵母细胞及颗粒细胞,而在膜细胞中表达较少,且随着卵泡发育进程KDM4B表达信号增强。体外培养结果显示,卵母细胞成熟率随着卵泡的发育程度上升,其中大卵泡组(91.30%)、中卵泡组(85.77%)成熟率显著高于小卵泡组(63.51%,P<0.05);RT-qPCR结果显示,KDM4B mRNA在各组卵泡细胞中均有表达,其中卵母细胞中表达量随卵泡发育呈下降趋势,且大卵泡组、中卵泡组显著低于小卵泡组(P<0.05),大卵泡组、中卵泡组间差异不显著(P>0.05)。卵丘颗粒细胞与壁层颗粒细胞中KDM4B mRNA表达规律基本一致,即随卵泡发育进程其表达均呈上升趋势,大卵泡组、中卵泡组表达极显著高于小卵泡组(P<0.01);此外,各组别的卵丘颗粒细胞中KDM4B的表达量约为同组中壁层颗粒细胞的2倍(P<0.01)。结果表明,KDM4B在牦牛卵泡发育过程中呈动态变化,参与了牦牛卵泡发育过程,其具体调控机制有待进一步研究。本研究旨在分析组蛋白去甲基化酶4B(histone demethylase 4B,KDM4B/JMJD2B)基因在牦牛不同发育阶段卵泡中的表达规律,探究其对牦牛卵泡发育成熟的影响,为深入探讨KDM4B在卵巢卵泡发育中的调控机理提供依据。
The aim of this study was to analyze the expression of histone demethylase 4 B(KDM4 B/JMJD2 B) gene in different development stages of yak follicles and to explore its effect on the development and maturation of yak follicles.The ovaries of yaks were collected, the expression and localization of KDM4 B protein in the follicles of yaks at different developmental stages were analyzed by immunohistochemical method.According to the diameter of yak follicles, the follicles were divided into three groups: large(≥7 mm),medium(3.0-6.9 mm) and small(≤2.9 mm) follicle group.The cumulus oocyte complex(cumulus-oocyte complex, COCs) and parietal granulosa cells were collected by syringe extraction.COCs were matured in vitro and the maturation rate of oocytes in each group was calculated.The total RNA of follicular oocytes,cumulus cells and granulosa cells were extracted and the relative expression of KDM4 BmRNA was detected by RT-qPCR method.The results showed that KDM4 Bprotein was expressed in all stages of yak follicles,it mainly located in granulosa cells,but less in theca cells.The expression signal of KDM4 Bwas increased along with the development of follicles.The results of in vitro culture showed that the oocyte maturation rate increased with the degree of follicular development,and the maturation rate of large(91.30%)and medium(85.77%)follicles was significantly higher than that of small follicles(63.51%,P<0.05).The results of RT-qPCR indicated that KDM4 B mRNA was expressed in all groups of follicular cells,and the expression in oocytes decreased with the development of follicles,and the expression in large and medium follicles was significantly lower than that in small follicles(P>0.05),but there was no significant difference between large and medium follicles(P<0.05).The expression of KDM4 BmRNA in cumulus granulosa cells and parietal granulosa cells increased with the development of follicles,and the expression of KDM4 Bin large and middle follicles was significantly higher than that in small follicles(P<0.01).In addition,the expression of KDM4 Bin cumulus granulosa cells was about twice as much as that in middle wall granulosa cells in the same group(P<0.01).The results suggest that KDM4 Bchanges dynamically in the process of yak follicular development and participates in the process of yak follicular development,and its specific regulation mechanism needs to be further studied.This study provides basic data for further exploring the regulatory mechanism of KDM4 Bin ovarian follicular development.
作者
马鸿程
熊显荣
海卓
闵星宇
郝振宇
李键
MA Hongcheng;XIONG Xianrong;HAI Zhuo;Min Xingyu;HAO Zhenyu;LI Jian(College of Animal Science and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation of Ministry of Education,Chengdu 610041,China;Key Laboratory of Animal Science of State Ethnic Affairs Commission,Southwest Minzu University,Chengdu 610041,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2021年第5期1016-1022,共7页
Chinese Journal of Veterinary Science
基金
国家“十三五”重点研发专项基金资助项目(2018YFD0502304)
西南民族大学研究生创新型科研基金资助项目(CX2020SZ59)。
