摘要
【目的】分析牦牛卵泡发育过程中组蛋白赖氨酸脱甲基酶1(lysine-specific histone demethylase 1A,KDM1A)基因的表达谱,探讨KDM1A对牦牛卵泡发育和卵母细胞成熟的影响。【方法】以牦牛卵泡为研究对象,根据卵泡直径的大小,将卵泡分为3组:大(6.0-9.0 mm)、中(3.0-5.9 mm)、小(1.0-2.9 mm)卵泡,收集各组别卵泡中卵丘-卵母细胞复合体(cumulus-oocyte complex,COCs)进行体外培养,对不同发育阶段卵泡中卵母细胞的体外成熟率进行统计并分析;分别提取各组卵泡卵母细胞及壁层颗粒细胞总RNA,通过实时荧光定量PCR(quantitative real-time RCR,RT-qPCR)方法,检测KDM1A基因在不同发育阶段的卵泡中mRNA相对表达量的变化;采用免疫组织化学技术检测KDM1A在牦牛卵泡发育过程中的细胞定位及表达规律,并结合体外成熟与RT-qPCR结果进行关联性分析。【结果】各组卵母细胞体外成熟率与卵泡直径的大小呈显著正相关关系,其伴随卵泡发育的进行呈明显的上升趋势,其中大、中、小卵泡组的体外成熟率依次分别为90.53%、88.10%、55.14%。RT-qPCR结果显示,KDM1A基因在牦牛卵泡发育过程中均有表达,且不同发育阶段的mRNA表达水平差异显著,其中大、中卵泡时期的卵母细胞中的mRNA相对表达量显著低于小卵泡时期(P<0.05),而在小卵泡时期的壁层颗粒细胞中的相对表达量极显著低于大、中卵泡时期(P<0.01),在大、中卵泡时期的卵母细胞及壁层颗粒细胞中该基因mRNA表达水平均无差异(P>0.05)。免疫组织化学研究发现KDM1A在大、中、小卵泡壁层颗粒细胞以及膜细胞中均表达,其蛋白表达趋势与RT-qPCR结果吻合,即随着发育时间的进行KDM1A表达水平呈上升趋势,其中在大卵泡时期KDM1A的表达量最高。【结论】在牦牛不同发育时期卵泡的卵母细胞及壁层颗粒细胞中KDM1A mRNA及蛋白的表达水平呈动态变化,提示KDM1A在卵泡发育及卵母细胞成熟过程中发挥重要作用。这可能与卵母细胞的减数分裂及颗粒细胞的增殖分化有关,本研究为进一步研究该基因在牦牛卵母细胞减数分裂过程中的作用机制提供基础数据。
【Objective】The aim of this study was to analyze the role of the lysine-specific histone demethylase 1A(KDM1A)in follicle development and oocyte maturation of yak.【Method】Taking yak follicles as research objects and according to the size of the follicles,they were divided into three groups:large-sized(6.0-9.0 mm),medium-sized(3.0-5.9 mm),and small-sized(1.0-2.9 mm)follicles.And the cumulus-oocyte complexes(COCs)were collected from each group and cultured in vitro.The maturation rate of oocytes was counted and analyzed.The total RNA was extracted from oocyte and granulosa cells in each group of follicles.The real time quantitative PCR(RT-qPCR)was used to detect the relative expression of KDM1A during follicular development.The cell localization and expression of KDM1A in yak follicle were detected by immunohistochemistry,and the correlation analysis was performed by in vitro maturation and RT-qPCR.【Result】The maturation rate of oocytes in vitro maturation was positively related to the size of follicles and showed a rising trend with progressing of follicular development.Meanwhile,the oocyte maturation rates of large-sized,medium-sized and small-sized follicle oocytes were 90.53%,88.10%and 55.14%,respectively.The result of RT-qPCR was found that KDM1A gene of yak was widely expressed during the development of follicles,and its expression level was significantly different in developmental stages of follicles.The relative expression of mRNA in the oocytes of the large and medium-sized follicle was significantly lower than small-sized follicles(P<0.05),but the relative expression of granulosa cells in the small-sized follicle was significantly lower than large and medium-sized(P<0.01),and there was no difference in mRNA expression levels between oocytes and granulosa cells in the large and middle-sized(P>0.05).The results of immunohistochemistry showed that KDM1A was expressed in granulosa cells and membrane cells of follicles,and its expression trend was consistent with RT-qPCR.The expression of KDM1A was the highest in the large follicle and increased with the development of follicles.【Conclusion】The expression levels of KDM1A mRNA and protein in oocytes and granulosa cells at the development of yak follicles were dynamic,which indicated that KDM1A played an important role in follicular development and oocyte maturation.It might be related to meiosis of oocytes and proliferation and differentiation of granulosa cells,and these results of study would provide a basic data for further research of the mechanism of KDM1A in the meiosis of yak oocytes.
作者
黄向月
熊显荣
韩杰
杨显英
王艳
王斌
李键
HUANG XiangYue;XIONG XianRong;HAN Jie;YANG XianYing;WANG Yan;WANG Bin;LI Jian(College of Life Science and Technology,Southwest Minzu University,Chengdu 610041;Key Laboratory of Ministry of Education for Qinghai-Tibet Plateau Animal Genetic Resources Reservation and Exploitation,Chengdu 610041)
出处
《中国农业科学》
CAS
CSCD
北大核心
2019年第24期4624-4631,共8页
Scientia Agricultura Sinica
基金
国家重点研发专项(2018YFD0502304)
牦牛遗传资源保护与利用创新团队(13CXTD01)
青藏高原生态畜牧业协同创新中心开放基金(QZGYXT05)
西南民族大学研究生创新型科研项目(CX2018SZ34)
