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2025年4月5日 星期六
农业生物技术学报  2019, Vol. 27 Issue (8): 1423-1433    DOI: 10.3969/j.issn.1674-7968.2019.08.010
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
TNF-α在牦牛卵母细胞及早期体外受精胚胎中的表达
许涛, 潘阳阳, 何翃闳, 李谷月, 陈平, 温泽星, 崔燕, 余四九*
甘肃农业大学 动物医学院/甘肃省牛羊胚胎工程技术研究中心,兰州 730070
The Expression of TNF-α in Yak (Bos grunniens) Oocytes and Preimplantation Embryos Produced by In vitro Fertilization
XU Tao, PAN Yang-Yang, HE Hong-Hong, LI Gu-Yue, CHEN Ping, WEN Ze-Xing, CUI Yan, YU Si-Jiu*
Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep & Goat, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
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摘要 肿瘤坏死因子α (tumor necrosis factor α, TNF-α)是一种促炎性辅助性T细胞(helper T cell, Th1)因子,能参与调节哺乳动物的排卵、胚胎发育和附植等生殖过程。本研究旨在通过检测TNF-α在牦牛(Bos grunniens)卵母细胞成熟及早期体外受精(in vitro fertilization, IVF)胚胎发育过程中的表达,探讨TNF-α在卵母细胞成熟与体外受精胚胎发育过程中的作用。本研究收集牦牛卵丘-卵母细胞复合体(cumulus-oocyte complexes, COCs),在体外进行成熟培养并受精,获得体外受精胚胎,并评估植入前各时期胚胎的发育能力。采用qRT-PCR、间接免疫荧光染色、Western blot等方法从基因和蛋白水平检测TNF-α在未成熟卵母细胞、成熟卵母细胞和植入前各时期胚胎中的表达与分布。结果表明:(1) 2~4细胞胚胎、5~8细胞胚胎和桑椹胚的发育率分别为(61.59±0.31)%、(53.76±0.22)%和(26.88±0.32)%,囊胚率为(16.12±0.23)%;(2) qRT-PCR结果表明,未成熟COCs、成熟COCs、桑椹胚和囊胚均可检测到TNF-α基因的表达,其相对表达水平由高到低依次为成熟COCs、未成熟COCs、囊胚和桑椹胚,2~4细胞胚胎和5~8细胞胚胎无表达;(3) Western blot结果显示,TNF-α蛋白相对表达水平由高到低依次为成熟COCs、未成熟COCs、囊胚和桑椹胚,2~4细胞胚胎和5~8细胞胚胎无表达;免疫荧光染色结果显示,TNF-α蛋白主要分布在COCs、桑椹胚和囊胚的细胞质中,在细胞核中表达较弱。本研究表明,TNF-α可能在牦牛卵母细胞的成熟、受精和胚胎发育过程中发挥作用,也可能参与囊胚的细胞凋亡和附植过程。本研究为揭示TNF-α在牦牛生殖过程中的作用机制提供了理论依据。
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许涛
潘阳阳
何翃闳
李谷月
陈平
温泽星
崔燕
余四九
关键词 肿瘤坏死因子α (TNF-α)牦牛体外受精胚胎发育    
Abstract:Tumor necrosis factor α (TNF-α) is a pro-inflammatory Th1 cytokine that is involved in the regulation of mammalian ovulation, embryonic development and implantation. The aim of this study was to investigate the role of TNF-α in the development of oocyte maturation and in vitro fertilization (IVF) embryos by detecting the expression of TNF-α in yak (Bos grunneins) oocyte maturation and early IVF embryo development. In this study, the cumulus-oocyte complex (COCs) were collected and matured in vitro and fertilized to obtain IVF embryos, and to evaluate the developmental ability of embryos at different stages of implantation. Methods of qRT-PCR, indirect immunofluorescence staining and Western blot were used to detect the expression and distribution of TNF-α in immature oocytes, mature oocytes and embryos at different stages. The results showed as follows: (1) The rates of embryos developed to stages of 2~4 cells, 5~8 cells, morulae were (61.59±0.31)%, (53.76±0.22)% and (26.88±0.32)%, respectively, and the blastocyst rate was (16.12±0.23)%. (2) qRT-PCR results showed that, TNF-α mRNA could be detected in immature COCs, mature COCs, morulae and blastocysts, the relative expression levels in the order of high to low was mature COCs, immature COCs, blastocysts and morulae. 2~4 cells and 5~8 cells embryos had no expression. (3) The result of indirect immunofluorescence and Western blot showed that the protein of TNF-α was mainly distributed in the cytoplasm of COCs and embryos, which was weaker in the nucleus. The protein of TNF-α could be detected in COCs, morula and blastocyst, the relative expression levels in the order of high to low was mature COCs, immature COCs, blastocysts and morulae, but 2~4 cells and 5~8 cells embryos had no expression. The results in this study indicated that TNF-α might play a role in the process of the maturation and senescence of the yak oocytes and might be involved in the process of blastocyst cell apoptosis and embryo implantation, which provides a theoretical basis for revealing the mechanism of TNF-α in yak reproduction.
Key wordsTumor necrosis factor alpha (TNF-α)    Yak (Bos grunneins)    In vitro Fertilization (IVF)    Embryo development
收稿日期: 2019-01-24     
ZTFLH:  S857.2  
基金资助:国家自然科学基金(No.31472244和No.31702311)、甘肃农业大学盛彤笙科技创新基金(No.GSAU-STS-1614)和甘肃省青年科技基金计划(No.17JR5RA141)
通讯作者: *,sjyu@163.com   
引用本文:   
许涛, 潘阳阳, 何翃闳, 李谷月, 陈平, 温泽星, 崔燕, 余四九. TNF-α在牦牛卵母细胞及早期体外受精胚胎中的表达[J]. 农业生物技术学报, 2019, 27(8): 1423-1433.
XU Tao, PAN Yang-Yang, HE Hong-Hong, LI Gu-Yue, CHEN Ping, WEN Ze-Xing, CUI Yan, YU Si-Jiu. The Expression of TNF-α in Yak (Bos grunniens) Oocytes and Preimplantation Embryos Produced by In vitro Fertilization. 农业生物技术学报, 2019, 27(8): 1423-1433.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.08.010     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I8/1423
 
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