中国农业科学

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最新录用:牦牛早期胚胎核旁斑点形成及对发育的影响

潘阳阳1,2王靖雷1,2王萌1,2王立斌1,2张倩1,2陈睿1张甜甜1崔燕1,2徐庚全1,2樊江峰1,2余四九1,2
  

  1. 1甘肃农业大学动物医学院,兰州,730070; 2甘肃省牛羊胚胎工程技术研究中心,兰州,730070
  • 发布日期:2022-08-01

Formation and Function of Paraspeckle During Pre-implantation Embryos Development in Yak (Bos grunniens)

PAN YangYang1,2, WANG JingLei1,2, WANG Meng1,2, WANG LiBin1,2, ZHANG Qian1,2, CHEN Rui1, ZHANG TianTian1, CUI Yan1,2, XU GengQuan1,2, FAN JiangFeng1,2, YU SiJiu1,2 #br#   

  1. 1College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070; 2Gansu Province Livestock Embryo Engineering Research Center, Lanzhou, 730070
  • Online:2022-08-01

摘要: 【目的】明确牦牛早期胚胎发育过程中核旁斑点形成的关键时期,确定其参与形成长链非编码RNALong non-coding RNALncRNAs,探索核旁斑点形成对后续胚胎发育能力的影响及调控机制。【方法】体外受精生产牦牛胚胎, DAPI染色标记结合核旁斑点结构蛋白1Paraspeckle Protein 1PSPC1mRNA实时荧光定量PCRReal-time fluorescence quantitative PCRqRT-PCR检测确定牦牛早期胚胎发育核旁斑点形成关键时期,免疫荧光技术验证胚胎PSPC1蛋白表达水平;qRT-PCR检测核旁斑点形成相关LncRNAs核旁斑点组装转录因子1Encoding nuclear paraspeckle assembly transcript 1NEAT1)、共激活因子相关的精氨酸甲基转移酶1Coactivator associated arginine methyltransferase 1CARM1)及54 kD核结合蛋白(Non-POU domain containing octamer-binding proteinp54nrbmRNA在各时期胚胎中表达水平;RNA干扰技术抑制合子PSPC1 mRNA水平,比较后续各阶段胚胎发育率,通过分析囊胚细胞总数、滋养层细胞数(Trophoblast cells,TE)、内细胞团数(Inner cell mass,ICM)评估囊胚质量;检测对照组和PSPC1 mRNA干扰组囊胚中B-细胞淋巴瘤/白血病-2原癌基因(B-celllymphoma/leukemia-2Bcl-2)和B细胞淋巴瘤/白血病基因伴随蛋白xB-cell lymphoma/leukemia associatedx proteinBax)表达水平。【结果】(1)在不同发育阶段胚胎细胞的细胞核均可观察到核旁斑点,但2-细胞和4-细胞时期胚胎细胞核中核旁斑点更为清晰,2-细胞至桑椹胚阶段PSPC1 mRNA呈现高水平表达,其中4-细胞到桑椹胚阶段PSPC1 mRNA水平最高,PSPC1蛋白荧光强度在此阶段最强。(2NEAT1CARM1p54nrb mRNA均在2-细胞到桑椹胚阶段呈现高水平表达,其中NEAT1p54nrb在4细胞时期水平最高,CARM12-细胞到桑椹胚3个阶段表达水平差异不显著(p > 0.05)。(3)PSPC1 mRNA干扰组桑椹胚与囊胚发育率均显著降低,且桑椹胚发育率降低幅度高于囊胚PSPC1 mRNA干扰组囊胚细胞总数显著低于对照组,其中以ICM细胞数降低为主,TE细胞数在两组之间差异不显著。(4PSPC1 mRNA干扰处理组囊胚中促细胞凋亡因子Bax mRNA和蛋白均显著增加,抑凋亡相关因子Bcl-2 mRNA和蛋白降低,且囊胚中内细胞团发生裂解。【结论】牦牛早期胚胎发育核旁斑点形成的关键时期为2-细胞至桑椹胚阶段,其中主要集中在4-细胞时期,且PSPC1、NEAT1CRAM1、p54nrb在核旁斑点形成时期呈高水表达。干扰牦牛合子PSPC1 mRNA导致后续胚胎发育能力降低,并通过诱导ICM凋亡降低囊胚质量,影响囊胚中细胞命运决定。


关键词: 牦牛, 核旁斑点, 细胞命运, 细胞凋亡, 长链非编码RNA(LncRNAs)

Abstract: 【ObjectiveThe aim of present study was to identify of paraspeckle formation stages during early embryonic development in yak (Bos grunniens). Furthermore, we also determined long non-coding RNAs (LncRNAs) involved in paraspeckle formation and study effects and regulatory mechanism of their formation on the subsequent developmental ability of yak embryos.MethodThe yak embryos were produced by in vitro fertilization (IVF), DAPI staining of embryonic nuclei combined with paraspeckle protein 1 (PSPC1) mRNA detection were done by quantitative real-time fluorescence PCR (qRT-PCR) at different stages in order to confirm paraspeckle formation. PSPC1 protein in embryos was verified by immunofluorescence technique. The levels of encoding nuclear paraspeckle assembly transcript 1(NEAT1), coactivator associated arginine methyl transferase 1(CARM1) and non-POU domain containing octamer-binding protein(p54nrb) mRNAs were also detected by qRT-PCR at different stages. The mRNA level of PSPC1 in zygote was inhibited by RNA interference technology, and the developmental rate of embryos in subsequent stages was compared. The blastocyst quality was evaluated by analyzing the number of total cells, trophoblast cells(TE) and inner cell mass (ICM). B-cell lymphoma/leukemia-2(Bcl-2) and b-cell lymphoma/leukemia associated X protein (Bax) in blastocysts form in the control and PSPC1 mRNA interference groups was detected.Result(1) Paraspeckle could be observed in the nuclei of embryos at all different stages; however, nuclei can be more clearly seen at 2-cell stage and 4-cell stage. The PSPC1 mRNA was higher in yak embryos from 2-cell to morula stage, which were highest in embryos at 4-cell embryos and morula. The fluorescence intensity of PSPC1 protein was strongest in embryos from those stages. (2) Levels of NEAT1, CARM1 and p54nrb mRNA were higher from 2-cell to morula stage than in other stages. NEAT1 and p54nrb were found to be highest in embryos at 4-cell stage, while CARM1 was not significantly different from 2-cell to morula stage (p > 0.05). (3) The developmental rates of morula and blastocyst in PSPC1 mRNA interference group were reduced, which was more significantly reduced in morula rate. Total number of blastocyst cells in PSPC1 mRNA interference group was significantly lower than that in the control group, which was mainly caused by ICM reduction. There was no significant difference in number of TE between the two groups. (4) The levels of Bax mRNA and protein were enhanced in blastocyst form PSPC1 mRNA interference group, while the levels of Bcl-2 mRNA and protein were reduced in blastocyst; and the cell lysis was observed in ICM.ConclusionResults in our present study show that paraspeckle formed at 2-cell to morula stage transition in the yak embryo, and was more prominent in 4-cell stage. The expression of PSPC1, NEAT1, CRAM1 and p54nrb in the stages of paraspeckle formation were on high levels. Interference with PSPC1 mRNA in yak zygotes resulted in decreased developmental ability of subsequent embryo. The blastocyst quality was also reduced by inducing apoptosis of inner cell mass, which was also involved in the regulation of cell fate determination in early embryo development.


Key words: yak, paraspeckles, cell fate, apoptosis, LncRNAs