牦牛早期胚胎核旁斑点形成及对后续发育的影响

doi:10.3864/j.issn.0578-1752.2023.06.014

摘要/Abstract

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RNA（Long non-coding RNA，LncRNAs），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1（paraspeckle Protein 1，PSPC1）mRNA实时荧光定量PCR（real-time fluorescence quantitative PCR，qRT-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1（encoding nuclear paraspeckle assembly transcript 1，NEAT1）、共激活因子相关的精氨酸甲基转移酶1（coactivator associated arginine methyltransferase 1，CARM1）及54 kD核结合蛋白（non-POU domain containing octamer-binding protein，p54nrb）mRNA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-2，Bcl-2）和B细胞淋巴瘤/白血病基因伴随蛋白x（B-cell lymphoma/leukemia associatedx protein，Bax）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。（2）NEAT1、CARM1及p54nrb mRNA均在2-细胞到桑椹胚阶段呈现高水平表达，其中NEAT1和p54nrb在4细胞时期水平最高，CARM1在2-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。（4）PSPC1 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、NEAT1、CRAM1、p54nrb在核旁斑点形成时期呈高水平表达。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:

【Objective】 The aim of present study was to identify of paraspeckle formation stages during the early embryonic development in yak (Bos grunniens). Furthermore, the long non-coding RNAs (LncRNAs) involved in paraspeckle formation were determined, and the effects and regulatory mechanism of their formation on the subsequent developmental ability of yak embryos were studied. 【Method】 The 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 could 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 was the highest in embryos at 4-cell embryos and morula. The fluorescence intensity of PSPC1 protein was the strongest in embryos from those stages. (2) The levels of NEAT1, CARM1 and p54nrb mRNA were higher from 2-cell to morula stage than that from 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. The 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. 【Conclusion】 The paraspeckle was formed at 2-cell to morula stage transition in the yak embryo, which 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, paraspeckle, cell fate, apoptosis, LncRNAs

潘阳阳, 王靖雷, 王萌, 王立斌, 张倩, 陈睿, 张甜甜, 崔燕, 徐庚全, 樊江峰, 余四九. 牦牛早期胚胎核旁斑点形成及对后续发育的影响[J]. 中国农业科学, 2023, 56(6): 1189-1203.

PAN YangYang, WANG JingLei, WANG Meng, WANG LiBin, ZHANG Qian, CHEN Rui, ZHANG TianTian, CUI Yan, XU GengQuan, FAN JiangFeng, YU SiJiu. Formation and Function of Paraspeckle During Pre-implantation Embryos Development in Yak[J]. Scientia Agricultura Sinica, 2023, 56(6): 1189-1203.

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基因 Gene	正义链序列 Sense sequence	反义链序列 Antisense sequence
siRNA1	AAAUGCUUGCUCUAGUAGCUC	GCUACUAGAGCAAGCAUUUUC
siRNA2	AGGUUUUGCUGCAAAUUCCAC	GGAAUUUGCAGCAAAACCUCC
siRNA3	UUCUCUUUCCUUAUGAUACUG	GUAUCAUAAGGAAAGAGAACA

基因 Gene	引物序列 Primer sequence（5'→3'）	T_m (℃)	产物大小 Amplicon size (bp)	GenBank登录号 GenBank accession No.
NEAT1	F:GAACTTGTCAATACCAGCAGC	56	291	XR_001351403.1
NEAT1	R: CAACAACTTTCCCGTCTTACC	56	291	XR_001351403.1
PSPC1	F :GGACATCACCGAGGACGACT	60	145	NM_001075277.1
PSPC1	R: GCCATCCAGCTCTGCTTTTG	60	145	NM_001075277.1
P54nrb	F:CTTTATCCGCTTGGAAACAC	55	209	NM_001046554.1
P54nrb	R:CCACAATGACTACAGCCCTC	55	209	NM_001046554.1
CARM1	F:GTCATTCATCATCACCCTG	50	274	XM_024994894.1
CARM1	R:AATCTTGTCCTTGAAGTCTGT	50	274	XM_024994894.1
Bax	F:TTTGCTTCAGGGTTTCATC	59	174	NM173894.1
Bax	R:CAGCTGCGATCATCCTCT	59	174	NM173894.1
Bcl-2	F: CTGCACCTGACGCCCTTCAC	62	236	NM001166486.1
Bcl-2	R: GCGTCCCAGCCTCCGTTGT	62	236	NM001166486.1
β-actin	F:CTTCAACACCCCTGCCAT	60	238	JF830811
β-actin	R: CTCGGCTGTGGTGGTGAAG	60	238	JF830811

组别 Group	4细胞胚胎数 No. of 4-cell embryos	桑葚胚（发育率） Morula (Development rate, %)	囊胚率（发育率） Blastocyst (Development rate, %)
Control	113	85（75.22 ± 3.32a）	35（41.17 ± 2.23%a）
siRNA1	115	67（58.26 ± 1.86b）	22（32.83 ± 1.54%b）

组别 Group	总细胞数量 Total no. of cells	TE细胞数量 No. of TE cells	ICM细胞数量 No. of ICM cells
Control	102.45 ± 3.08a	81.40 ± 5.02a	21.07 ± 0.58a
siRNA1	98.80 ± 2.65b	80.00 ± 1.56a	18.80 ± 1.45b