基于Smart-seq2探究玻璃化冷冻对猪孤雌激活囊胚基因表达的影响

doi:10.11843/j.issn.0366-6964.2024.09.018

摘要/Abstract

摘要：

旨在探究玻璃化冷冻对猪孤雌激活(parthenogenetic activation, PA)囊胚基因表达的影响。本研究以猪PA囊胚为研究对象，根据试验处理分为新鲜组、玻璃化冷冻组Ⅰ和玻璃化冷冻组Ⅱ，随后从每组挑选3枚形态良好的囊胚，利用Smart-seq2单细胞全长转录组测序技术进行转录组测序分析。结果显示，玻璃化冷冻组Ⅰ与新鲜组相比，共鉴定到772个差异表达基因(differential expression genes，DEGs)，GO和KEGG富集分析结果显示，这些DEGs与细胞脂质代谢过程、细胞葡萄糖稳态、MAPK信号通路、PI3K-Akt信号通路等相关；玻璃化冷冻组Ⅱ与新鲜组相比，共鉴定到1 613个DEGs，主要与糖异生、代谢途径、氨基酸生物合成等相关；玻璃化冷冻组Ⅱ与玻璃化冷冻组Ⅰ相比，鉴定到822个DEGs，主要与丙酮酸代谢过程、N-聚糖生物合成、细胞衰老等相关。综上，本研究基于Smart-seq2单细胞全长转录组测序技术揭示了玻璃化冷冻对猪PA囊胚脂质代谢、能量代谢、MAPK信号通路等相关基因表达的影响。

关键词: Smart-seq2, 玻璃化冷冻, 猪, 孤雌激活, 囊胚

Abstract:

The aim of this study was to investigate the effect of vitrification on gene expression in porcine parthenogenetic activation (PA) blastocysts. In this study, porcine PA blastocysts were divided into fresh, vitrification group Ⅰ and vitrification group Ⅱ according to the experimental treatments, and then 3 well-morphologised blastocysts were selected from each group and analysed by transcriptome sequencing using Smart-seq2 single-cell full-length transcriptome sequencing technology. As a result, compared with the fresh group, a total of 772 differential expression genes (DEGs) were identified in the vitrification group Ⅰ. GO and KEGG enrichment analysis results showed that these DEGs were related to cellular lipid metabolic process, cell glucose homeostasis, MAPK signaling pathway, PI3K-Akt signaling pathway, etc. Compared with the fresh group, a total of 1 613 DEGs were identified in the vitrification group Ⅱ, which mainly related to gluconeogenesis, metabolic pathways, biosynthesis of amino acids, etc. Compared with the vitrification group Ⅰ, 822 DEGs were identified in the vitrification group Ⅱ, which mainly involved in pyruvate metabolic process, N-Glycan biosynthesis, cellular senescence, etc. In summary, this study revealed the effects of vitrification on the expression of genes related to lipid metabolism, energy metabolism, MAPK signalling pathway and other related genes in porcine PA blastocysts based on Smart-seq2 single-cell full-length transcriptome sequencing technology.

Key words: Smart-seq2, vitrification, pig, parthenogenetic activation, blastocyst

中图分类号:

S828.3

杨柏高, 龙熙, 张亮, 徐皆欢, 戴建军, 赵学明, 潘红梅. 基于Smart-seq2探究玻璃化冷冻对猪孤雌激活囊胚基因表达的影响[J]. 畜牧兽医学报, 2024, 55(9): 3936-3946.

Baigao YANG, Xi LONG, Liang ZHANG, Jiehuan XU, Jianjun DAI, Xueming ZHAO, Hongmei PAN. Exploring the Effect of Vitrification on Gene Expression in Porcine Parthenogenetic Blastocysts by Smart-seq2[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3936-3946.

图/表 8

图 1

图 2

表 1

表 2

表 3

图 3

图 4

图 5

参考文献 50

1	DU X Z , ZHUAN Q R , CHENG K R , et al. Cryopreservation of porcine embryos: recent updates and progress[J]. Biopreserv Biobank, 2021, 19 (3): 210- 218. doi: 10.1089/bio.2020.0074
2	XU H X , WANG X G , TAO R X , et al. Optimal stage for cryotop vitrification of porcine embryos[J]. Cell Reprogram, 2022, 24 (3): 132- 141. doi: 10.1089/cell.2022.0001
3	ALMIÑANA C , DUBUISSON F , BAUERSACHS S , et al. Unveiling how vitrification affects the porcine blastocyst: clues from a transcriptomic study[J]. J Anim Sci Biotechnol, 2022, 13 (1): 46. doi: 10.1186/s40104-021-00672-1
4	WIESAK T , GORYSZEWSKA-SZCZUREK E . Effect of vitrification on the expression of genes in porcine blastocysts derived from in vitro matured oocytes[J]. Syst Biol Reprod Med, 2022, 68 (4): 239- 246. doi: 10.1080/19396368.2022.2072788
5	CUELLO C , MARTINEZ C A , CAMBRA J M , et al. Effects of vitrification on the blastocyst gene expression profile in a porcine model[J]. Int J Mol Sci, 2021, 22 (3): 1222. doi: 10.3390/ijms22031222
6	SANTORO F , CHIEN K R , SAHARA M . Isolation of human ESC-derived cardiac derivatives and embryonic heart cells for population and single-cell RNA-seq analysis[J]. STAR Protoc, 2021, 2 (1): 100339. doi: 10.1016/j.xpro.2021.100339
7	YANG T , YUAN X , XUE Q S , et al. Comparison of symmetrical and asymmetrical cleavage 2-cell embryos of porcine by Smart-seq2[J]. Theriogenology, 2023, 210, 221- 226. doi: 10.1016/j.theriogenology.2023.07.029
8	FAN X Y , TANG D , LIAO Y H , et al. Single-cell RNA-seq analysis of mouse preimplantation embryos by third-generation sequencing[J]. PLoS Biol, 2020, 18 (12): e3001017. doi: 10.1371/journal.pbio.3001017
9	QIAO Y B , REN C , HUANG S S , et al. High-resolution annotation of the mouse preimplantation embryo transcriptome using long-read sequencing[J]. Nat Commun, 2020, 11 (1): 2653. doi: 10.1038/s41467-020-16444-w
10	PICELLI S , FARIDANI O R , BJÖRKLUND Å K , et al. Full-length RNA-seq from single cells using Smart-seq2[J]. Nat Protoc, 2014, 9 (1): 171- 181. doi: 10.1038/nprot.2014.006
11	LI Z S , LIU Y L , MA T , et al. Smart-seq2 technology reveals a novel mechanism that zearalenone inhibits the in vitro maturation of ovine oocytes by influencing TNFAIP6 expression[J]. Toxins (Basel), 2023, 15 (10): 617. doi: 10.3390/toxins15100617
12	JIA B Y , XIANG D C , QUAN G B , et al. Transcriptome analysis of porcine immature oocytes and surrounding cumulus cells after vitrification and in vitro maturation[J]. Theriogenology, 2019, 134, 90- 97. doi: 10.1016/j.theriogenology.2019.05.019
13	XU T T , LIU C X , ZHANG M Y , et al. Vitrification of pronuclear zygotes perturbs porcine zygotic genome activation[J]. Animals (Basel), 2022, 12 (5): 610.
14	KAJDASZ A , WARZYCH E , DEREBECKA N , et al. Lipid stores and lipid metabolism associated gene expression in porcine and bovine parthenogenetic embryos revealed by fluorescent staining and RNA-seq[J]. Int J Mol Sci, 2020, 21 (18): 6488. doi: 10.3390/ijms21186488
15	LIPINSKA P , PAWLAK P , WARZYCH E . Species and embryo genome origin affect lipid droplets in preimplantation embryos[J]. Front Cell Dev Biol, 2023, 11, 1187832. doi: 10.3389/fcell.2023.1187832
16	MASEK M , ETARD C , HOFMANN C , et al. Loss of the Bardet-Biedl protein Bbs1 alters photoreceptor outer segment protein and lipid composition[J]. Nat Commun, 2022, 13 (1): 1282. doi: 10.1038/s41467-022-28982-6
17	ROUABHI M , GUO D F , MORGAN D A , et al. BBSome ablation in SF1 neurons causes obesity without comorbidities[J]. Mol Metab, 2021, 48, 101211. doi: 10.1016/j.molmet.2021.101211
18	YAN L , RUST B M , PALMER D G . Time-restricted feeding restores metabolic flexibility in adult mice with excess adiposity[J]. Front Nutr, 2024, 11, 1340735. doi: 10.3389/fnut.2024.1340735
19	WANG Y , MCNUTT M C , BANFI S , et al. Hepatic ANGPTL3 regulates adipose tissue energy homeostasis[J]. Proc Natl Acad Sci U S A, 2015, 112 (37): 11630- 11635. doi: 10.1073/pnas.1515374112
20	KLID S , MAYMÓ-MASIP E , ALGABA-CHUECA F , et al. The ANGPTL3-4-8 axis in normal gestation and in gestational diabetes, and its potential involvement in fetal growth[J]. Int J Mol Sci, 2023, 24 (3): 2486. doi: 10.3390/ijms24032486
21	PAWLAK P , MALYSZKA N , SZCZERBAL I , et al. Fatty acid induced lipolysis influences embryo development, gene expression and lipid droplet Formation in the porcine cumulus cells[J]. Biol Reprod, 2020, 103 (1): 36- 48. doi: 10.1093/biolre/ioaa045
22	FRAGOULI E , WELLS D . Mitochondrial DNA assessment to determine oocyte and embryo viability[J]. Semin Reprod Med, 2015, 33 (6): 401- 409. doi: 10.1055/s-0035-1567821
23	BARBE A , KUROWSKA P , RAME C , et al. Adipolin (C1QTNF12) is a new adipokine in female reproduction: expression and function in porcine granulosa cells[J]. Reproduction, 2023, 167 (1): e230272.
24	OSBAK K K , COLCLOUGH K , SAINT-MARTIN C , et al. Update on mutations in glucokinase (GCK), which cause maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemic hypoglycemia[J]. Hum Mutat, 2009, 30 (11): 1512- 1526. doi: 10.1002/humu.21110
25	ZHANG Y L , GUO L Y , HAN S , et al. Adult mesenchymal stem cell ageing interplays with depressed mitochondrial Ndufs6[J]. Cell Death Dis, 2020, 11 (12): 1075. doi: 10.1038/s41419-020-03289-w
26	XIANG D C , JIA B Y , ZHANG B , et al. Astaxanthin supplementation improves the subsequent developmental competence of vitrified porcine zygotes[J]. Front Vet Sci, 2022, 9, 871289. doi: 10.3389/fvets.2022.871289
27	LI G L , FLODBY P , LUO J , et al. Knockout mice reveal key roles for claudin 18 in alveolar barrier properties and fluid homeostasis[J]. Am J Respir Cell Mol Biol, 2014, 51 (2): 210- 222. doi: 10.1165/rcmb.2013-0353OC
28	SOMFAI T . Vitrification of immature oocytes in pigs[J]. Anim Sci J, 2024, 95 (1): e13943. doi: 10.1111/asj.13943
29	JØRGENSEN J P , LAURIDSEN A M , KRISTENSEN P , et al. Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor[J]. J Mol Biol, 2006, 360 (5): 1043- 1052. doi: 10.1016/j.jmb.2006.06.011
30	MO X H , WU G Q , YUAN D S , et al. Leukemia inhibitory factor enhances bovine oocyte maturation and early embryo development[J]. Mol Reprod Dev, 2014, 81 (7): 608- 618. doi: 10.1002/mrd.22327
31	WANG S H , HAO J , ZHANG C , et al. KLF17 promotes human naive pluripotency through repressing MAPK3 and ZIC2[J]. Sci China Life Sci, 2022, 65 (10): 1985- 1997. doi: 10.1007/s11427-021-2076-x
32	DE SOUZA D K , SALLES L P , CAMARGO R , et al. Effects of PI3K and FSH on steroidogenesis, viability and embryo development of the cumulus-oocyte complex after in vitro culture[J]. Zygote, 2018, 26 (1): 50- 61. doi: 10.1017/S0967199417000703
33	PEREZ Y , SHORER Z , LIANI-LEIBSON K , et al. SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome[J]. Brain, 2017, 140 (4): 928- 939. doi: 10.1093/brain/awx013
34	JEON Y , YOON J D , CAI L , et al. Effect of zinc on in vitro development of porcine embryos[J]. Theriogenology, 2015, 84 (4): 531- 537. doi: 10.1016/j.theriogenology.2015.04.006
35	WANG J , DA C L , SU Y , et al. MKNK2 enhances chemoresistance of ovarian cancer by suppressing autophagy via miR-125b[J]. Biochem Biophys Res Commun, 2021, 556, 31- 38. doi: 10.1016/j.bbrc.2021.02.084
36	LYNCH A M , ZHU Y Y , LUCAS B G , et al. TES-1/Tes and ZYX-1/Zyxin protect junctional actin networks under tension during epidermal morphogenesis in the C. elegans embryo[J]. Curr Biol, 2022, 32 (23): 5189- 5199.e6. doi: 10.1016/j.cub.2022.10.045
37	CAI T Y , BAI J J , TAN P , et al. Zyxin promotes hepatocellular carcinoma progression via the activation of AKT/mTOR signaling pathway[J]. Oncol Res, 2023, 31 (5): 805- 817. doi: 10.32604/or.2023.029549
38	ZHANG N , YU X Q , XIE J X , et al. New insights into the role of ferritin in iron homeostasis and neurodegenerative diseases[J]. Mol Neurobiol, 2021, 58 (6): 2812- 2823. doi: 10.1007/s12035-020-02277-7
39	NING N N , SHANG Z Q , LIU Z P , et al. A novel microtubule inhibitor promotes tumor ferroptosis by attenuating SLC7A11/GPX4 signaling[J]. Cell Death Discov, 2023, 9 (1): 453. doi: 10.1038/s41420-023-01713-6
40	CHEN S M , WU Y R , GAO Y , et al. Allosterically inhibited PFKL via prostaglandin E2 withholds glucose metabolism and ovarian cancer invasiveness[J]. Cell Rep, 2023, 42 (10): 113246. doi: 10.1016/j.celrep.2023.113246
41	PEI Y F , LV S N , SHI Y , et al. RAB21 controls autophagy and cellular energy homeostasis by regulating retromer-mediated recycling of SLC2A1/GLUT1[J]. Autophagy, 2023, 19 (4): 1070- 1086. doi: 10.1080/15548627.2022.2114271
42	QI J J , ZHANG S X , QU H X , et al. Lysine-specific demethylase 1 (LSD1) participate in porcine early embryonic development by regulating cell autophagy and apoptosis through the mTOR signaling pathway[J]. Theriogenology, 2024, 224, 119- 133. doi: 10.1016/j.theriogenology.2024.05.010
43	HARA T , KIN A , AOKI S , et al. Resveratrol enhances the clearance of mitochondrial damage by vitrification and improves the development of vitrified-warmed bovine embryos[J]. PLoS One, 2018, 13 (10): e0204571. doi: 10.1371/journal.pone.0204571
44	ZHEN Y , STENMARK H . Autophagosome biogenesis[J]. Cells, 2023, 12 (4): 668. doi: 10.3390/cells12040668
45	AFZAL A , SARFRAZ M , LI G L , et al. Taking a holistic view of PEST-containing nuclear protein (PCNP) in cancer biology[J]. Cancer Med, 2019, 8 (14): 6335- 6343. doi: 10.1002/cam4.2465
46	ZHUANG W X , ZHANG L , ZHENG Y , et al. USP3 deubiquitinates and stabilizes the adapter protein ASC to regulate inflammasome activation[J]. Cell Mol Immunol, 2022, 19 (10): 1141- 1152. doi: 10.1038/s41423-022-00917-7
47	DONG P Z , FU H , CHEN L , et al. PCNP promotes ovarian cancer progression by accelerating β-catenin nuclear accumulation and triggering EMT transition[J]. J Cell Mol Med, 2020, 24 (14): 8221- 8235. doi: 10.1111/jcmm.15491
48	LI N , XIONG R , LI G R , et al. PM_2.5 contributed to pulmonary epithelial senescence and ferroptosis by regulating USP3-SIRT3-P53 axis[J]. Free Radical Biol Med, 2023, 205, 291- 304. doi: 10.1016/j.freeradbiomed.2023.06.017
49	KOWALCZYK A , GBADAMOSI O , KOLOR K , et al. Evolutionary rate covariation identifies SLC30A9 (ZnT9) as a mitochondrial zinc transporter[J]. Biochem J, 2021, 478 (17): 3205- 3220. doi: 10.1042/BCJ20210342
50	EN A , TAKANASHI S , OKAZAKI R , et al. A mutation in SLC30A9, a zinc transporter, causes an increased sensitivity to oxidative stress in the nematode Caenorhabditis elegans[J]. Biochem Biophys Res Commun, 2022, 634, 175- 181. doi: 10.1016/j.bbrc.2022.09.107

基因ID Gene ID	基因名称 Gene name	log₂(FC)	P值 P value	上调或下调 Up or Down
ENSSSCG00000003333	C1QTNF12	20.68	8.76×10^-14	UP
ENSSSCG00000049746	ENSSSCG00000049746	6.06	6.16×10^-8	UP
ENSSSCG00000052366	ENSSSCG00000052366	9.46	7.49×10^-8	UP
ENSSSCG00000061727	ENSSSCG00000061727	5.22	1.79×10^-7	UP
ENSSSCG00000059571	ENSSSCG00000059571	9.77	2.80×10^-7	UP
ENSSSCG00000032135	ENSSSCG00000032135	-7.10	1.16×10^-7	Down
ENSSSCG00000008294	ACTG2	-8.19	3.51×10^-7	Down
ENSSSCG00000011657	CLDN18	-7.29	2.41×10^-5	Down
ENSSSCG00000022774	NOP53	-3.09	3.67×10^-5	Down
ENSSSCG00000038718	ADRM1	-4.35	5.93×10^-5	Down

基因ID Gene ID	基因名称 Gene name	log₂(FC)	P值 P value	上调或下调 Up or Down
ENSSSCG00000045128	ENSSSCG00000045128	6.11	2.10×10^-28	UP
ENSSSCG00000008801	SLC30A9	4.32	4.49×10^-27	UP
ENSSSCG00000041449	ENSSSCG00000041449	5.41	2.77×10^-24	UP
ENSSSCG00000013448	MKNK2	4.68	2.05×10^-23	UP
ENSSSCG00000055081	ENSSSCG00000055081	7.55	1.98×10^-21	UP
ENSSSCG00000036649	FAM151A	-4.30	2.03×10^-19	Down
ENSSSCG00000007984	MRPL28	-3.25	5.21×10^-18	Down
ENSSSCG00000016461	ZYX	-3.31	2.16×10^-16	Down
ENSSSCG00000003153	FTL	-4.07	2.45×10^-16	Down
ENSSSCG00000028197	PFKL	-3.74	1.15×10^-14	Down

基因ID Gene ID	基因名称 Gene name	log₂(FC)	P值 P value	上调或下调 Up or Down
ENSSSCG00000059025	RAB21	2.93	4.92×10^-6	UP
ENSSSCG00000036491	PCNP	2.79	1.76×10^-5	UP
ENSSSCG00000031337	SNX18	3.18	1.89×10^-5	UP
ENSSSCG00000008801	SLC30A9	3.03	2.45×10^-5	UP
ENSSSCG00000028737	USP3	2.86	3.49×10^-5	UP
ENSSSCG00000049267	ENSSSCG00000049267	-21.72	1.57×10^-11	Down
ENSSSCG00000051983	ENSSSCG00000051983	-21.68	1.81×10^-11	Down
ENSSSCG00000038832	U2	-21.63	2.49×10^-11	Down
ENSSSCG00000063160	ENSSSCG00000063160	-21.63	2.49×10^-11	Down
ENSSSCG00000051866	ENSSSCG00000051866	-21.61	2.73×10^-11	Down

[1]	田晶晶, 王晓庆, 李棉燕, 王海玲, 吴启钿, 王立贤, 张龙超, 赵福平. 北京黑猪全基因组ROH检测和选择信号分析[J]. 畜牧兽医学报, 2024, 55(9): 3833-3842.
[2]	陈栋, 周文譞, 赵真坚, 申琦, 余杨, 崔晟頔, 王俊戈, 陈子旸, 禹世欣, 陈佳苗, 王翔枫, 吴平先, 郭宗义, 王金勇, 唐国庆. 基于计算机视觉技术的猪肌内脂肪含量和眼肌面积测定系统的研发[J]. 畜牧兽医学报, 2024, 55(9): 3843-3852.
[3]	陈南珠, 李俊良, 余大为, 周心仪, 王晶, 邹惠影, 杜卫华. 猪MKRN3基因的印记表达和DNA甲基化状态分析[J]. 畜牧兽医学报, 2024, 55(9): 3853-3863.
[4]	任聪, 张虎, 王钰明, 解竞静, 萨仁娜, 赵峰. 仿生消化法估测生长猪饲料有效能的准确性及可加性研究[J]. 畜牧兽医学报, 2024, 55(9): 3988-4000.
[5]	高力国, 申翰钦, 陈诒全, 陈胜, 蔺文成, 陈峰. 猪轮状病毒重组VP6*蛋白的原核表达及间接ELISA检测方法的建立[J]. 畜牧兽医学报, 2024, 55(9): 4021-4028.
[6]	朋璐, 张衡, 庞思琪, 乔竹林, 张小芬, 谭臣, 宋云峰, 周锐, 黎璐. 利用大蜡螟幼虫和小鼠感染模型筛选猪链球菌血清2、3和9型三价灭活疫苗候选菌株[J]. 畜牧兽医学报, 2024, 55(9): 4077-4090.
[7]	付艺乾, 梁东阁, 王铭洋, 潘佳佳, 杨彦宾, 曾磊, 康相涛. 干扰素调节因子敲减细胞系的构建及其对猪伪狂犬病病毒增殖的影响[J]. 畜牧兽医学报, 2024, 55(9): 4100-4109.
[8]	彭宁, 梁雅旭, 龙菲, 余东明, 钟翔. 白藜芦醇对轮状病毒感染猪肠上皮细胞IPEC-J2的抑制效应[J]. 畜牧兽医学报, 2024, 55(9): 4213-4225.
[9]	冯露, 田宏, 郑海学, 石正旺, 罗俊聪, 张晓阳, 尉娟娟, 周静, 廖焕程, 王婉莹. 基于酶促重组酶扩增的非洲猪瘟病毒检测方法[J]. 畜牧兽医学报, 2024, 55(9): 4226-4231.
[10]	夏振涛, 王楠, 王婉洁, 周期律, 黄雷, 牟玉莲. pAPN基因敲除的IPEC-J2介导的TGEV感染特征分析[J]. 畜牧兽医学报, 2024, 55(8): 3395-3407.
[11]	王怡, 高娟, 胡悦旻, 杨跃飞, 范博钧, 鞠辉明. 短期血清饥饿胁迫对猪骨骼肌卫星细胞代谢及自噬发生的影响[J]. 畜牧兽医学报, 2024, 55(8): 3408-3417.
[12]	杨程, 刘野, 程宁, 王凯月, 李欣蕾, 孙久英, 韩俊平, 李文军, 王欢欢, 邵笑, 程雪娇, 孙英峰. 一株PRRSV-2谱系1.8与1.5重组毒株的基因组特征分析[J]. 畜牧兽医学报, 2024, 55(8): 3570-3578.
[13]	李跃, 张长春, 刘光裕, 高梦源, 符超俊, 邢家宝, 徐思佳, 邝麒元, 刘静, 高校鹏, 王衡, 龚浪, 张桂红, 孙彦阔. 宏转录组测序技术在一起仔猪病毒性腹泻疾病诊断中的运用及分析[J]. 畜牧兽医学报, 2024, 55(8): 3579-3589.
[14]	吕林丹, 牟豪, 胡霞, 刘明妮, 李绍梅, 李星, 宋振辉, 杨柳. 猪传染性胃肠炎病毒S基因RAA检测方法的建立与初步应用[J]. 畜牧兽医学报, 2024, 55(8): 3590-3599.
[15]	鲜婷婷, 刘彦, 曹忻, 冯涛. 母猪子宫内膜炎阴道菌群与血清促炎细胞因子的变化及其相关性分析[J]. 畜牧兽医学报, 2024, 55(8): 3688-3698.