基于scWGBS探究玻璃化冷冻对猪孤雌激活囊胚基因组甲基化水平的影响

doi:10.11843/j.issn.0366-6964.2025.01.021

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (1): 222-231.doi: 10.11843/j.issn.0366-6964.2025.01.021

基于scWGBS探究玻璃化冷冻对猪孤雌激活囊胚基因组甲基化水平的影响

杨柏高¹^,²(), 徐皆欢³, 张亮¹, 龙熙¹, 戴建军³, 赵学明²^,*, 潘红梅¹^,*()

1. 重庆市畜牧科学院, 重庆 402460
2. 中国农业科学院北京畜牧兽医研究所, 北京 100193
3. 上海市农业科学院畜牧兽医研究所, 上海 201106

收稿日期:2024-07-30 出版日期:2025-01-23 发布日期:2025-01-18
通讯作者: 赵学明,潘红梅 E-mail:Yangbaigao915@163.com;panhm_2118@163.com
作者简介:杨柏高(1991-)，男，重庆人，博士，主要从事动物繁殖研究，E-mail: Yangbaigao915@163.com
基金资助:
重庆市技术创新与应用发展专项重点项目(cstc2021jscx-dxwtBX0004)；重庆市人民政府与中国农业科学院战略合作资助项目(重庆地方猪胚胎冷冻保存技术研究与猪育种新材料创制，23310)

Exploring the Effect of Vitrification on Genome Methylation Level of Porcine Parthenogenetic Activation Blastocysts by scWGBS

YANG Baigao¹^,²(), XU Jiehuan³, ZHANG Liang¹, LONG Xi¹, DAI Jianjun³, ZHAO Xueming²^,*, PAN Hongmei¹^,*()

1. Chongqing Academy of Animal Sciences, Chongqing 402460, China
2. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
3. Institute of Animal Science and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China

Received:2024-07-30 Online:2025-01-23 Published:2025-01-18
Contact: ZHAO Xueming, PAN Hongmei E-mail:Yangbaigao915@163.com;panhm_2118@163.com

摘要/Abstract

摘要：

旨在探究玻璃化冷冻对猪孤雌激活(parthenogenetic activation, PA)囊胚基因组甲基化水平的影响。以猪第6天PA囊胚为研究对象，分别采用常规玻璃化冷冻液(玻璃化冷冻Ⅰ组)和改良玻璃化冷冻液(玻璃化冷冻Ⅱ组)进行玻璃化冷冻-复苏试验，每组选择3枚复苏形态良好的囊胚用于单细胞全基因组甲基化测序(single cell whole-genome bisulfite sequencing，scWGBS)，分析囊胚基因组甲基化水平变化。结果显示，与新鲜组相比，玻璃化冷冻Ⅰ组猪PA囊胚的基因组整体甲基化水平极显著降低(18% vs. 20.9%, P < 0.01)，而玻璃化冷冻Ⅱ组显著升高(21.5% vs. 20.9%, P < 0.05)。与新鲜组相比，在玻璃化冷冻Ⅰ组共鉴定到2 717个差异甲基化区域(differential methylation region, DMR)，而在玻璃化冷冻Ⅱ组共鉴定到1 964个DMRs。基因功能富集分析结果显示，玻璃化冷冻导致胚胎发育、ATP代谢、MAPK信号通路、Wnt信号通路等生物学过程相关基因的甲基化水平发生紊乱。在玻璃化冷冻Ⅱ组中，Hippo信号通路、PI3K-Akt信号通路和mTOR信号通路等生物学过程更为活跃。综上所述，玻璃化冷冻改变猪PA囊胚基因组甲基化水平，其可能通过扰乱胚胎发育、ATP代谢、MAPK信号通路、Wnt信号通路等相关基因的甲基化水平进而损害猪PA囊胚冻后发育。此外，Hippo信号通路、PI3K-Akt信号通路和mTOR信号通路可作为缓解玻璃化冷冻损伤的候选途径。

关键词: 猪, 孤雌激活, 囊胚, 玻璃化冷冻, scWGBS

Abstract:

This study aimed to investigate the effect of vitrification on genome methylation level of porcine parthenogenetic activation (PA) blastocysts. The porcine PA blastocysts on day 6 were used for vitrification-thawing experiments using conventional vitrification solution (vitrification group Ⅰ) and improved vitrification solution (vitrification group Ⅱ). Three blastocysts with good morphology were selected from each group for single cell whole-genome bisulfite sequencing (scWGBS) to analyze the genomic methylation levels changes in blastocysts. The results showed that compared with the fresh group, the genomic methylation levels of porcine PA blastocysts in the vitrification group Ⅰ were significantly reduced (18% vs. 20.9%, P < 0.01), while the vitrification group Ⅱ were significantly increased (21.5% vs. 20.9%, P < 0.05). Compared with the fresh group, a total of 2 717 differential methylation regions (DMRs) were identified in the vitrification group Ⅰ, and a total of 1 964 DMRs were identified in the vitrification group Ⅱ. Gene function enrichment analysis showed that vitrification caused disturbances in the methylation levels of genes related to biological processes such as embryonic development, ATP metabolism, MAPK signaling pathway, and Wnt signaling pathway. In the vitrification Ⅱ group, biological processes such as the Hippo signaling pathway, PI3K-Akt signaling pathway, and mTOR signaling pathway were more active. In summary, vitrification changes the genomic methylation level of porcine PA blastocysts, which may damage the development of porcine PA blastocysts by disrupting the methylation levels of genes related to embryonic development, ATP metabolism, MAPK signaling pathway, and Wnt signaling pathway, etc. In addition, the Hippo signaling pathway, PI3K-Akt signaling pathway, and mTOR signaling pathway can be used as candidate pathways to alleviate vitrification injury.

Key words: pig, parthenogenetic activation, blastocyst, vitrification, scWGBS

中图分类号:

S828.2

杨柏高, 徐皆欢, 张亮, 龙熙, 戴建军, 赵学明, 潘红梅. 基于scWGBS探究玻璃化冷冻对猪孤雌激活囊胚基因组甲基化水平的影响[J]. 畜牧兽医学报, 2025, 56(1): 222-231.

YANG Baigao, XU Jiehuan, ZHANG Liang, LONG Xi, DAI Jianjun, ZHAO Xueming, PAN Hongmei. Exploring the Effect of Vitrification on Genome Methylation Level of Porcine Parthenogenetic Activation Blastocysts by scWGBS[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 222-231.

图/表 3

参考文献 40

1	TANG C , HU W Q . Epigenetic modifications during embryonic development: gene reprogramming and regulatory networks[J]. J Reprod Immunol, 2024, 165, 104311. doi: 10.1016/j.jri.2024.104311
2	YANG M , TAO X , SCOTT K , et al. Evaluation of genome-wide DNA methylation profile of human embryos with different developmental competences[J]. Hum Reprod, 2021, 36 (6): 1682- 1690. doi: 10.1093/humrep/deab074
3	YAN R , CHENG X , GU C , et al. Dynamics of DNA hydroxymethylation and methylation during mouse embryonic and germline development[J]. Nat Genet, 2023, 55 (1): 130- 143. doi: 10.1038/s41588-022-01258-x
4	ZHANG M Y , WU X Q , GUO T L , et al. Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos[J]. Theriogenology, 2024, 226, 378- 386. doi: 10.1016/j.theriogenology.2024.06.021
5	UYSAL F , SUKUR G , BOZDEMIR N , et al. Antioxidant supplementation may effect DNA methylation patterns, apoptosis, and ROS levels in developing mouse embryos[J]. Histochem Cell Biol, 2024, 162 (3): 215- 224. doi: 10.1007/s00418-024-02286-w
6	SABER Y H A , IBRAHIM S , MAHMOUD K G M , et al. Expression profile of viability and stress response genes as a result of resveratrol supplementation in vitrified and in vitro produced cattle embryos[J]. Mol Biol Rep, 2024, 51 (1): 692. doi: 10.1007/s11033-024-09614-2
7	ZHU L , SUN L W , LIU W W , et al. Long-term storage does not affect the DNA methylation profiles of vitrified-warmed human embryos[J]. Mol Reprod Dev, 2024, 91 (8): e23713. doi: 10.1002/mrd.23713
8	YU B , SMITH T H , BATTLE S L , et al. Superovulation alters global DNA methylation in early mouse embryo development[J]. Epigenetics, 2019, 14 (8): 780- 790. doi: 10.1080/15592294.2019.1615353
9	ZHANG Z J , XU J W , LYU S J , et al. Whole-genome DNA methylation dynamics of sheep preimplantation embryo investigated by single-cell DNA methylome sequencing[J]. Front Genet, 2021, 12, 753144. doi: 10.3389/fgene.2021.753144
10	BOLGER A M , LOHSE M , USADEL B . Trimmomatic: a flexible trimmer for Illumina sequence data[J]. Bioinformatics, 2014, 30 (15): 2114- 2120. doi: 10.1093/bioinformatics/btu170
11	GUO W L , FIZIEV P , YAN W H , et al. BS-Seeker2:a versatile aligning pipeline for bisulfite sequencing data[J]. BMC Genomics, 2013, 14 (1): 774. doi: 10.1186/1471-2164-14-774
12	GUO W L , ZHU P , PELLEGRINI M , et al. CGmapTools improves the precision of heterozygous SNV calls and supports allele-specific methylation detection and visualization in bisulfite-sequencing data[J]. Bioinformatics, 2018, 34 (3): 381- 387. doi: 10.1093/bioinformatics/btx595
13	BU D C , LUO H T , HUO P P , et al. KOBAS-i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment analysis[J]. Nucleic Acids Res, 2021, 49 (W1): W317- W325. doi: 10.1093/nar/gkab447
14	GOUIL Q , KENIRY A . Latest techniques to study DNA methylation[J]. Essays Biochem, 2019, 63 (6): 639- 648. doi: 10.1042/EBC20190027
15	YAO J F , GENG L X , HUANG R F , et al. Effect of vitrification on in vitro development and imprinted gene Grb10 in mouse embryos[J]. Reproduction, 2017, 154 (3): 197- 205. doi: 10.1530/REP-16-0480
16	NGUYEN H T , SOMFAI T , NGUYEN B X , et al. Oocyte and embryo cryopreservation in porcine in vitro production systems[J]. Indian J Vet Sci Biotechnol, 2024, 20 (4): 1- 5.
17	PÉREZ-GÓMEZ A , GONZÁLEZ-BRUSI L , FLORES-BOROBIA I , et al. PPARG is dispensable for bovine embryo development up to tubular stages[J]. Biol Reprod, 2024, 111 (3): 557- 566. doi: 10.1093/biolre/ioae083
18	CHEN C , HU J H , LING K . The role of primary cilia-associated phosphoinositide signaling in development[J]. J Dev Biol, 2022, 10 (4): 51. doi: 10.3390/jdb10040051
19	YAN S , THIENTHANASIT R , CHEN D Q , et al. CHD7 regulates cardiovascular development through ATP-dependent and-independent activities[J]. Proc Natl Acad Sci U S A, 2020, 117 (46): 28847- 28858. doi: 10.1073/pnas.2005222117
20	AU J , REQUENA D F , RISHIK H , et al. Role of autocrine bone morphogenetic protein signaling in trophoblast stem cells[J]. Biol Reprod, 2022, 106 (3): 540- 550. doi: 10.1093/biolre/ioab213
21	KEYAN K S , SALIM S , GOWDA S , et al. Control of TGFβ signalling by ubiquitination independent function of E3 ubiquitin ligase TRIP12[J]. Cell Death Dis, 2023, 14 (10): 692. doi: 10.1038/s41419-023-06215-y
22	LEWIS E P , AL KHAZAL F , WILBANKS B , et al. Mouse developmental defects, but not paraganglioma tumorigenesis, upon conditional Complex Ⅱ loss in early Sox10⁺ cells[J]. FASEB Bioadv, 2024, 6 (9): 327- 336. doi: 10.1096/fba.2024-00056
23	HEMKEMEYER S A , LIU Z J , VOLLMER V , et al. The RhoGAP-myosin Myo9b regulates ocular lens pit morphogenesis[J]. Dev Dyn, 2022, 251 (11): 1897- 1907. doi: 10.1002/dvdy.522
24	SHAN M M , ZOU Y J , PAN Z N , et al. Kinesin motor KIFC1 is required for tubulin acetylation and actin-dependent spindle migration in mouse oocyte meiosis[J]. Development, 2022, 149 (5): dev200231. doi: 10.1242/dev.200231
25	FU Y J , ZHANG X Y , WU H B , et al. HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway[J]. Front Immunol, 2024, 14, 1258074. doi: 10.3389/fimmu.2023.1258074
26	HIGA R , ROSARIO F J , POWELL T L , et al. Inhibition of MTOR signaling impairs rat embryo organogenesis by affecting folate availability[J]. Reproduction, 2021, 161 (4): 365- 373. doi: 10.1530/REP-20-0603
27	YU W J , CHEN C Z , PENG Y X , et al. Schisanhenol improves early porcine embryo development by regulating the phosphorylation level of MAPK[J]. Theriogenology, 2021, 175, 34- 43. doi: 10.1016/j.theriogenology.2021.08.019
28	SIDRAT T , REHMAN Z U , JOO M D , et al. Wnt/β-catenin pathway-mediated PPARδ expression during embryonic development differentiation and disease[J]. Int J Mol Sci, 2021, 22 (4): 1854. doi: 10.3390/ijms22041854
29	SUN J C , WANG L , MATTHEWS R C , et al. CCND2 modified mRNA activates cell cycle of cardiomyocytes in hearts with myocardial infarction in mice and pigs[J]. Circ Res, 2023, 133 (6): 484- 504. doi: 10.1161/CIRCRESAHA.123.322929
30	NAKANOH S , KADIWALA J , PINTE L , et al. Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells[J]. PLoS One, 2022, 17 (11): e0269122. doi: 10.1371/journal.pone.0269122
31	HAN Z H , RAO J S , GANGWAR L , et al. Vitrification and nanowarming enable long-term organ cryopreservation and life-sustaining kidney transplantation in a rat model[J]. Nat Commun, 2023, 14 (1): 3407. doi: 10.1038/s41467-023-38824-8
32	KURZELLA J , MISKEL D , RINGS F , et al. Mitochondrial bioenergetic profiles of warmed bovine blastocysts are typically altered after cryopreservation by slow freezing and vitrification[J]. Theriogenology, 2024, 214, 21- 32. doi: 10.1016/j.theriogenology.2023.10.002
33	MOGAS T , GARCÍA-MARTÍNEZ T , MARTÍNEZ-RODERO I . Methodological approaches in vitrification: Enhancing viability of bovine oocytes and in vitro-produced embryos[J]. Reprod Domes Anim, 2024, 59 (S3): e14623. doi: 10.1111/rda.14623
34	ANISA-ANNUR S , WAN-HAFIZAH W , NOR-ASHIKIN M , et al. Effect of coenzyme Q10 supplementation on post-vitrification mouse embryo development[J]. Asian Pac J Reprod, 2024, 13 (3): 126- 132. doi: 10.4103/apjr.apjr_136_23
35	WU Z M , GUAN K L . Hippo signaling in embryogenesis and development[J]. Trends Biochem Sci, 2021, 46 (1): 51- 63. doi: 10.1016/j.tibs.2020.08.008
36	XIONG X R , YANG M Z , HAI Z , et al. Maternal Kdm2a-mediated PI3K/Akt signaling and E-cadherin stimulate the morula-to-blastocyst transition revealing crucial roles in early embryonic development[J]. Theriogenology, 2023, 209, 60- 75. doi: 10.1016/j.theriogenology.2023.06.017
37	ZHOU D J , LI X H , LEE S H , et al. GRK2 is critical for the cleavage of the porcine embryo by regulating HSP90 and the AKT pathway[J]. Reproduction, 2024, 168 (4): e230463.
38	ZHAO X Y , CAI X X , ZHU H Y , et al. 27-Hydroxycholesterol inhibits trophoblast fusion during placenta development by activating PI3K/AKT/mTOR signaling pathway[J]. Arch Toxicol, 2024, 98 (3): 849- 863. doi: 10.1007/s00204-023-03664-4
39	CUI C , WU C C , WANG J , et al. Leucine supplementation during late gestation globally alters placental metabolism and nutrient transport via modulation of the PI3K/AKT/mTOR signaling pathway in sows[J]. Food Funct, 2022, 13 (4): 2083- 2097. doi: 10.1039/D1FO04082K
40	LIAO W Y , DENG X , CHEN G D , et al. MiR-150-5p contributes to unexplained recurrent spontaneous abortion by targeting VEGFA and downregulating the PI3K/AKT/mTOR signaling pathway[J]. J Assist Reprod Genet, 2024, 41 (1): 63- 77. doi: 10.1007/s10815-023-02959-w

[1]	孙雅雯, 陈思颍, 李伉, 冷璇, 王栋, 庞云渭. 猪卵母细胞玻璃化冷冻损伤的缓解策略[J]. 畜牧兽医学报, 2025, 56(1): 36-44.
[2]	张素, 孙丽芳, 李兰兰, 吴琳娇, 陈磊清, 吴允昆. 非洲猪瘟病毒结构蛋白与宿主蛋白相互作用研究进展[J]. 畜牧兽医学报, 2025, 56(1): 95-106.
[3]	周显珊, 黄世会, 牛熙, 冉雪琴, 王嘉福. 皱皮香猪泛素化连接酶2基因结构变异的差异表达研究[J]. 畜牧兽医学报, 2025, 56(1): 136-146.
[4]	吴平先, 王俊戈, 刁淑琪, 柴捷, 查琳, 郭宗义, 陈红跃, 龙熙. 基于填充测序数据的荣昌猪群体遗传结构和选择信号分析[J]. 畜牧兽医学报, 2025, 56(1): 147-158.
[5]	阳文攀, 刘相杰, 罗冬香, 陈梦会, 谢瑛, 方跃鑫, 林婷燕, 李爱民, 李文静, 邓政, 丁能水. 基于芯片数据的长白猪繁殖性状基因组选择研究[J]. 畜牧兽医学报, 2025, 56(1): 213-221.
[6]	范定坤, 张涛, 焦帅, 陆伟, 付域泽, 杨宏, 屠焰, 石玲元, 张乃锋. 基于斜率比法评价断奶仔猪对高温烧结法磷酸三钙的相对生物学利用率[J]. 畜牧兽医学报, 2025, 56(1): 269-280.
[7]	蔡云凤, 李宏, 黄小铭, 何庆, 丁振宇, 余婉婷, 罗施乐, 陈方志, 王乃东, 杨毅, 湛洋. 猪圆环病毒2型病毒样颗粒三倍轴区域嵌合猪细小病毒抗原表位的展示策略及免疫原性[J]. 畜牧兽医学报, 2025, 56(1): 307-318.
[8]	曾苗苗, 杨小曼, 张鑫, 刘大凯, 时洪艳, 张记宇, 张燎原, 陈建飞, 冯廷帅, 李修文, 石达, 冯力. 猪急性腹泻综合征冠状病毒N蛋白间接ELISA抗体检测方法的建立及初步应用[J]. 畜牧兽医学报, 2025, 56(1): 319-326.
[9]	曾焱, 欧祥龙, 闫晓阳, 刘灿, 廖永洪. 猪胸膜肺炎放线杆菌OmpD、LppB蛋白的原核表达及免疫原性分析[J]. 畜牧兽医学报, 2025, 56(1): 343-352.
[10]	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.
[11]	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.
[12]	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.
[13]	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.
[14]	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.
[15]	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.

基于scWGBS探究玻璃化冷冻对猪孤雌激活囊胚基因组甲基化水平的影响

Exploring the Effect of Vitrification on Genome Methylation Level of Porcine Parthenogenetic Activation Blastocysts by scWGBS

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 3

参考文献 40

相关文章 15

编辑推荐

Metrics

本文评价